# Patent

## Title
Methods and compositions for obtaining high-resolution crystals of membrane proteins

## Abstract

The invention describes compositions and method useful for the crystallization of membrane proteins.

## Background
RELATED APPLICATIONS
This invention claims the benefit of U.S. provisional applications 60/999,951, filed Oct. 22, 2007; U.S. provisional application 61/000,325, filed Oct. 24, 2007; U.S. provisional application 61/060,107, filed Jun. 9, 2008; and U.S. provisional application 61/194,961, filed Oct. 1, 2008, each of which is incorporated herein by reference, in its entirety, for all purposes.


STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant Nos. GM73197 awarded by the National Institutes of Health; GM74691 and GM62411 awarded by the Protein Structure Initiative; Y1-CO-1020 awarded by the National Cancer Institute; and yl-GM-1104 awarded by the National Institute of General Medical Sciences. The government has certain rights in this invention.


Coordinates and structure factors have been deposited in the Protein Data Bank with identification code 2RH1.
REFERENCE TO SEQUENCE LISTING
This application contains a computer-readable Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 21, 2010, is named “14675_US_Sequence Listing.txt”, lists 9 sequences, and is 12 kb in size.
BACKGROUND OF THE INVENTION
Naturally occurring polypeptides or proteins often fold into complex, three-dimensional shapes that determine both chemical and physiological functionality. Thus a thorough understanding of proteins necessarily involves a detailed representation of their spatial topography. The field of protein crystallography has flourished over the last 20 years resulting in a rapid increase in the knowledge bas of protein structure enabling great strides in other disciplines including biochemistry, pharmaceutical development and cell biology. However, the structural biology field has largely been restrained to working with protein that is naturally soluble in aqueous media, or made soluble by incorporation into surfactant micelles. The present invention provides methods and compositions that allow for the study of membrane-embedded proteins (i.e., integral membrane proteins) in a more natural membrane bilayer environment. The present invention enable a more detailed analysis of important classes of membrane-embedded polypeptides that play key roles in a variety of cellular processes including energy and signal transduction.
SUMMARY OF THE INVENTION
Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for all purposes and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
In one aspect the invention provides compositions, e.g., useful for the crystallization of membrane protein. In some embodiments, the composition is suitable for lipidic cubic phase crystallization. In some embodiments the compositions contain 10-60% v/v of a polyethylene glycol, 0.01-0.5 M of a salt, 1-20% v/v of an organic compound, and 1-50% w/w of a lipid additive in a host lipid. In some embodiments of the compositions, the protein to be crystallized is present at a concentration of 1 to 100 mg/mL. In some embodiments, the protein to be crystallized is present at a concentration of 50 mg/mL.
In some embodiments of the compositions of the inventions, the polyethylene glycol is PEG or modified PEG at a molecular size of 10-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-2000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-1000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEGcorn oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).
In some embodiments of the compositions, the salt is a sulfate salt. In some embodiments, the sulfate salt is sodium sulfate. In some embodiments, the salt is present at a concentration of 0.1-0.5 M. In some embodiments, the salt is present at a concentration of 0.1-0.2 M. In some embodiments, the compositions of the invention contain a buffer. In some embodiments, the buffer is present at a concentration of 0.01-0.5 M. In some embodiments, the buffer is present at a concentration of 0.1-0.2 M. In some embodiments, the buffer is present at a concentration of 0.1 M. In some embodiments, the buffer is Bis-tris propane. In some embodiments, the buffer has a pH 6.5-7.0. In some embodiments of the compositions, an organic compound is present at a concentration of 1-10% v/v. In some embodiments, the organic compound is present at a concentration of 5-7% v/v. In some embodiments, the organic compound is 1,4-butanediol.
In some embodiments of the compositions, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include but are not limited to cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments, the host lipid is monoolein.
In some embodiments of this aspect, the composition of the invention comprises 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid.
In another aspect the invention includes compositions suitable for lipidic cubic phase crystallization. In some embodiments, the compositions of the inventions contain a lipid additive. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.
In another aspect the invention includes methods for crystallization of membrane proteins. In some embodiments, the method for crystallization of membrane proteins comprises adding a lipid additive to a lipidic cubic phase. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.
In another aspect, the invention provides for methods of crystallization of a protein. In some embodiments of this aspect, the method comprises, providing said protein in a lipidic cubic phase composition, filling a plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic) with said composition, placing said plate containing said composition under conditions suitable for crystallization of said protein and detecting the presence of a crystal of said protein in said plate. In some embodiments, the method further comprises covering said plate with a second plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic).
In some embodiments of the methods, the protein is a non-colored protein. In some embodiments, the protein is a G protein-coupled receptor (GPCR). In some embodiments, the protein comprises a β2AR, a CXCR4, or a human adenosine A2A receptor. In some embodiments the protein comprises a stabilizing point mutation or a T4 lysozyme fusion or both.
In some embodiments of the methods, the lipidic cubic phase composition comprises a lipid additive. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.
In some embodiments of the methods, the first plate and second plate are made of glass. In some embodiments, the plate allows for the control of crystallization conditions, such as the humidity and temperature of said lipidic cubic phase composition.
In some embodiments of the methods, the crystals are harvested directly from the plate. In some embodiments of the methods, the crystals are harvested between the cubic and the sponge phase of the lipidic cubic phase composition. In some embodiments of the methods, the crystals are harvested directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen.
In another aspect the invention provides methods for screening a crystal present in a liquid cubic phase composition. In some embodiments, the method comprises exposing the composition to a first beam and determining a change of the first beam, exposing the composition to a second beam and determining a change of the second beam, and identifying an area where the crystal is present in said composition. Examples of changes in the beams, include but are not limited to, change in direction and/or intensity of the beams. In some embodiments the crystals are non-colored.
In some embodiments of the methods, the first beam and second beam are attenuated. In some embodiments the beams are attenuated 10 times. In some embodiments, the first beam is a slitted 100×25 μm beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to a third beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to up to ten extra beams. In some embodiments, the exposure of composition to the beams is 2 seconds. In some embodiments, the beams are beams of visible light.
In another aspect the invention includes a crystal of a membrane protein. In some embodiments, the invention includes a crystal of a G protein-coupled receptor (i.e., a “GPCR”) non-covalently bound to a ligand. In some embodiments, the extracellular domain of said crystalline GPCR is resolvable by X-Ray crystallography. In some embodiments, the ligand is a diffusible ligand.
In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of crystal is greater than 40×20×5 μm. In some embodiments, volume of the crystal is estimated assuming that each of the stated dimensions are orthogonal so that the volume estimate is the product of the dimensions. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, X-ray crystallographic analysis can be carried out to determine the structure of a protein comprising said crystal.
In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystal diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.
In some embodiments, the G protein-coupled receptor is a β2AR protein, a CXCR4 protein, or a human adenosine A2A receptor protein.
In another aspect the invention provides for a crystal of β2AR. In some embodiments, the structure of an extracellular domain of said crystal is capable of being resolved by X-ray crystallography. In some embodiments, the crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341 and an acetamide molecule bound to Cys2656.27, a diffusible ligand, up to 10 molecules a lipid additive, up to five salt ions and up to 10 butanediol molecules. In some embodiments, the lipid additive is cholesterol. In some embodiments, the crystal comprises three cholesterol molecules. In some embodiments, the salt ion is a sulfate ion. In some embodiments, the crystal comprises two sulfate ions. In some embodiments, the diffusible ligand is carazol. In some embodiments the crystal comprises two butanediol molecules. In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of the crystal is greater than 40×20×5 μm. In some embodiments the volume of the crystal is estimated by assuming that each dimension is orthogonal to the other dimensions so that the volume is the product of the three lengths. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, the structure of a β2AR protein can be determined from said crystal using X-ray crystallographic analysis. In some embodiments, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystals diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.
In another embodiment, the invention provides a composition for lipidic cubic phase crystallization of a membrane protein, comprising a polyethylene glycol or modified polyethylene glycol; 0.01-1M of a salt; a host lipid; a lipid additive, wherein said lipid additive is present at 10-60% v/v ratio relative to the host lipid; a buffer; and 1 to 100 mg/ml of a membrane protein. In a related embodiment, the polyethylene glycol is PEG or modified PEG, wherein said PEG or modified PEG has an average molecular weight of 200-20,000, 400-8000, or 400-2000. In yet another related embodiment, the PEG or modified PEG in the composition has an average molecular weight of 400. In another related embodiment, the salt is selected from the group consisting of a sulfate salt, a citrate salt, a malonate salt, a tartrate salt, an acetate salt, and a formate salt. In certain embodiments of the composition, the salt is present at a concentration of 0.1-0.2 M. In another related embodiment, the buffer is present at a concentration of 0.05-0.5 M in the composition. In certain embodiments, the buffer is Bis-tris propane or sodium citrate. In other related embodiments of the composition, the buffer has a pH between 4.5-8.0.
In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the composition further comprising an alcohol present at a concentration of 1-10% v/v or 5-7% v/v. In certain embodiments, the alcohol is a diol or triol. In other embodiments, the alcohol is 1,4-butanediol or 2,6-hexanediol.
In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w in a host lipid or 8-10% w/w in a host lipid. In yet another related embodiment of the composition, the lipid additive is selected from the group consisting of 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPC”), Asolectin, and a sterol. In still other embodiments, the lipid additive is a sterol. In related embodiments, the lipid additive is cholesterol.
In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the host lipid is selected from the group consisting of monopalmitolein, monovaccenin and monoolein. In a related embodiment, the host lipid is monoolein. In still another related embodiment, the membrane protein to be crystallized in said composition is present at a concentration of 1 to 100 mg/mL. In yet another embodiment, the memrane protein to be crystallized in said composition is present at a concentration of 40-60 mg/mL.
In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein membrane protein is a G-protein coupled receptor, such as a β2AR protein, a human adenosine A2A receptor protein, a CXCR4-T4L protein, or a β2AR-T4L protein. In related embodiments, the G-protein coupled receptor is a protein comprising or consisting of a β2AR(E122W), a β2AR(E122W)-T4L, a human adenosine A2A receptor-T4L, a CXCR4-T4L or β2AR-T4L. In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the composition comprises a ligand selected from the group consisting of carazolol, timolol, alprenolol, and clenbutorol.
In another embodiment, the invention provides a method of generating crystals of a membrane protein comprising: mixing a lipid additive with a host lipid to form a lipid mixture, wherein said lipid additive is selected from the group consisting of a sterol, DOPE, DOPE-Me, DOPC, and Asolectin, and wherein said lipid additive is 5 to 20% w/w in said host lipid; and combining said lipid mixture with a membrane protein solution under conditions suitable for the formation of a lipidic cubic phase composition. In a related embodiment of the method, said protein is a non-colored protein. In a related embodiment, the amount of said lipid additive is 8 to 10% w/w in said lipid. In another related embodiment, the invention further comprises: filling a plate with said lipidic cubic phase composition, wherein said plate is compatible with imaging; placing said plate containing said lipic cubic phase composition under conditions suitable for crystallization of said protein; and detecting the presence of a crystal of said protein in said plate. In another embodiment, the method further comprises covering said plate with a second plate.
In a related embodiment of the method of generating crystals of a membrane protein, the protein is a GPCR. In yet another related embodiments, the protein comprises a β2AR. In yet another related embodiment, the β2AR protein is selected from the group consisting of β2AR(E122W), β2AR(E122W)-T4L, and β2AR-T4L. In yet another related embodiment, the GPCR is a human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.
In yet another related embodiment of method of generating crystals of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w or 8-10% w/w in a host lipid. In yet another related embodiment of the method, the second plate comprises a glass. In yet another related embodiment, the method further comprises harvesting crystals directly from said plate. Another related embodiment of the method comprises harvesting crystals from between the cubic and the sponge phase of said lipidic cubic phase composition. In another related embodiment, the method comprises harvesting crystals directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen. In yet another related embodiment, the method comprising a step of soaking into said crystal a diffusable ligand or candidate ligand.
The invention also provides a method of screening a crystal of a GPCR present in a liquid cubic phase composition comprising: preparing a liquid cubic phase composition comprising a GPCR protein, a host lipid, and a lipid additive; exposing said composition to a first X-ray beam and determining a change in direction or intensity of said first X-ray beam; exposing said composition to a second beam and determining a change in direction or intensity of said second X-ray beam; identifying an area where said GPCR crystal is present in said composition; and exposing said identified area to at least a third X-ray beam. In a related embodiment, the first beam is a slitted 100×25 μm beam. In another related embodiment, the crystal is colorless. In yet another embodiment, the GPCR crystal is β2AR(E122W)-T4L, β2AR(E122W), β2AR, or β2AR-T4L protein. In related embodiments, the crystal is human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.
In another embodiment, the invention provides a crystal of a human β2AR protein wherein the extracellular loop ECL2 of said β2AR is sufficiently ordered to produce interpretable electron density in a crystallographically-derived electron density map. In yet another related embodiment, each β2AR molecule in said crystal comprises three non-covalently bound cholesterol molecules and at least one salt ion. In yet another related embodiment, the at least one salt ion is a sulfate ion. In yet another related embodiment, each β2AR molecule in said crystal said crystal comprises two sulfate ions. In yet another related embodiment, each β2AR molecule in said crystal said crystal further comprises carazol. In yet another related embodiment of the crystal, each β2AR molecule in said crystal comprises between 1 and 10 butanediol molecules. In yet another related embodiment of the crystal, the volume of said crystal exceeds 15×5×1 μm, 30×5×5 μm or 40×20×5 μm. In yet another related embodiment, the specific surface area of the crystal is 0.0001-5 m2/g. In yet another related embodiment, the crystal is crystallized using liquid cubic phase crystallization. In yet another related embodiment, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In yet another related embodiment, the crystal diffracts to a resolution of 2.0 to 10.0 Å, 2.0 to 5.0 Å, or 2.2 to 2.4 Å. In yet another related embodiment, the structure of said crystal is solved and refined at a resolution higher than about 3.2, higher than about 2.8, or higher than about 2.4 Å.
In yet another embodiment, the invention provides a crystalline form of a human β2AR protein having an atomic arrangement of coordinates comprising the β2AR coordinates set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance).
In another embodiment, the invention provides a crystalline form of a human β2AR protein, where said form has unit cell dimensions of a=106.3 Angstroms, b=169.2 Angstroms, and c=40.2 Angstroms. In a related embodiment, said β2AR protein is β2AR-T4L. In another related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.
In another embodiment, the invention provides a crystalline form of a human β2AR protein, wherein said space group of said crystalline form is C2. In a related embodiment, said β2AR protein is β2AR-T4L. In another related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.
In another embodiment, the invention provides a crystalline form of a human β2AR protein, wherein said crystalline form diffracts X-rays to a resolution of 2.4 Angstroms. In a related embodiment, said β2AR protein comprises a point mutation that stabilizes said β2AR protein. In another related embodiment, said β2AR protein is β2AR-T4L. In a related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.
In another embodiment, the invention provides a crystalline form of a human β2AR protein wherein each β2AR molecule in said crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341, an acetamide molecule bound to Cys2656.27, a diffusible ligand, one to ten molecules of a lipid additive, one to five salt ions and one to ten butanediol molecules.
In another embodiment, the invention provides a method of identifying a compound that binds to a ligand binding site of a human β2AR protein, comprising comparing a set of three-dimensional structures representing a set of candidate compounds with a three-dimensional molecular model of said ligand binding site, comprising: receiving a three-dimensional model of a ligand binding site on said human β2AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand-binding residues, wherein said atomic co-ordinates are taken from Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance); determining, for each of the set of compound three-dimensional models, a plurality of distance values indicating distances between said atomic co-ordinates of said candidate compound of the set of candidate compounds and said atomic coordinates of said ligand-binding residues comprising said ligand binding site; determining, for each of the set of compound three-dimensional models, a binding strength value based on the plurality of distance values determined for the compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human β2AR protein and a compound represented by the compound three-dimensional model; and storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values. In a related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of Y199, A200, S204, T118, V117, W286, Y316, F290, F289, N293, W109, F193, and Y308. In another related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of W109, V117, T118, F193, Y199, A200, W286, F289, F290, Y316. In another related embodiment of the method of identifying a compound that binds to a ligand binding site of a human β2AR protein, said binding strength value is based on one or more of a hydrogen bonding strength, a hydrophobic interaction strength, or a Coulombic interaction binding strength. In another related embodiment, one or more of said receiving, determining, or storing steps is carried out using a commercially-available software program. In yet another related embodiment, the commercially-available software program is selected from the group consisting of DOCK, QUANTA, Sybyl, CHARMM, AMBER, GRID, MCSS, AUTODOCK, CERIUS II, Flexx, CAVEAT, MACCS-3D, HOOK, LUDI, LEGEND, LeapFrog, Gaussian 92, QUANTA/CHARMM, Insight II/Discover, and ICM. In yet another related embodiment, the method further comprising the step of contacting a human β2AR protein with a molecule comprising an identified candidate compound. In yet another related embodiment, the molecule further comprises a moiety capable of competitively displacing a ligand from said human β2AR protein, wherein said ligand binds to said ligand binding site of said human β2AR protein. In yet another related embodiment, the method further comprising characterizing a binding interaction between said human β2AR protein and said molecule comprising said identified candidate compound, and storing a result of said characterizing. In yet another related embodiment, said characterization comprises determining an activation of a function of said human β2AR protein, an inhibition of a function of said human β2AR protein, an increase in expression of said human β2AR protein, a decrease in expression of said human β2AR protein, a displacement of a ligand bound to said ligand binding site, or a stability measure for said human β2AR protein.
In another embodiment, the invention provides a method for selecting a library of potential modulators of β2AR to be screened, comprising calculating a structure of a first potential modulator using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance), correlating said structure of said first potential modulator with a library of modulators identified as comprising said structure said first potential modulator, and storing or transmitting information about the identified library.
In yet another embodiment, the invention provides a method of solving the structure of a crystalline form of a protein, comprising: using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) to solve the structure of the crystalline form of a test protein, wherein said test protein has significant amino acid sequence homology to any functional domain of β2AR; and transmitting or storing data descriptive of the structure of said test protein.
In another embodiment, the invention provides a method of identifying from a set of candidate compound three-dimensional models a compound that binds to a ligand binding site of a GPCR or β2AR protein comprising: receiving a three-dimensional model of a ligand binding site on said GPCR or β2AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand binding residues; determining, for each candidate compound of the set of candidate compound three-dimensional models, a plurality of distance and angle values indicating distances and angles between atomic co-ordinates of said candidate compound of the set of candidate compound three-dimensional models and said ligand binding site comprising atomic coordinates of said ligand-binding residues; determining, for each of the set of candidate compound three-dimensional models, a binding strength value based on the plurality of distance and angle values determined for the candidate compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human GPCR or β2AR protein and a compound represented by the compound three-dimensional model; storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values; searching a database of small organic molecules for compounds exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on the binding strength values; and identifying the set of small organic molecules exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on binding strength values as likely to also bind to the GPCR or β2AR the database of small organic molecules is the available chemicals database. In a related embodiment, the shape, chemistry or electrostatic similarity is determined using a program selected from the group consisting of BROOD (openeye), EON (openeye), ROCS (openeye), ISIS Base, and SciFinder.
In another embodiment, the invention provides a method of identifying a ligand that binds to a membrane protein comprising: preparing a lipid meso phase, wherein said lipid meso phase composition comprises (1) a host lipid; (2) said membrane protein; (3) a lipid additive selected from the group consisting of consisting of a sterol, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin, wherein said lipid additive is 1 to 50% w/w in a lipid host; subjecting said lipid meso phase to humidity and temperature conditions to grow crystals of said membrane protein; contacting said membrane protein crystals with diffusible ligands or a mixture of diffusible ligands; determining the three-dimensional structure of said diffusible ligand contacted membrane protein crystals by X-ray crystallography to obtain an electron density map; and identifying bound ligands by inspection of the electron density map. In a related embodiment, the ligands are substantially insoluble in water.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be Provided by the Office upon request and payment of the necessary fee.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1. Crystals of β2AR-T4L obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. FIG. 1a shows β2AR-T4L crystals in the crystallization mixture drop (upper left) and in the loop. FIG. 1b shows crystals in the “sponge” phase.
FIG. 2. Before (top) and after (bottom) images of a lipidic cubic phase crystal harvested directly from a well in a previously sealed glass sandwich plate, according to the method described herein (see, e.g., Example 1).
FIG. 3. Diffraction pattern (2.8 Å resolution) from β2AR-T4L crystals grown in lipid cubic phase. The crystal size was approximately 25×5×5 Å; space group C2 (a=106.8 Å, b=169.5 Å, c=40.5 Å; β=105.3°, α=γ=90°. Beam diameter was 10 μm, exposure 10 s, oscillation: 1°.
FIG. 4. Gallery of crystals of various GPCRs obtained using LCP/cholesterol mixtures and in combination with a variety of ligands. The top panel corresponds to non-optimized initial hits, whereas the bottom panel shows diffraction quality for optimized crystals. From left to right, β2AR-T4L (bound to carazolol), diffracted to 2.4 Å resolution; β2AR(E122W)T4L (bound to carazolol), diffracted to 3.5 Å resolution; β2AR(E122W)T4L (bound to alprenolol), diffracted to 3.5 Å resolution; β2AR(E122W)T4L (bound to timolol), diffracted to 2.8 Å resolution; β2AR(E122W) (bound to carazolol); β2AR(E122W)T4L (bound to clenbuterol), diffracted to 6 Å, anisotropic; human A2A adenosine receptor-T4L (bound to ZM241385), diffracted to 2.6 Å resolution.
FIG. 5. A. β2AR-T4L Crystals grown from bicelle conditions. B. Diffraction image from bicelle grown microcrystals of β2AR-T4L recorded using 10 μm minibeam on 23ID-B beamline at APS. Black circle is drawn at resolution 3.5 Å.
FIG. 6. A. Microcrystals of β2AR-T4L grown in lipidic mesophase. B. Diffraction image from lipidic cubic phase grown microcrystals of β2AR-T4L recorded using a 10 μm minibeam on 23ID-B beamline at APS. The white circle is drawn at resolution 2.2 Å.
FIG. 7. Detailed representation of the carazolol binding site in β2AR-T4L. FIG. 7A, B, and C are representations of the electron density of the ligand binding site at three different orientations. Residues are labeled by their Ballesteros-Weinstein numbers as superscripts. Electon density is contoured at 1σ from a 2 Fo−Fc difference map. Both B and C are generated by rotating the field of view 90° about the y-axis clockwise and counterclockwise respectively.
FIG. 8. Electron density of: A. Cholesterol molecules shown with a Fo−Fc electron density contoured at 2σ omitting the lipid from phase calculation, palmitic acid is also shown. B. Helix-kinked region with 2Fo—Fc electron density contoured at 1.5σ.
FIG. 9. Overall fold of the β2AR-T4L fusion with its predicted orientation in the plasma membrane and key intramolecular interactions. A. Stereoview of the overall fold of β2AR-T4L. The receptor and T4L are colored gray and green, respectively. Carazolol is colored blue and the lipid molecules bound to the receptor are colored yellow. B. The receptor is aligned to a rhodopsin model that was positioned in a lipid membrane (boundaries indicated by horizontal black lines) as found in the orientations of proteins in membranes (OPM) database (M. A. Lomize et al., Bioinformatics 22, 623 (2006)). T4L is fused internally into the third intracellular loop of β2AR and maintains minimal intramolecular packing interactions by tilting away from the receptor. C. Specific intramolecular interactions between β2AR and T4L are represented.
FIG. 10. Crystal packing interactions in the lipidic mesophase crystallized β2AR-T4L. A. There are four main contact areas, two of which are mediated by T4L in the plane of the membrane with itself through a two-fold symmetry axis and translation. The third interaction is normal to the membrane plane between T4L and lumen exposed loops of β2AR. The fourth interaction is generated by the two-fold symmetry axis, packing one receptor to receptor in the plane of the membrane. B. The receptor crystal packing interface is composed mainly of lipids with two cholesterol molecules and two palmitic acid molecules forming the majority of the interactions. A network of ionic charge interactions exists on the cytoplasmic end of the interface forming the only inter-receptor protein contacts. C. Comparison between β2AR-T4L and rhodopsin (PDB ID Code 2135) parallel receptor association interface. Helices I (blue) and VIII (magenta) are highlighted in both structures. Only one monomer is shown for each receptor representation along with helices I′ and VIII′ only from the opposing symmetry related molecule. The rhodopsin interface is twisted significantly relative to β2AR-T4L resulting in a significant offset from the parallel orientation required for a physiological dimer interface. β2AR-T4L associated monomers are in a highly parallel orientation.
FIG. 11. Surface representation of β2AR colored by calculated charge from red (−10 kbT/ec) to blue (+10 kbT/ec) using a dielectric constant of 70. A. Three main areas of interest are indicated. The binding site cleft is negatively charged as is a groove between helices III, IV and V. The third region is an overall positive charge in the region of the ionic lock and DRY motif on the cytoplasmic face. The overall result is a highly polarized molecule that may utilize its negative charge to facilitate binding of catecholamine ligands. The presence of a negative charge in the groove between helices III, IV and V is unexpected as it is in the middle of the lipid membrane. This charge may be partially derived from the presence of an unpaired glutamate at position 1223.41. The effective charge in this region is likely greater than shown here due to its location in the low dielectric environment of the lipid membrane. B. View rotated 90° from A. Showing both the negatively charged binding site cleft (top) and positively charged cytoplasmic face (bottom). Poisson-Boltzmann electrostatics were calculated using the program APBS (Baker et al., Proc Natl Acad Sci USA, 98, 10037 (2001)) as implemented in Pymol (The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org). Pymol was used exclusively in the preparation of all figures.
FIG. 12. Comparison of the extracellular sides of β2AR-T4L and rhodopsin. A. The N-terminus is missing from the experimental density in the β2AR-T4L structure and is not shown. ECL2 is shown in green and contains a short α-helix and two disulfide bonds (yellow). The intraloop disulfide bond constrains the tip of ECL2 which interacts with ECL1. The second disulfide bond links ECL2 with helix III. There is one interaction between ECL2 and carazolol (blue) through Phe1935.32. The entire loop is held out of the ligand binding site by a combination of the rigid helical segment and the two disulfide bonds. B. In contrast, ECL2 (green) in rhodopsin assumes a lower position in the structure that occludes direct access to the retinal-binding site and forms a small β-sheet in combination with the N-terminal region (magenta) directly above the bound retinal (pink).
FIG. 13. Ligand binding characterization and comparison to rhodopsin. A. A view looking down on the plane of the membrane from the extracellular surface showing a detailed representation of the carazolol binding site in β2AR-T4L. Carazolol is shown as sticks with carbon atoms colored yellow. β2AR-T4L residues contributing to carazolol binding are shown in green and labeled. Electron density is contoured at 5σ from an Fo−Fc omit map calculated without the contribution of carazolol. B. Binding orientation comparison between 11-cis-retinal in rhodopsin and carazolol in β2AR-T4L. Van der Waals' surfaces for carazolol and retinal are represented as dots to accentuate the close packing interactions. Retinal in the all-cis conformation (pink), binds deep in the active site of rhodopsin as compared to carazolol (blue), packing its β-ionone ring between Tyr2686.51 and Phe2125.47 (cyan), blocking movement of Trp2656.48 (magenta) into the space. The β-ionone ring of trans-retinal in activated rhodopsin would not block Trp2656.48 from rotating into the space allowing a rotameric shift into its proposed active form. C. There are four residues involved in the toggle switch mechanism of β2AR-T4L as shown. Phe2906.52 (magenta) is sandwiched between Phe2085.47 (tan) and Phe2896.51 (tan) forming a ring-face aromatic interaction. Like rhodopsin, an activation step is thought to occur by a rotameric change of Trp2866.48 (magenta) which would displace Phe2906.52. Carazolol is shown to interact extensively with the sandwich motif as shown: however, few interactions are seen with Trp2866.48. The 6.52 position in β2AR-T4L is occupied by Phe2906.52 as opposed to Ala2696.52 in rhodopsin where the β-ionone ring replaces an aromatic protein side chain in forming the sandwich interactions. The aromatic character of the sandwich is otherwise maintained by Phe2896.51 and Phe2085.47 in β2AR-T4L.
FIG. 14. Comparison of β2AR-T4L helical orientations with rhodopsin (PDB ID Code 1U19). A. β2AR-T4L is rendered as a ribbon trace colored with a blue to red spectrum corresponding to observed distances between Cα positions in the two structures (RMSD 2.7 Å between all residues in the transmembrane region). Helix II shows very little movement, whereas the entire lengths of helices III, IV, V shift significantly. Helix VIII and loops were not included in the comparison and are colored in tan. B. Movements of helices I and V of rhodopsin (grey) are shown relative to β2AR-T4L. C. Movements of helices III, IV and VI. D. Ligand binding site representation. Carazolol is shown with yellow carbons. Entire helices are assigned a single designation based on their divergence from the rhodopsin position in the area of the ligand binding site as shown. Helix I is highly divergent, Helices II and VI are similar to rhodopsin. Helices IV and VII are moderately constant. Helices III and V are moderately divergent.
FIG. 15. Affinity curves for adrenergic ligands binding to β2AR-T4L and wildtype β2AR. Saturation curves for the antagonist [3H]DHA is shown at left, next to competition binding curves for the natural ligand (−)-Epinephrine and the high-affinity synthetic agonist Formoterol. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described above.
FIG. 16. Comparison of the proteolytic stability between the wild-type β2AR and β2AR-T4L in a limited trypsin proteolysis assay. TPCK-trypsin was added to carazolol-bound, purified, dodecylmaltoside-solubilized receptor at a 1:1000 ratio (wt:wt), and samples were analyzed by SDS-PAGE. Intact β2AR-T4L (56.7 kD) and FLAG-tagged wild-type β2AR (47.4 kD) migrate similarly as  ̃55 kD bands. Markers are Biorad low-range SDS-PAGE protein standards.
FIG. 17. Stability comparison of unliganded β2AR365 and β2AR-T4L. For dodecylmaltoside-solubilized receptor preparations, maintenance of the ability to specifically bind [3H]DHA after incubation at 37° C. is taken as a measure of stability.
FIG. 18. Superimposed Cα traces of the receptor component of β2AR-T4L (in blue) and β2AR365 (in yellow). Common modeled transmembrane helix regions 41-58, 67-87, 108-137, 147-164, 204-230, 267-291, 312-326, 332-339 were used in the superposition by the program Lsqkab (The CCP4 Suite, Acta Crystallogr D Biol Crystallogr 50, 760 (1994)) (RMSD=0.8 Å).
FIG. 19. Carazolol dissociation from β2AR365. Dodecylmaltoside-solubilized carazolol-bound receptor (at 50 μM) was dialyzed in a large volume of buffer containing 300 micromolar alprenonol as a competing ligand, and aliquots were removed from the dialysis cassette at different time points. Remaining bound carazolol was measured (in a relative sense) by collecting fluorescence emission with excitation at 330 nm and emission from 335 to 400 nm. For each carazolol fluorescence measurement, data was normalized for the protein concentration in the dialysis cassette (measured with the Bio-Rad Protein DC kit). The Y-axis represents carazolol fluorescence emission Intensity (in cps) at 341 nm. The exponential decay of carazolol concentration in the receptor dialysis cassette was fit using Graphpad Prism software, giving a half-life of 30.4 hrs.
FIG. 20. Comparison of β1 and β2AR sequences. After aligning the β1 and β2AR sequences, positions that have different amino acids between the two receptors were mapped onto the high-resolution structure of β2ART4L (shown as red sticks). The carazolol ligand is shown as green sticks (with nitrogens in blue and oxygens in red). Highlighted residues Ala852.56, Ala922.63 and Tyr3087.35 are homologous to amino acids Leu1102.56, Thr1172.63 and Phe3597.35 of the PAR, which were shown to be primarily responsible for its selectivity over β2AR for the compound RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)). In the β2AR-T4L structure, only Tyr3087.35 faces the ligand, while Ala852.56 lies at the interface between helices II and III. Of all the divergent amino acids, only Tyr3087.35 is found within 4 Å of any atom of carazolol.
FIG. 21. Design and optimization of the β2AR-T4L fusion protein A. The sequence of the region of the β2AR targeted for insertion of a crystallizable domain is shown (SEQ ID NO:2), and the positions of the junctions between the receptor and T4L (in red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. Peptide ‘LNKYADWT’ disclosed as SEQ ID NO 3 B Immunofluorescence images of HEK293 cells expressing selected fusion constructs. Panels on the left shows M1 anti-FLAG signal corresponding to antibody bound to the N-terminus of the receptor. Panels on the right show the same signal merged with blue emission from DAPI (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3 and D1, while C3 and D5 are retained in the endoplasmic reticulum.
FIG. 22. Functional characterization of β2AR-T4L. A. Affinity competition curves for adrenergic ligands binding to β2AR-T4L and wild-type β2AR. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described in the methods section of Example 4. B. β2AR-T4L is still able to undergo ligand-induced conformational changes. Bimane fluorescence spectra (excitation at 350 nm) of detergent-solubilized β2AR-T4L and wild-type β2AR truncated at 365, labeled under conditions that selectively modify Cys2656.27 (see methods section of Example 4), were measured after incubating unliganded receptor with compounds for 15 min at room temperature. The cartoon illustrates that the observed changes in fluorescence can be interpreted as a movement of the bimane probe from a more buried, hydrophobic environment to a more polar, solvent-exposed position.
FIG. 23. A. Side-by-side comparison of the crystal structures of the β2AR-T4L fusion protein and the complex between β2AR365 and a Fab fragment. The receptor component of the fusion protein is shown as a blue cartoon (with modeled carazolol as red spheres), while the receptor bound to Fab5 is in yellow. B. Differences in the environment surrounding Phe2646.26 (shown as spheres) for the two proteins. C. The analogous interactions to the “ionic lock” between the E(D)RY motif and Glu2476.30 seen in rhodopsin (right panel, darkened) are broken in both structures of the β2AR (left panel). Pymol (W. L. DeLano, The PyMOL Molecular Graphics System (2002) on the World Wide Web http://www.pymol.org) was used for the preparation of all figures.
FIG. 24. Schematic representation of the interactions between β2AR-T4L and carazolol at the ligand binding pocket. Residues shown have at least one atom within 4 Å of the ligand in the 2.4 Å resolution crystal structure.
FIG. 25. The ligand binding pocket of β2AR-T4L with carazolol bound. A. Residues within 4 Å of the ligand are shown as sticks, with the exception of A200, N293, F289, and Y308. Residues that form polar contacts with the ligand (distance cutoff 3.5 Å) are in green, other residues are gray (in all panels, oxygens are colored red and nitrogens are blue). B. Same as panel A, except that the ligand is oriented with its amine facing out of the page. W109 is not shown. C. Packing interactions between carazolol and all residues within 5 Å of the ligand. View is from the extracellular side of the membrane. Carazolol is shown as yellow spheres, receptor residues are shown as sticks within van der Waals dot surfaces. D. Model of (−)-isoproterenol (magenta sticks) in the ligand binding pocket observed in the crystal structure. A model of the agonist with optimal bond lengths and angles was obtained from the PRODRG server (Schuettelkopf, et al., Acta Crystallogr D Biol Crystallogr D60, 1355 (2004)), and the dihedral angles were adjusted to the values observed in the homologous atoms of bound carazolol (16-22 in FIG. 24). The one remaining unaccounted dihedral in (−)-isoproterenol was adjusted in order to place the catechol ring in the same plane as the C16—C15—O14 plane in carazolol. Residues known to specifically interact with agonists are shown as green sticks.
FIG. 26. Packing interactions in the β2AR that are likely to be modulated during the activation process. A. On the left, residues previously demonstrated to be CAMs (Rasmussen et al., Mol Pharmacol 56, 175 (1999); Tao, et. al., Mol Endocrinol 14, 1272 (2000); Jensen et al., J Biol Chem 276, 9279 (2001); Shi et al., J Biol Chem 277, 40989 (2002); Zuscik, et. al., (1998)) or UCMs (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987); Chung, et al., J Biol Chem 263, 4052 (1988); Moro, et. al., J Biol Chem 269, 6651 (1994); Green, et. al., J Biol Chem 268, 23116 (1993); Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)) are shown as van der Waals spheres mapped onto a backbone cartoon of the β2AR-T4L structure. On the right, residues that are found within 4 Å of the CAMs Leu1243.43 and Leu2726.34 are shown as yellow spheres or dot surfaces. A vertical cross-section through the structure illustrates that these surrounding residues connect the CAMs on helices III and VI with the UCMs on helix VII through packing interactions. B. In both β2AR-T4L (blue) and rhodopsin (purple), a network of ordered water molecules is found at the interface between the transmembrane helices at their cytoplasmic ends. C. Network of hydrogen bonding interactions between water molecules and β2AR-T4L residues (sidechains as blue sticks), notably the UCMs on helix VII (orange cartoon).



DETAILED DESCRIPTION OF THE INVENTION
The invention described herein provides methods and compositions for generating crystal structures of membrane proteins that diffract to resolutions as low as 2 to 3 Ångstroms. In one embodiment, the methods involve the crystallization of proteins in a lipidic cubic phase, wherein the host lipid comprises an additive, e.g., a sterol, such as cholesterol. The invention also provides the crystallized membrane proteins themselves, wherein the crystallized membrane proteins include GPCRs or modified GPCRs. The crystallized proteins can also include bound ligands, natural agonists, antagonists, and/or allosteric effectors. The invention additionally provides methods of using the 3-dimensional structures of the proteins (obtained from the crystals) to screen for novel ligands, drugs, and other useful molecules that affect the conformation and/or activity of the proteins in vitro or in vivo.
More specifically, the invention provides particular crystal forms of GPCRs diffracting to high resolutions. GPCRs have been grouped into five classes (Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation, with class A GPCRs, including β2AR, being the largest and most studied. β2AR agonists are used in the treatment of asthma and preterm labor (DeLano, The PyMOL Molecular Graphics System (2002) on World Wide Web at pymol.org). The crystal forms provided by the invention include several diffraction-quality class A GPCR crystals, including crystals comprising β2AR and crystals comprising the human adenosine A2A receptor.
The invention provides a three-dimensional structure of a human β2AR protein comprising a T4-lysozyme (T4L) in place of the third intracellular loop (β2AR-T4L′′) that has been solved in the presence of carazolol (2-propanol, 1-(9H-carbazol-4-yloxy)-3-[(1-methylethyl)amino] at 2.4 Å resolution. Additional class A GPCR structures make it possible to correlate sequence differences between GPCRs, e.g., between rhodopsin and β2AR, with emperically determined structural differences and extrapolate to other class A GPCRs. Highlighting interactions that constrain class A receptors into each of the two observed states allows a more comprehensive analysis of structural divergence and, therefore, more accurate models. Furthermore, GPCR structures provide an alternative signaling state on which to base homology models that will be more relevant For virtual ligand screening and structure-based drug design (Bissantz, et. al, Proteins 50, 5 (2003); Gouldson et al., Proteins 56, 67 (2004)).
DEFINITIONS
Terms used in the claims and specification are defined as set forth below unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
As used herein, the term “binding site” or “binding pocket” refers to a region of a protein that binds or interacts with a particular compound.
As used herein, the terms “binding” or “interaction” refers to a condition of proximity between a chemical entity, compound, or portions thereof, with another chemical entity, compound or portion thereof. The association or interaction can be non-covalent—wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it can be covalent.
As used herein, the term “residue” refers to an amino acid that is joined to another by a peptide bond. Residue is referred to herein to describe both an amino acid and its position in a polypeptide sequence.
As used herein, the term “surface residue” refers to a residue located on a surface of a polypeptide. In contrast, a buried residue is a residue that is not located on the surface of a polypeptide. A surface residue usually includes a hydrophilic side chain. Operationally, a surface residue can be identified computationally from a structural model of a polypeptide as a residue that contacts a sphere of hydration rolled over the surface of the molecular structure. A surface residue also can be identified experimentally through the use of deuterium exchange studies, or accessibility to various labeling reagents such as, e.g., hydrophilic alkylating agents.
As used herein, the term “polypeptide” refers to a single linear chain of 2 or more amino acids. A protein is an example of a polypeptide.
As used herein, the term “homolog” refers to a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, can apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication.
As used herein, the term “conservation” refers to a high degree of similarity in the primary or secondary structure of molecules between homologs. This similarity is thought to confer functional importance to a conserved region of the molecule. In reference to an individual residue or amino acid, conservation is used to refer to a computed likelihood of substitution or deletion based on comparison with homologous molecules.
As used herein, the term “distance matrix” refers to the method used to present the results of the calculation of an optimal pairwise alignment score. The matrix field (i,j) is the score assigned to the optimal alignment between two residues (up to a total of i by j residues) from the input sequences. Each entry is calculated from the top-left neighboring entries by way of a recursive equation.
As used herein, the term “substitution matrix” refers to a matrix that defines scores for amino acid substitutions, reflecting the similarity of physicochemical properties, and observed substitution frequencies. These matrices are the foundation of statistical techniques for finding alignments.
As used herein, the term “pharmacophore” refers to an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger or block a biological response. A pharmacophore can be used to design one or more candidate compounds that comprise all or most of the ensemble of steric and electronic features present in the pharmacophore and that are expected to bind to a site and trigger or block a biological response.
As used herein, the term “G-protein coupled receptor” (or “GPCR”) refers to a member of a family of heterotrimeric guanine-nucleotide binding protein (“G-protein”) coupled receptors (Pierce, et al., Nat. Rev. Mol. Cell. Biol. 3:630 (2002)). GPCRs share a common structural signature of seven membrane-spanning helices with an extra-cellular N terminus and an intracellular C terminus. The family has been grouped into at least five classes (designated A, B, C, D, E, etc.; see, e.g., Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation. When used without a descriptive limitation, the term “a G-protein couple receptor” includes GPCRs with native amino acid sequences as well as genetically engineered or otherwise mutated GPCR proteins. Mutated GPCR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a GPCR referred to herein that comprises a point mutation is β2ARE122W. An example of a GPCR referred to herein that comprises an inserted T4 lysozyme sequence is the human A2a receptor-T4L.
Adrenergic receptors in the class A or amine group are some of the most thoroughly investigated GPCRs (Kobilka, Annu Rev Neurosci 15, 87 (1992); Caron, et al., Recent Prog Horm Res 48, 277 (1993); Strosberg, Protein Sci 2, 1198 (1993); Hein, et al., Trends Cardiovasc Med 7, 137 (1997); Rohrer, J Mol Med 76, 764 (1998); Xiang, et al., Adrenergic Receptors, 267 (2006)), and are composed of two main subfamilies, α and β, which differ in tissue localization and ligand specificity, as well as in G protein coupling and downstream effector mechanisms (Milligan, et al., Biochem Pharmacol 48, 1059 (1994)). Some representative class A receptors include the human A2A adenosine receptor and the beta-2 adrenergic receptor. The term “beta-2 adrenergic receptor” (or “β2AR” or “β2AR”) refers to a class A GPCR that responds to diffusable hormones and neurotransmitters and resides predominantly in smooth muscles throughout the body. When used without a descriptive limitation, the term “β2AR” includes β2ARs with native amino acid sequences as well as genetically engineered or otherwise mutated β2AR proteins. Mutated β2AR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a β2AR referred to herein that comprises a point mutation is β2ARE122W. An example of a β2AR referred to herein that comprises an inserted T4 lysozyme sequence is the human adenosine receptor β2ARE122W-T4L.
The term “diffracts to a resolution of xx-yy Angstroms” means that diffraction data exceeding a predetermined signal to noise ratio can be obtained within the stated resolution range. In some embodiments, that diffraction data can be obtained using synchrotron radiation. Also, in some embodiments, that diffraction data can be obtained following freezing of the crystal in liquid nitrogen.
As used herein, the term “atomic co-ordinates” refers to a set of three-dimensional co-ordinates for atoms within a molecular structure. In one embodiment, atomic-co-ordinates are obtained using X-ray crystallography according to methods well-known to those of ordinarily skill in the art of biophysics. Briefly described, X-ray diffraction patterns can be obtained by diffracting X-rays off a crystal. The diffraction data are used to calculate an electron density map of the unit cell comprising the crystal; said maps are used to establish the positions of the atoms (i.e., the atomic co-ordinates) within the unit cell. Those of skill in the art understand that a set of structure co-ordinates determined by X-ray crystallography contains standard errors. In other embodiments, atomic co-ordinates can be obtained using other experimental biophysical structure determination methods that can include electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In yet other embodiments, atomic co-ordinates can be obtained using molecular modeling tools which can be based on one or more of ab initio protein folding algorithms, energy minimization, and homology-based modeling. These techniques are well known to persons of ordinary skill in the biophysical and bioinformatic arts, and are described in greater detail below.
Atomic co-ordinates for binding pockets, such as, e.g., the ligand binding pocket of β2AR, and/or other agonist/antagonist binding sites of the present invention are intended to encompass those co-ordinates set out in the .pdb file (Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) incorporated into this specification, as well as co-ordinates that are substantially equivalent. Substantially equivalent co-ordinates are those that can be related to a reference set of co-ordinates by transformation reflecting differences in the choice of origin or inter-axis angels for one or more axes used to define the coordinate system. Operationally, co-ordinates are “substantially equivalent” when the structures represented by those co-ordinates can be superimposed in a manner such that root mean square deviations (RMSD) of atomic positions for the structures differs by less than a predetermined threshold. In some embodiments that threshold is less than about 5 Angstroms, or less than about 4 Angstroms, or less than about 3 Angstroms, or less than about 2 Angstroms, or less than about 1 Angstrom, or less than about 0.9 Angstrom, or less than about 0.8 Angstrom, or less than about 0.7 Angstrom, or less than about 0.6 Angstrom, or less than about 0.5 Angstrom, or less than about 0.4 Angstrom, or less than about 0.3 Angstrom. Preferably, co-ordinates are considered “substantially equivalent” when the RMSD is less than about 1 Angstrom. Methods for structure superpositioning and RMSD calculations are well known to those of ordinary skill in the art, and can be carried out using programs such as, e.g., the programs listed in Table 5 below.
Structural similarity can be inferred from, e.g., sequence similarity, which can be determined by one of ordinary skill through visual inspection and comparison of the sequences, or through the use of well-known alignment software programs such as CLUSTAL (Wilbur et al., J. Proc. Natl. Acad. Sci. USA, 80, 726 730 (1983)) or CLUSTALW (Thompson et al., Nucleic Acids Research, 22:4673 4680 (1994)) or BLAST® (Altschul et al., J Mol. Biol., October 5; 215(3):403 10 (1990)), a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. CLUSTAL W is available at the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/); BLAST is available from the National Center for Biotechnology website (http://www.ncbi.nlm.nih.gov/BLAST/). A residue within a first protein or nucleic acid sequence corresponds to a residue within a second protein or nucleic acid sequence if the two residues occupy the same position when the first and second sequences are aligned.
The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence co-ordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI web-site).
The term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. See Fahy E. Subramaniam S et al., “A comprehensive classification system for lipids,” J. Lipid Res. 46 (5):839-861 (2005)). Sterols are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. Sterols can include, e.g., cholesterol or cholesteryl hemisuccinate (“CHS”).
The term “atomic co-ordinates for residues” refers to co-ordinates for all atoms associated with a residue, or for some of the atoms such as, e.g., side chain atoms.
The term “atomic co-ordinates of a candidate compound” refers to co-ordinates for all atoms comprising the compound or a subset of atoms comprising the compound.
The term “characterizing a binding interaction” refers to characterizing any observable property of a first molecule and determining an whether there is a change in that observable property after contacting the first molecule with a second molecule under conditions in which said first and second molecules can potentially bind.
The term “antagonist” refers to molecules that bind to and block the active site of a protein, but do not affect the equilibrium between inactive and active states. In contrast, an “agonist” is a ligand that shifts the equilibrium to an active receptor state. An “inverse agonist” is a ligand that acts to reduce the basal activity of a receptor through interactions that shift the equilibrium to more of an inactive state.
Ballesteros-Weinstein numbering appears in the text and Figures as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated X.50, where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Method for Producing Diffraction Quality Crystals of Membrane Proteins
In one aspect, the present invention discloses a modified lipidic cubic mesophase method for crystallizing proteins (see, e.g., Cherezov et al., Biophysical J., v. 83, 3393-3407 (2002)). The novel method described herein yields diffraction quality crystals of membrane proteins and is particularly useful for generating crystals of G-protein coupled receptor proteins (“GPCRs”). The method has now been successfully applied to diverse members of this important family of proteins, yielding crystals that diffract to resolutions in the 2.5 Å range. Among other advantages, this method allows diffraction-quality crystals of membrane proteins to be generated in the absence of any stabilizing antibodies bound to the protein in the crystal.
The LCP/sterol crystallization method described herein includes a step of mixing a solution containing the protein of interest with a host lipid or a host lipid mixture that includes a lipid additive. Given the teaching provided herein, one skilled in the art will recognize that a variety of host lipids may suffice for the generation of a cubic mesophase, e.g., hydrated monounsaturated monoacylglycerols such as monoolein, monopalmitolein, and/or monovacennin. The host lipid 1-monoolein is a preferred host lipid for certain applications of the method. In embodiments utilizing a lipid mixture, a lipid additive that is distinct from the host lipid is included, e.g., monounsaturated monoacylglycerols or other hydrophobic molecules known to interact with membranes or membrane-associated proteins such as, 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPC”), Asolectin, or a sterol (e.g., cholesterol, ergosterol, etc.). An example of a lipid mixture for GPCR crystallization is one comprising cholesterol as lipid additive in a ratio between 1 and 50% w/w relative to the host lipid, more preferably between 5 and 20%, and even more preferably between 8 and 12%. The protein mixture may include ligands of physiological interest and/or ligands that stabilize the protein. In the case of GPCRs, the ligands may include various agonists and antagonists known to the artisan, including well-known agonists such as carazolol (an inverse agonist), timolol, and other molecules including, without limitation, Examples of ligands, include but are not limited to carazolol, light and olfactory stimulatory molecules; adenosine, bombesin, bradykinin, endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), glucagon, acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin releasing hormone (TRH), and oxytocin).
A typical concentration of protein in the protein mixture is 25-75 mgs/ml but this concentration may vary according to protein identity and purification methods. As will be recognized by the skilled artisan, the concentration must be high enough to result in a degree of insolubility sufficient for nucleation to occur after a precipitation solution is combined with the protein-laden lipid solution; on the other hand, concentrations of protein that are too high may prevent the orderly growth of high-quality crystals.
The lipid mixture is preferably combined with the protein mixture and homogenized, e.g., using a syringe mixer, spontaneously yielding a homogenous cubic phase. Typically, the lipid mixture is added to the protein solution at a ratio of 1:1, 3:2, 4:2 w/w lipid:protein, but this ratio may be varied by the skilled artisan as desired, depending on various parameters, e.g., the concentration of protein in the protein mixture. The protein-laden lipidic cubic phase preparation thus obtained is then combined with precipitation solution (also referred to as crystallization solution) on or in an appropriate surface or container, e.g., a glass sandwich plate with wells where the mixed solutions can incubate while crystallization occurs. A typical volume of the protein-laden lipidic cubic phase used in the method is between 10 and 100 mL, with 40 to 60 mL preferred in certain embodiments. A typical volume of precipitation solution is 20 to 100 times greater, e.g., for a 20 mL volume of protein-laden lipidic cubic phase, approximately 1 μL of precipitation solution would be added to initiate crystallization.
The precipitation solution used in the crystallization method is an appropriately buffered solution (i.e., buffered to approximate the physiological conditions of the native protein) comprising polyethylene glycol, a salt, and optionally a small soluble molecule such as an alcohol.
With respect to the polyethylene glycol in the precipitation solution, useful PEG molecules include PEG 300, PEG 400, PEG 550, PEG 550 mme, PEG 1000, and PEG 1500, as well as other PEG molecules with average molecular weights less than 2000. In certain embodiments, larger average molecular weight PEG molecules (up to 20,000) or modified PEG molecules may be preferred. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEGcorn oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).
PEG may be present in the crystallization solution in concentrations between 10-60% v/v, and most typically between 20-40% v/v. The preferred concentration will vary depending on the average molecular weight of PEG utilized, i.e., 10-60% v/v of PEG will be preferred for PEG<1000 whereas 10-30% w/v will be preferred for PEG>1000 (larger average molecular weight PEG formulations are described in % w/v rather than % v/v).
With respect to the salt used in the method, an optimum cation can usually be found for a given crystal. Both sodium and lithium sulfate have proven useful for obtaining high resolution proteins of GPCRs. Again, the concentrations may be varied up to 1M, with lower concentrations of approximately 50-200 mM typically preferred. Other organic salts, e.g., citrate, malonate, tartrate, formate and acetate, may also be screened for their effects on crystal formation. In certain embodiments, the precipitation solution additionally comprises a small organic molecule such as an alcohol, a diol or a triol, e.g., a hexanediol, a butanediol, or derivative thereof. These molecules may be present in the precipitation solution in various concentrations, as appropriate, but typically in the range of 1-20% v/v, more typically in the 5-10% v/v range. In certain embodiments, preferred combinations of lipid additives (in the protein-laden lipidic cubic phase mixture) and small molecules (in the precipitation solution) yield optimal results. Examples of such combinations include 1,4-butanediol in combination with DOPE or cholesterol, and 2,6-hexanediol in combination with cholesterol.
In optimizing the conditions from micro-crystals to larger crystals for a given system (e.g., a protein/ligand system), the choice and concentration of a specific sterol(s) and specific lipid(s), as well the pH, buffer system, salt, and salt concentration may be varied, as in other types of crystallization formats. As noted above, small organic additives, especially alcohols and diols such as 1,4 butanediol, 1,6 hexanediol, etc., can be particularly useful in generating large diffraction quality crystals. Also, due to the membrane fluidity-altering properties of cholesterol and other sterols, sterol and precipitant concentration should be treated as dependent variables. For example, increasing concentrations of cholesterol in monoolein serve to rigidify the membranes, potentially slowing diffusion of the membrane protein within the lipid matrix. Conversely, increasing concentrations of PEG 400 swell the cubic phase, thereby increasing the lattice parameter of the matrix and speeding diffusion within the lipid. The former scenario would slow the rate of crystallization while the latter would increase the rate. The two effects should therefore be balanced for optimal nucleation and also for optimal growth of large, well-ordered crystals that diffract to a high resolution.
The mixing of the protein-laden lipidic cubic phase solution and the precipitation solution typically occurs at room temperatures. After set-up, the plates containing the mixed crystallization solutions can be monitored as often as desired for the appearance of crystal growth. One skilled in the art will recognize that further optimization of these conditions may be desirable, for example, to maximize the size and number of diffraction quality crystals that are obtained. In making determinations as to the preferred molecules and conditions for crystallization, the skilled artisan may rely on well-known phase diagrams and other previously determined physical constants, in addition to the novel methodology and Examples described herein. For certain lipid mixtures, pre-screening their phase behavior by microscope visualization and/or by X-ray prior to being mixed with the protein solution may facilitate the process of optimization. An in meso crystallization robot and automatic imager combined with multiple 96-well optimization screens can be used to run thousands of trials in a relatively facile manner.
It also possible to achieve additional stabilization of proteins and improve the yield of diffraction-quality crystals using the LCP/sterol method described herein by modification of the protein. For example, an unstable region of the protein may be replaced or stabilized by incorporation of a portion of a stable protein, e.g., a T4 lysozyme, whose structure is previously known but which does not (when fused) significantly affect the biochemical activity of the protein of interest. For example, the ECL2 and ECL3 regions of a β2AR can be stabilized by such modifications, as described herein (Examples 3 and 4). Other modifications include one or more point mutations that do not significantly alter the properties of the protein of interest except to increase its stability and/or tendency to crystallize well. For example, β2AR(E122W) comprises an E122W point mutation and yields crystals with the LPC/sterol method. Analogous residues in other GPCRs could be modified in the same way. One advantage of the LCP method applied to both modified and unmodified proteins, as noted above, is that it allows (but does not preclude) the crystallization of proteins in the absence of heterologous proteins, such as antibodies, that may not be of interest to the crystallographer.
Method of Ligand Screening by Lipidic Cubic Phase Crystallization
Aspects of the lipidic cubic phase crystallization methodology described above can be modified for the purpose of determining low affinity lipid binding sites within integral membrane proteins through co-crystallization trials within a lipidic cubic phase matrix. In this method various lipids of different composition are incorporated at a variety of concentrations into monoolein, wherein the monoolein is solubilized in chloroform or heated to its fluid isotropic phase. Crystal growth is then assessed by visual inspections and diffraction data collected on any crystalline material within the experiment. Because the lipid is low affinity the method requires an environment conducive for free exchange of lipid from annular to non-annular protein binding sites. The presence of interpretable electron density not associated with crystal packing interfaces allows the inference of specific binding sites for a particular lipid within the context of the membrane protein in a membrane environment. Because the binding occurs within a membrane the complicating factor of detergent partitioning is eliminated and thermodynamics of association are more realistic. The method thus allows one to characterize in detail previously inaccessible regions of membrane proteins, as well as describe and exploit binding interactions that might otherwise remain undetected. Furthermore, the technique can be applied to ligand binding studies where the ligand occupies a site on the membrane protein that is juxtaposed to the lipid plane relying on partitioning into the aqueous phase to enable saturation of the site. This limits the exposure of the protein to harsh organic co-solvents and may also find utility for soluble proteins that crystallize within the lipidic cubic phase solvent channels.
By wan of example, existing crystallization conditions for a protein can be utilized as a starting point for screening novel ligands to the β2-adrenergic receptor. In the first instance, cholesterol solubilized in chloroform can be incorporated into chloroform-solubilized monoolein at weight ratio of 10%. After drying and desiccating the mixture, protein at 30-80 mg/mL can incorporated at 2/3 volume ratio and used for crystallization trials. A similar protocol was used for other lipid like molecules, including cholesteryl hemisuccinate and a variety of other cholesterol analogs. In each case protein was incorporated into the resulting mixture and screened for crystallization. Binding of the novel ligand to the receptor is indicated by diffraction quality crystals and ultimately by three-dimensional structural data. By incorporating analogues of cholesterol we are able to map out the binding specificities based on the unique structural features of their respective sterol rings and polar moieties and, if their incorporation led to diffraction quality crystals, the interactions between the protein and cholesterol analogue are determined.
This method of ligand screening is not limited to lipid-like molecules, as we can use the lipidic cubic phase as a host for other highly hydrophobic molecules that act at orthosteric binding sites. One problem with structure based or fragment based design of novel ligands is the hydrophobicity often associated with potential drug leads. This is a problem in aqueous based crystallization schemes because the solubility of the ligand is often less than 1 mM and unless there is a slow off rate from the protein of interest the binding site will be in a ligand depleted state at crystallization conditions which often involve protein concentrations between 0.5 and 1 mM. One can attempt to co-solubilize the hydrophobic ligand in aqueous miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). However, these often interfere with the stability or crystallization properties of the protein and their usefulness is not general. Therefore, this method allows one to incorporate the hydrophobic ligand directly into the lipidic cubic phase where its accessibility to the protein will be limited by the partitioning between the lipid and aqueous phase and/or the accessibility of the binding.
Additional guidance relating to these methods is provided by the working and prophetic examples of protein crystallization presented herein.
The Crystal Structure of Human β2AR Bound to Carazolol and Uses Thereof
G-protein coupled receptors are cell surface receptors that indirectly transduce extracellular signals to downstream effectors, e.g., intracellular signaling proteins, enzymes, or channels. G-protein coupled receptor membrane proteins are grouped into one of at least 6 classes (i.e., A, B, C, D, E, and F). An example of a mammalian G-protein coupled receptor is the β2A receptor, a receptor in the Class A subfamily of GPCRs.
Class A GPCRs function in a variety of physiological processes such as vasodilation, bronchodilation, neurotransmitter signaling, stimulation of endocrine secretions, gut peristalsis, development, mitogenesis, cell proliferation, cell migration, immune system function, and oncogenesis. Accordingly, class A GPCRs can be used as screening targets to identify modulators of these processes which can then function to ameliorate diseases associated with these processes, e.g., cancer and autoimmunity.
The 2.4 Angstrom structure of β2AR bound to carazolol, described herein (PDB coordinates appear in Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model for rationally designing pharmacophore and/or candidate compounds, either de novo or by modification of known compounds. As noted below, the multiple ligand binding sites in this structure include amino acids that are highly conserved across a large number of class A G protein coupled receptors (GPCRs) indicating that the 2.4 Angstrom structure of β2AR can be used for the rational designing of ligands (e.g., therapeutic compounds) that bind to this receptor and others. Pharmacophore and candidate compounds identified through the use of the crystal structure co-ordinates will have utility as pharmaceuticals due to their ability to alter the structure and/or binding properties of β2AR. Pharmacophores and candidate compounds can be determined according to any method known in the art, including the methods described in U.S. Pat. No. 5,888,738 to Hendry, and the methods described in U.S. Pat. No. 5,856,116 to Wilson et al. the disclosures of which both are incorporated by reference in their entirety for all purposes.
The structure data provided herein can be used in conjunction with computer-modeling techniques to develop models of sites on the human β2AR or related GPCRs selected by analysis of the crystal structure data. The site models characterize the three-dimensional topography of site surface, as well as factors including van der Waals contacts, electrostatic interactions, and hydrogen-bonding opportunities. Computer simulation techniques can be used to map interaction positions for functional groups including protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups can be designed into a pharmacophore or candidate compound with the expectation that the candidate compound will specifically bind to the site. Pharmacophore design thus involves a consideration of the ability of the candidate compounds falling within the pharmacophore to interact with a site through any or all of the available types of chemical interactions, including hydrogen bonding, van der Waals, electrostatic, and covalent interactions, although, in general, and preferably, pharmacophores interact with a site through non-covalent mechanisms.
The ability of a pharmacophore or candidate compound to bind to the human β2AR can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target with sufficient binding energy (i.e., binding energy corresponding to a dissociation constant with the target on the order of 10−2M or tighter) can be synthesized and tested for their ability to bind to the human β2AR using binding assays or functional assays known to those of skill in the art. The computational evaluation step thus avoids the unnecessary synthesis of compounds that are unlikely to bind β2AR or one or more of its constitutive binding sites, or the related binding sites of another GPCR with adequate affinity.
A human β2AR or candidate compound(s) can be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on β2AR or binding site thereof, including, but not limited to a binding pocket of the human β2AR. One skilled in the art can use one of several methods to screen chemical entities or fragments for their ability to associate with one or more of these human β2AR binding sites. For example, increased affinity and specificity may be designed into caffeine and other xanthine molecules by combining interactions with both xanthine and non-xanthine binding sites.
The process can begin by visual inspection of, for example a target site on a computer screen, based on the human β2AR co-ordinates, or a subset of those co-ordinates (e.g., binding pocket residues V117, T118, F193, Y199, A200, W286, F289, F290, and Y316), as set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Selected fragments or chemical entities can then be positioned in a variety of orientations or “docked” within a target site of the human β2AR as defined from analysis of the crystal structure data Docking can be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.), ICM (Molsoft, San Diego, Calif.), and AMBER (University of California, San Francisco).
Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules,” J. Med. Chem., 28, pp. 849 857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method,” Proteins: Structure, Function and Genetics, 11, pp. 29 34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S, and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing,” Proteins: Structure, Function, and Genetics, 8, pp. 195 202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (Kuntz, I. D., et al. “A Geometric Approach to Macromolecule-Ligand Interactions,” J. Mol. Biol., 161, pp. 269 288 (1982)); DOCK is available from University of California, San Francisco, Calif.; CERIUS II (available from Molecular Simulations, Inc., San Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470 489 (1996)).
After selecting suitable chemical entities or fragments, they can be assembled into a single compound. Assembly can proceed by visual inspection of the relationship of the fragments to each other on a three-dimensional image of the fragments in relation to the human β2AR or its binding sites or those of a related GPCR receptor structure or portion thereof displayed on a computer screen. Visual inspection can be followed by manual model building using software such as the Quanta or Sybyl programs described above.
Software programs also can be used to aid one skilled in the art in connecting the individual chemical entities or fragments. These include, but are not limited to CAVEAT (Bartlett, P. A., et al. “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules” In “Molecular Recognition in Chemical and Biological Problems,” Special Publ, Royal Chem. Soc., 78, pp. 182 196 (1989)); CAVEAT is available from the University of California, Berkeley, Calif.; 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.); this area is reviewed in Martin, Y. C., “3D Database Searching in Drug Design,” J. Med. Chem., 35:2145 2154 (1992)); and HOOK (available from Molecular Simulations Inc., San Diego, Calif.).
As an alternative to building candidate pharmacophores or candidate compounds up from individual fragments or chemical entities, they can be designed de novo using the structure of the β2AR, its constituent ligand binding pocket, or the homologous cavities in a related GPCR, optionally, including information from co-factor(s) or known activators or inhibitor(s) that bind to the target site. De novo design can be implemented by programs including, but not limited to LUDI (Bohm, H. J., “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors,” J. Comp. Aid. Molec. Design, 6, pp. 61 78 (1992)); LUDI is available from Molecular Simulations, Inc., San Diego, Calif.; LEGEND (Nishibata, Y., and Itai, A., Tetrahedron 47, p. 8985 (1991); LEGEND is available from Molecular Simulations, San Diego, Calif.; and LeapFrog (available from Tripos Associates, St. Louis, Mo.).
The functional effects of known β2AR also can be altered through the use of the molecular modeling and design techniques described herein. This can be carried out by docking the structure of the known ligand on a human A2A adenosine receptor or a model structure of one or more binding sites of the human β2AR (e.g., the binding pocket described herein) and modifying the shape and charge distribution of the ligand or protein model structure to optimize the binding interactions between the ligand and protein. The modified structure can be synthesized or obtained from a library of compounds and tested for its binding affinity and/or effect on ribosome function. Of course, where the crystal structure of a complex between a human β2AR (or subunit thereof) and a ligand is known, comparisons between said complex and the structures of the present invention can be made to gain additional information about alterations in human β2AR conformation that occur upon ligand binding. This information can be used in design of optimized ligands. Compounds that interfere or activate human β2AR function (e.g., by interacting with a binding pocket) are especially well suited for the docking, co-crystallization, and optimization applications of the present invention.
Additional molecular modeling techniques also can be employed in accordance with the invention. See, e.g., Cohen, N. C., et al. “Molecular Modeling Software and Methods for Medicinal Chemistry,” J. Med. Chem., 33, pp. 883 894 (1990); Hubbard, Roderick E., “Can drugs be designed?” Curr. Opin. Biotechnol. 8, pp. 696 700 (1997); and Afshar, et al. “Structure-Based and Combinatorial Search for New RNA-Binding Drugs,” Curr. Opin. Biotechnol. 10, pp. 59 63 (1999).
Following pharmacophore or candidate compound design or selection according to any of the above methods or other methods known to one skilled in the art, the efficiency with which a candidate compound falling within the pharmacophore definition binds to the human β2AR or its ligand binding site, or alternatively binds to a related GPCR or homologous portions thereof, can be tested and optimized using computational evaluation. A candidate compound can be optimized, e.g., so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. These repulsive electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. It is preferred that the sum of all electrostatic interactions between the candidate compound and the human β2AR, including its ligand binding site when the candidate compound is bound to the target make a neutral or favorable contribution to the binding enthalpy or free energy.
Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include, but are not limited to Gaussian 92, revision C (Frisch, M. J., Gaussian, Inc., Pittsburgh, Pa. (1992)); AMBER, version 4.0 (Kollman, P. A., University of California at San Francisco, (1994)); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. (1994)); and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif. (1994)). These programs can be run, using, e.g., a Silicon Graphics workstation, Indigo, 02-R10000 or IBM RISC/6000 workstation model 550. Other hardware and software combinations can be used to carry out the above described functions, and are known to those of skill in the art. In general, the methods described herein, particularly computer-implemented methods, comprise a step of recording or storing data onto a medium, wherein the medium can include a computer-readable medium. Additionally, or alternatively, the methods comprise a step of reporting or communicating the data to a user of interest, e.g., an operator of the device and/or computer that is employed in the method; or the computer can perform an additional useful task, e.g., alert the operator of the computer that a function has been completed, upon completing one or more determining steps of the method.
Once a pharmacophore or candidate compound has been optimally selected or designed, as described above, substitutions can then be made in some of its atoms or side groups to improve or modify its binding properties. Generally, initial substitutions are conservative in that the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Components known in the art to alter conformation should be avoided in making substitutions. Substituted candidates can be analyzed for efficiency of fit to the human β2AR (or one or more binding sites of the humanβ2AR) using the same methods described above.
Assays
Any one of a number of assays of function known to those of skill in the art can be used to determine the biological activity of candidate compounds.
Candidate compound interaction with the human β2AR (or one or more binding sites of human β2AR) or to a related GPCR or portion thereof can be evaluated using direct binding assays including filter binding assays, such as are known to those skilled in the art. Binding assays can be modified to evaluate candidate compounds that competitively inhibit the binding of, e.g., known human β2AR binding compounds including xanthine and xanthine-based compounds such as theophylline, theobromine and caffeine. These and other assays are described in International Publication WO 00/69391, the entire disclosure of which is incorporated by reference in its entirety for all purposes. Methods of assaying for modulators of ligand binding and signal transduction include in vitro ligand binding assays using GPCRs, such as human β2AR (or one or more binding sites selected from the binding pockets I, II and III of the human β2AR), portions thereof such as the extracellular domain, or chimeric proteins comprising one or more domains of a GPCR, oocyte GPCR expression or tissue culture cell GPCR expression, either naturally occurring or recombinant; membrane expression of a GPCR, either naturally occurring or recombinant; tissue expression of a GPCR; expression of a GPCR in a transgenic animal, etc.
As noted above, GPCRs and their alleles and polymorphic variants are G-protein coupled receptors that participate in signal transduction and are associated with cellular function in a variety of cells, e.g., neurons, immune system cells, kidney, liver, colon, adipose, and other cells. The activity of GPCR polypeptides can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding, (e.g., radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP3, DAG, or Ca2+), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like. Such assays can be used to test for inhibitors and activators of a GPCR. In particular, the assays can be used to test for compounds that modulate natural ligand-induced GPCR activity, for example, by modulating the binding of the natural ligand to the receptor and/or by modulating the ability of the natural ligand to activate the receptor. Typically in such assays, the test compound is contacted with the GPCR in the presence of the natural ligand. The natural ligand can be added to the assay before, after, or concurrently with the test compound. The results of the assay, for example, the level of binding, calcium mobilization, etc. is then compared to the level in a control assay that comprises the GPCR and natural ligand in the absence of the test compound.
Screening assays of the invention are used to identify modulators that can be used as therapeutic agents, e.g., antagonists of GPCR activity. For example, carazolol is a known high-affinity inverse agonist of human β2AR.
The effects of test compounds upon the function of the GPCR polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the GPCRs and natural ligand-mediated GPCR activity. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca2+, IP3 or cAMP.
For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27 (1991); Bourne et al., Nature 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92 (1998).
Modulators of GPCR activity are tested using GPCR polypeptides, either recombinant or naturally occurring. The protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring. For example, neurons, cells of the immune system, adipocytes, kidney cells, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein or others as generally known in the art. Signal transduction can also be examined in vitro with soluble or solid state reactions, using a chimeric molecule such as an extracellular domain of a receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain covalently linked to the transmembrane and or cytoplasmic domain of a receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.
Ligand binding to a human β2AR (or one or more binding sites thereof) or a chimeric protein derivative can be tested in a number of formats. For example, binding can be performed in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Typically, in an assay of the invention, the binding of the natural ligand to its receptor is measured in the presence of a candidate modulator. Alternatively, the binding of the candidate modulator can be measured in the presence of the natural ligand. Often, competitive assay that measure the ability of a compound to compete with binding of the natural ligand to the receptor are used. Binding can be measured by assessing GPCR activity or by other assays: binding can be tested by measuring e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape) changes, or changes in chromatographic or solubility properties.
Receptor-G-protein interactions can also be used to assay for modulators. For example, in the absence of GTP, binding of an activator such as the natural ligand will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors. For example, a ligand can be added to the human β2AR and G protein in the absence of GTP to form a tight complex. Inhibitors can be identified by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.
An activated or inhibited G-protein will in turn alter the properties of downstream effectors such as proteins, enzymes, and channels. The classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream consequences such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization e.g., by IP3 can also be examined. Thus, modulators can be evaluated for the ability to stimulate or inhibit ligand-mediated downstream effects. In other examples, the ability of a modulator to activate a GPCR expressed in adipocytes in comparison to the ability of a natural ligand, can be determined using assays such as lipolysis (see, e.g., WO01/61359).
Activated GPCRs become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators will promote the transfer of 32P from gamma-labeled GTP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G-proteins. The kinase/arrestin pathway plays a key role in the desensitization of many GPCR receptors. Modulators can therefore also be identified using assays involving beta-arrestin recruitment. Beta-arrestin serves as a regulatory protein that is distributed throughout the cytoplasm in unactivated cells. Ligand binding to an appropriate GPCR is associated with redistribution of beta-arrestin from the cytoplasm to the cell surface, where it associates with the GPCR. Thus, receptor activation and the effect of candidate modulators on ligand-induced receptor activation, can be assessed by monitoring beta-arrestin recruitment to the cell surface. This is frequently performed by transfecting a labeled beta-arrestin fusion protein (e.g., beta-arrestin-green fluorescent protein (GFP)) into cells and monitoring its distribution using confocal microscopy (see, e.g., Groarke et al., J. Biol. Chem. 274(33):23263-69 (1999)).
Receptor internalization assays can also be used to assess receptor function. Upon ligand binding, the G-protein coupled receptor—ligand complex is internalized from the plasma membrane by a clathrin-coated vesicular endocytic process; internalization motifs on the receptors bind to adaptor protein complexes and mediate the recruitment of the activated receptors into clathrin-coated pits and vesicles. Because only activated receptors are internalized, it is possible to detect ligand-receptor binding by determining the amount of internalized receptor. In one assay format, cells are transiently transfected with radiolabeled receptor and incubated for an appropriate period of time to allow for ligand binding and receptor internalization. Thereafter, surface-bound radioactivity is removed by washing with an acid solution, the cells are solubilized, and the amount of internalized radioactivity is calculated as a percentage of ligand binding. See, e.g., Vrecl et al., Mol. Endocrinol. 12:1818-29 (1988) and Conway et al., J. Cell Physiol. 189(3):341-55 (2001). In addition, receptor internalization approaches have allowed real-time optical measurements of GPCR interactions with other cellular components in living cells (see, e.g., Barak et al., Mol. Pharmacol. 51(2)177-84 (1997)). Modulators can be identified by comparing receptor internalization levels in control cells and cells contacted with candidate compounds. For example, candidate modulators the human β2AR are assayed by examining their effects on receptor internalization upon binding of the natural ligand.
Another technology that can be used to evaluate GPCR-protein interactions in living cells involves bioluminescence resonance energy transfer (BRET). A detailed discussion regarding BRET can be found in Kroeger et al., J. Biol. Chem., 276(16):12736-43 (2001).
Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([35S]GTPγS) binding to G-proteins can also be used as an assay for evaluating modulators of GPCRs. [35S]GTPγS is a radiolabeled GTP analog that has a high affinity for all types of G-proteins, is available with a high specific activity and, although unstable in the unbound form, is not hydrolyzed when bound to the G-protein. Thus, it is possible to quantitatively assess ligand-bound receptor by comparing stimulated versus unstimulated [35S]GTPγS binding utilizing, for example, a liquid scintillation counter. Inhibitors of the receptor-ligand interactions would result in decreased [35S]GTPγS binding. Descriptions of [35S]GTPγS binding assays are provided in Traynor and Nahorski, Mol. Pharmacol. 47(4):848-54 (1995) and Bohn et al., Nature 408:720-23 (2000).
The ability of modulators to affect ligand-induced ion flux can also be determined. Ion flux can be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing a GPCR. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al., Pflügers. Archiv. 391:85 (1981). Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.
Preferred assays for G-protein coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors and the natural ligands disclosed herein as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage are monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that can be employed are those disclosed in the Molecular Probes 1997 Catalog. For G-protein coupled receptors, promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of a wide range of receptors to signal transduction pathways in heterologous cells.
Receptor activation by ligand binding typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions. Activation of some G-protein coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G-protein coupled receptor function. Cells expressing such G-protein coupled receptors can exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it can be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
Other assays can involve determining the activity of receptors which, when activated by ligand binding, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting downstream effectors such as adenylate cyclase. There are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187 (1990)). In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it can be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
In one embodiment, changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) can be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994) can be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.
In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates are separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of buffer control (which can or can not contain an agonist).
In another embodiment, transcription levels can be measured to assess the effects of a test compound on ligand-induced signal transduction. A host cell containing the protein of interest is contacted with a test compound in the presence of the natural ligand for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions can be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription can be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest can be detected using northern blots or their polypeptide products can be identified using immunoassays. Alternatively, transcription based assays using reporter genes can be used as described in U.S. Pat. No. 5,436,128, herein incorporated by reference. The reporter genes can be, e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, beta-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).
The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it can be compared with the amount of transcription in a substantially identical cell that lacks the protein of interest. A substantially identical cell can be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the protein of interest.
Samples that are treated-with a potential GPCR inhibitor or activator are compared to control samples comprising the natural ligand without the test compound to examine the extent of modulation. Control samples (untreated with activators or inhibitors) are assigned a relative GPCR activity value of 100. Inhibition of a GPCR is achieved when the GPCR activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a GPCR is achieved when the GPCR activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.
In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.
Certain screening methods involve screening for a compound that modulates the expression of the GPCRs described herein, or the levels of natural ligands, e.g., ASP and stanniocalcins. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing the GPCR or ligand and then detecting an increase or decrease in expression (either transcript or translation product). Such assays are typically performed with cells that express the endogenous GPCR or ligand. Expression can be detected in a number of different ways. As described herein, the expression levels of the protein in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of the GPCR or protein ligand. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques (see above). Alternatively, protein can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to the protein.
Other cell-based assays are reporter assays conducted with cells that do not express the protein. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, beta-glucuronidase, CAT (chloramphenicol acetyl transferase), luciferase, beta-galactosidase and alkaline phosphatase.
In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either modulates the activity of the promoter by binding to it or triggers a cascade that produces a molecule that modulates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of the GPCR or ligand and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.
In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100-1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention.
The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the signal transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and are appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
Modulators
Inhibitors and/or activators identified according to the methods of the invention can be provided from libraries of compounds available from a number of sources or can be derived by combinatorial chemistry approaches known in the art. Such libraries include but are not limited to the available Chemical Director, Maybridge, and natural product collections. In one embodiment of the invention libraries of compounds with known or predicted structures can be docked to the human β2AR structures of the invention. In another embodiment, the libraries for ligands binding to the ligand binding site can include carazolol and related compounds. In another embodiment, the libraries can include a linker component or moiety. In some embodiments, the linker can include from about 10-22 atoms and can include one or more of C, O, N, S, and/or H atoms. In another embodiment, the libraries can include a ligand binding site (also known as the ligand, agonist, or antagonist binding pocket) component or moiety. In some embodiments, the libraries can include drug-like molecules, i.e., molecules having structural attributes of one or more compounds known to bind to and/or affect a physiologic function of a GPCR.
In some embodiments, the invention includes compounds that can be tested as modulators of GPCR activity. Compounds tested as modulators of GPCRs can be any small chemical compound or biological entity. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps. The assays are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or ligand libraries are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Russell & Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
It is noted that modulators that compete with the binding and/or activity of the known ligands for to human β2AR can be used to treat various diseases including, but not limited to, coronary artery disease, atherosclerosis, thrombosis, obesity, diabetes, stroke, and other diseases.
In one embodiment, a modulator binds to a site on a GPCR, e.g., a human β2AR. In one aspect, the site is a carazolol binding site. In a related aspect, the site is a ligand binding site. In another aspect, the modulator has a first moiety that binds to a binding site. In another aspect, the first moiety is connected to a linker. In another aspect, the first moiety and the linker are connected to at least one additional moiety that binds to a site other than that bound by the first moiety. In another aspect, the two or more moieties are not connected by a linker and are both present in a composition.
Computer-Based Modeling of β2AR
Protein-ligand docking aims to employ principles by which protein receptors, e.g., human β2AR, recognize, interact, and associate with molecular substrates and compounds to predict the structure arising from the association between a given compound and a target protein of known three-dimensional structure.
In protein-ligand docking, the search algorithm can allow the degrees of freedom of the protein-ligand system to be sampled sufficiently as to include the true binding modes. Three general categories of algorithms have been developed to address this problem of ligand flexibility: systematic methods; random or stochastic methods; and simulation methods.
Systematic search algorithms attempt to explore all degrees of freedom in a molecule. These algorithms can be further divided into three types: conformational search methods, fragmentation methods, and database methods.
In conformational search methods, all rotatable bonds in the ligand are systematically rotated through 360° using a fixed increment, until all possible combinations have been generated and evaluated. As the number of structures generated increases immensely with the number of rotatable bonds (combinatorial explosion), the application of this type of method, in its purest form, is very limited.
Fragmentation methods use two different approaches to incrementally grow the ligands into the active site. One approach is by docking the several fragments into a site and linking them covalently to recreate the initial ligand (“the place-and-join approach”). Another approach is by dividing the ligand into a rigid core-fragment that is docked in first place and flexible regions that are subsequently and successively added (“the incremental approach”). DOCK (see above) is an example of s docking programs that use a fragmentation search method.
Database methods using libraries of pre-generated conformations or conformational ensembles to address the combinatorial explosion problem. A example of a docking program using database methods is FLOG which generates a small set of 25 database conformations per molecule based on distance geometry, that are subsequently subject to a rigid docking protocol.
Random search algorithms sample the conformational space by performing random changes to a single ligand or a population of ligands. At each step, the alteration performed is accepted or rejected based on a predefined probability function. There are three basic types of methods based on random algorithms: Monte Carlo methods (MC), Genetic Algorithm methods (GA), and Tabu Search methods.
Simulation methods employ a rather different approach to the docking problem, based on the calculation of the solutions to Newton's equations of motion. Two major types exist: molecular dynamics (MD) and pure energy minimization methods.
Scoring functions normally employed in protein-ligand docking are generally able to predict binding free energies within 7-10 kJ/mol and can be divided into three major classes: force field-based, empirical, and knowledge-based scoring functions.
In force-field based scoring, standard force fields quantify the sum of two energies: the interaction energy between the receptor and the ligand, and the internal energy of the ligand. The energies are normally accounted through a combination of a van der Waals with an electrostatic energy terms. A Lennard-Jones potential is used to describe the van der Waals energy term, whereas the electrostatic term is given by a Coulombic formulation with a distance-dependent dielectric function that lessens the contribution from charge-charge interactions.
Empirical scoring functions are based on the idea that binding energies can be approximated by a sum of several individual uncorrelated terms. Experimentally determined binding energies and sometimes a training set of experimentally resolved receptor-ligand complexes are used to determine the coefficients for the various terms by means of a regression analysis.
Knowledge-based scoring functions focus on following the rules and general principles statistically derived that aim to reproduce experimentally determined structures, instead of binding energies, trying to implicitly capture binding effects that are difficult to model explicitly. Typically, these methods use very simple atomic interactions-pair potentials, allowing large compound databases to be efficiently screened. These potentials are based on the frequency of occurrence of different atom-atom pair contacts and other typical interactions in large datasets of protein-ligand complexes of known structure. Therefore, their derivation is dependent on the information available in limited sets of structures.
Consensus Scoring combines the information obtained from different scores to compensate for errors from individual scoring functions, therefore improving the probability of finding the correct solution. Several studies have demonstrated the success of consensus scoring methods in relation to the use of individual functions schemes.
Using the Protein-ligand docking methods described above, a predicted association can be made between a selected chemical library compound (see above for examples) and the binding sites in the human β2AR structure described in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). These methods will therefore allow the generation of a binding profile for any known compound in any of the binding sites or cavities of the human β2AR based on the simulated docking of the compound.
In another embodiment, a form of computer-assisted drug design is employed in which a computer system is used to generate a three-dimensional structure of the candidate class A GPCR based on the structural information encoded by the amino acid sequence. This will allow use of the methods described above to identify candidate compounds based on their ability to dock in one or more of the predicted GPCR structure binding sites. In one aspect, the input amino acid sequence of the GPCR interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the class A GPCR. The models of the class A GPCR structure are then examined to identify the position and structure of the binding sites, e.g., a binding pocket. The position and structure of the predicted binding site(s) is then used to identify various compounds that modulate ligand-receptor binding using the methods described above.
The three-dimensional structural model of the GPCR is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a GPCR polypeptide into the computer system. The amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein. At least 10 residues of the amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the GPCR is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art. Any method of protein structure modeling such as ab-initio modeling, threading or sequence-sequence based methods of fold recognition. In one embodiment, the AS2TS system of protein structure modeling is used. In other embodiments, a sequence alignment in combination with a threshold protein sequence similarity to determine a set of protein sequences for which to model protein structure is used. In one aspect, sequence alignments are generated for the set of sequences to be modeled with sequences of proteins with solved empirical structure in a protein structure databank known to one of skill in the art. If the sequences to be modeled have a sufficient similarity to one or more sequences with known protein structure, then the three dimensional structure of the sequence can be modeled.
The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the GPCR of interest. In one embodiment, software can look at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as “energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
In another embodiment, protein structure alignments can be used to determine the structure of GPCRs using the known structure of the β2AR (Appendix I) (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Protein structure alignments preferably are sets of correspondences between spatial co-ordinates of sets of carbon alpha atoms which form the ‘backbone’ of the three-dimensional structure of polypeptides, although alignments of other backbone or side chain atoms also can be envisioned. These correspondences are generated by computationally aligning or superimposing two sets of atoms order to minimize distance between the two sets of carbon alpha atoms. The root mean square deviation (RMSD) of all the corresponding carbon alpha atoms in the backbone is commonly used as a quantitative measure of the quality of alignment. Another quantitative measure of alignment is the number of equivalent or structurally aligned residues.
In another embodiment, a GPCR structure is calculated based on the solved structure of the human β2AR by computationally aligning or superimposing two sets of atoms to minimize distance between the two sets of carbon alpha atoms (i.e., the alpha carbon atoms of the human β2AR and an unknown GPCR structure), followed by one or more of simulated annealing and energy minimization. The result of this calculation is a computed structure for a GPCR that provides atomic co-ordinates for the alpha carbon backbone as well as side chain atoms.
A variety of methods for generating an optimal set of correspondences can be used in the present invention. Some methods use the calculation of distance matrices to generate an optimal alignment. Other methods maximize the number of equivalent residues while RMSD is kept close to a constant value.
In the calculation of correspondences, various cutoff values can be specified to increase or decrease the stringency of the alignment. These cutoffs can be specified using distance in Angstroms. Depending on the level of stringency employed in the present invention, the distance cutoff used is less than 10 Angstroms or less than 5 Angstroms, or less than 4 Angstroms, or less than 3 Angstroms. One of ordinary skill will recognize that the utility of stringency criterion depends on the resolution of the structure determination.
In another embodiment of the present invention, the set of residue-residue correspondences are created using a local-global alignment (LGA), as described in US Patent Publication Number 2004/0185486. In this method, a set of local superpositions are created in order to detect regions which are most similar. The LGA scoring function has two components, LCS (longest continuous segments) and GDT (global distance test), established for the detection of regions of local and global structure similarities between proteins. In comparing two protein structures, the LCS procedure is able to localize and superimpose the longest segments of residues that can fit under a selected RMSD cutoff. The GDT algorithm is designed to complement evaluations made with LCS searching for the largest (not necessary continuous) set of ‘equivalent’ residues that deviate by no more than a specified distance cutoff.
Using the protein structure alignments described above, the structure of human β2AR in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model on which to discern the structure of other GPCRs and/or their predicted ligand-binding sites.
Once the GPCR structure has been generated, a binding pocket can be identified by the computer system. Computational models seek to identify the regions by characterization of the three dimensional structure of the GPCR. Some methods of identifying a binding pocket use triangulation such as weighted Delaunay triangulation to determine pocket volumes (castP). Other methods use spheres to determining protein pocket volumes (Q-site-finder, UniquePocket). Conserved binding-site identification seeks to identify conserved regions such as a binding pocket through associating the residues which form the aforementioned regions with conserved residues in homologous protein sequences or structures, e.g., through the use of sequence alignments.
One method of identifying a binding pocket in a GPCR entails filling the three dimensional protein structures with spheres, creating a “negative image” of the structure. A cutoff distance, such as 8 Angstroms, is used to determine spheres which interact with residues. Spheres are labeled as conserved or not-conserved based on their interaction with residues which form a conserved binding site. The conserved spheres are clustered based on their three dimensional co-ordinates to identify a set of spheres with interact with conserved residues and are proximal in three dimensional space forming a cluster. Three-dimensional structures for potential compounds are generated by entering chemical formulas of compounds. The three-dimensional structure of the potential compound is then compared to that of the GPCR protein ligand-binding site(s) (e.g., a binding pocket) to identify compounds that bind to the GPCR binding site(s). Binding affinity between the GPCR binding site(s) and the compound is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
It should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and can not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the claims.
Reference will now be made in detail to particularly preferred embodiments of the invention. Examples of the preferred embodiments are illustrated in the following Examples section.
EXAMPLES
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols. A and B (1992).
Example 1
Crystallization of a β2AR Protein Using a Lipidic Cubic Phase/Sterol Method
This Example describes the generation of diffraction-quality crystals of a β2AR protein, specifically crystals of β2AR-T4L, a fusion protein of human β2AR with T4 lysozyme, bound to carazolol. A detailed description of the protein and its synthesis is provided in Example 4. Briefly, T4 lysozyme was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography.
LCP Crystallization Setup
Protein solution (30 mg/ml) was mixed with a host lipid or lipid mixture typically in 2:3 ratio by volume ratio using a syringe mixer (Cheng, et al., Chem Phys Lipids 95, 11 (1998). Upon mixing ( ̃100 passages, 2-3 min) the sample spontaneously formed homogeneous transparent cubic phase, which was directly used in crystallization trials. Robotic trials were performed using an in meso crystallization robot (Cherezov, et al., Acta Cryst D 60, 1795 (2004)). Six-well glass sandwich plates (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov, J Appl Cryst 36, 1372 (2003)), were filled with 25 or 50 mL protein-laden lipidic cubic phase drops overlaid by 800 μL of precipitant solution in each well and sealed with a glass coverslip. Manual setups were performed in Impact microbatch plates (Hampton Research cat# HR3-293), Innovaplate SD-2 sitting drop plates (Hampton Research cat# HR3-083) or VDX48 hanging drop plates (Hampton Research cat# HR3-275). Modified repetitive syringe dispenser (Cherezov, et al., J Appl Cryst 38, 398 (2005)), coupled with a gas-tight 10 μL syringe was used to deliver 70 mL of cubic phase drops per well to which 1-2 μL of precipitant solution was added with a microvolume pipette. Reservoirs of the Innovaplate and VDX48 plates were filed with 50 and 100 μL of precipitant respectively. All operations starting from mixing lipid and protein were performed at room temperature ( ̃21-23° C.). After setup, plates were transferred into an automatic incubator/imager (Rocklmager 1000, Formulatrix Inc.) maintained at 20° C. Plates were imaged every 12 hours for the first 3 days, then every day until 7 days and after that on the 10th and on the 14th day.
Initial Hits
Initial trials were performed using protein solution at a concentration of 30 mg/mL mixed with monoolein as a host lipid against a set of 6 commercial screens (Index HT, SaltRxHT and MemFac HT from Hampton Research, JCSG+ and MbClass from Nextal, and MemSys&MemStart from Molecular Dimensions) set up in duplicates. Initial hits detected in three different wells contained extremely small, <5 μm, needle-like birefringent crystal showers. The detection of such small colorless protein crystals in LCP was made feasible by specially developed glass sandwich plates with optimized optical properties (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov et al., J Appl Cryst 36, 1372 (2003)). Hit conditions were similar by chemical composition containing 30% v/v PEG 400 as a precipitant, low concentration of Li sulfate and a buffer at pH 6.5 or 7.5.
Optimization
Crystal condition optimization is well known to practioners in the art of x-ray crystallography. What follows is a specific example of a generally applicable optimization approach in which one or more of the crystallization mixture components is systematically varied in concentration or substituted by a chemical analog. Initial rounds of optimization were focused on varying concentration of the main precipitant, PEG 400, buffer pH and identity, and salt identity and concentration. As a result, Li sulfate was replaced with Na sulfate and useful concentration and pH ranges were established. Crystals were still rather small reaching  ̃15×5×1 μm in size.
Further, lipid and soluble additives were searched for and optimized simultaneously. Three different host lipids (monopalmitolein, monovaccenin and monoolein), five lipid additives to monoolein host (DOPE, DOPE-Me, DOPC, Asolectin and cholesterol) and 96-well soluble additives screen were tried in different combinations along with the previously found basic crystallization conditions. One of the soluble additives, 1,4-butanediol, stood out, but only when it was used in combination with lipid additives, DOPE or cholesterol. When DOPE was used as an additive the crystals grew as thin plates (40×7×2 μm), while when cholesterol was used the crystals grew as small rods (30×5×5 μm). DOPE was dropped out in favor of cholesterol in the subsequent optimization steps.
Final optimization required fine tuning concentrations of all components (protein, PEG 400, Na sulfate, 1,4-butanediol, cholesterol, buffer pH). At the final stages of optimization, higher concentrations of protein, e.g., 50 mg/mL protein solution, were preferred. Decreasing the volume of lipidic cubic phase per trial from 50 to 20 mL consistently produced larger crystals. The best crystals (40×20×5 μm; FIG. 1) were obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. Thus, in another aspect, the invention provides a method of directly adding a lipid additive (e.g., cholesterol, DOPE) to the host lipid prior to combining with the protein mixture, thereby significantly improving the size and quality of LCP grown crystals. Previously, exogenous lipid had been added only to the protein solution prior to combining the protein solution with the host lipid (Luecke, et al., Science 293, 1499 (2001)).
To limit the range of precipitant and additives concentrations used for screening, previously published phased diagrams were used, e.g., for monoolein (Qiu, et al., Biomaterials 21, 223 (2000)), and monovaccenin (Qiu, et al., J. Phys. Chem. B 102, 4819 (1998)), and the effects on monoolein phase behavior of soluble (Cherezov, et al., Biophys J 81, 225 (2001)), and lipid additives (Cherezov, et al., Biophys J 83, 3393 (2002)). Certain lipid mixtures required pre-screening their phase behavior by microscope visualization and by X-ray, prior to being mixed with the protein for crystallization trials. Thirty-three 96-well optimization screens were used in combinations with different lipid mixtures, resulting in over 15,000 trials. This throughput was made feasible through the use of an in meso crystallization robot and automatic imager.
Typically, the best crystals grown under lipidic cubic phase conditions appear at the boundary between the cubic and the sponge phase (Cherezov, et al., J Mol Biol 357, 1605 (2006); Wadsten, et al., J Mol Biol 364, 44 (2006)). When crystals are close to the phase boundary, equally good crystals are obtained in either phase.
Crystal Harvesting
Crystals were harvested directly from the glass sandwich plates (FIG. 2) because this method provided results superior to those obtained with microbatch or vapor diffusion plates. These plates have been specifically designed to perform optimally only at the screening and optimization stages (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov and M. Caffrey, J Appl Cryst 36, 1372 (2003)). No harvesting has been previously attempted from them, due to the difficulties in separating glass slides strongly bound by a high performance double sticky tape. Thus, in another aspect, the invention disclosed herein provides a special technique for opening individual wells and harvesting crystals from them. A corner of a capillary cutting stone (in this instance, from Hampton Research) was used to scratch the top glass around the perimeter of the well. Gently pressing the glass slide just outside the scratch allows propagation of the scratch through the depth of the glass. The glass slide was then broken in one point just outside of the scratched perimeter using a sharp needle. This hole was used to lift up the glass slide and expose the cubic phase for harvesting. An extra drop of  ̃5 μL of precipitant was added to the well to reduce dehydration. Using this technique, it was possible to open up and harvest crystals successfully from more than 80% of attempted wells.
Crystals were scooped directly from the lipidic cubic phase using 30 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease the unwanted background. Attempts to dissolve the lipids, either by increasing concentration of PEG400 or using a mineral oil, typically resulted in decreasing the diffraction power of the crystals.
Data Collection
During screening for diffraction of these crystals at APS beamline GM/CA CAT (FIG. 3), the crystals themselves could not be observed in the mounted loops (FIG. 1a). Therefore, a systematic screening of the loop material with varying beams was conducted to identify the crystal in the loop. Optimization of the diffraction with a low x-ray dose was then used to center the crystals and eventually allow for complete data collection using the 10 μm×10 μm minibeam setup at GM/CA CAT. The complete data set is then compared to data filtered by a sigma cutoff (see Table 1). All of the data was used in structure solution and refinement.















TABLE 1






# Observed
# Unique








Resolution
refl
refl
Redundancy
Completeness
Rsym
I/SIGMA
R-meas
Rmrgd-F















Signal/noise −3















10
3352
333
10.1
87.40%
6.60%
23.25
6.90%
2.70%


8
3591
354
10.1
99.40%
7.60%
22.6
8.00%
2.80%


6
10480
1003
10.4
99.60%
9.90%
20.01
10.50%
4.00%


3
126008
11968
10.5
99.80%
13.30%
14.01
14.00%
6.60%


2.8
33158
3130
10.6
100.10%
38.00%
6.42
39.90%
18.70%


2.7
19702
1893
10.4
99.70%
49.50%
4.96
52.10%
24.90%


2.6
23772
2275
10.4
99.90%
60.20%
4.07
63.40%
30.10%


2.5
14108
2558
5.5
99.30%
58.90%
2.69
65.10%
51.80%


2.4
14672
3060
4.8
99.10%
67.80%
2.18
75.70%
62.80%


Total
248843
26574
9.4
99.50%
12.70%
9.62
13.40%
11.40%







Signal/noise 0















10
3352
333
10.1
87.40%
6.60%
23.25
6.90%
2.70%


8
3591
354
10.1
99.40%
7.60%
22.6
8.00%
2.80%


6
10480
1003
10.4
99.60%
9.90%
20.01
10.50%
4.00%


3
125628
11923
10.5
99.40%
13.30%
14.06
14.00%
6.50%


2.8
32679
3077
10.6
98.40%
37.60%
6.54
39.50%
17.70%


2.7
19346
1849
10.5
97.40%
48.80%
5.08
51.30%
23.40%


2.6
23201
2210
10.5
97.00%
58.90%
4.2
62.00%
28.00%


2.5
13461
2406
5.6
93.40%
56.40%
2.88
62.30%
45.90%


2.4
13833
2827
4.9
91.50%
64.10%
2.4
71.50%
54.30%


Total
245571
25982
9.5
97.30%
12.60%
9.85
13.30%
10.80%









Example 2
Using the LCP/Sterol Method to Generate Additional Membrane Protein Crysals
In addition to the β2AR-T4L/carazolol structure (Examples 1, 3, and 4), the LCP/sterol matrix has successfully been used to crystallize a diversity of receptor-ligand systems.
1. β2AR-T4L(E122W) A thermally-stabilized construct of β2AR-T4L comprising an E122W mutation has been crystallized in the presence of both agonist and antagonist ligands including: alprenolol, timolol, clenbuterol and carazolol. For lipidic cubic phase (LCP) crystallization of β2AR-T4L(E122W), robotic trials were performed using an in meso crystallization robot (Cherezov et al., 2004). Glass sandwich plates in 96-well format (Cherezov and Caffrey, 2003; Cherezov et al., 2004) were filled with 25 or 50 mL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature ( ̃21-23° C.). Crystals were obtained in 28% (v/v) PEG 400, 0.3 M potassium formate, 0.1 M Bis-tris propane pH 7.0 and saturating concentrations of ligand (e.g., 2 mM in the case of timolol) using 10% (w/w) cholesterol in monoolein as the host lipid. Diffraction data were collected on all four ligand complexes (see FIG. 4), and structures were determined for alprenolol (3.2 Å), timolol (2.8 Å), and carazolol (2.8 Å).
2. A2AR-T4L The applicability of the monoolein cholesterol system in the crystallization of non-biogenic amine receptors has also been demonstrated with the structural determination of the human A2A adenosine receptor (A2AR-T4L) bound to a high affinity selective antagonist, ZM241385, to 2.6 Å resolution. See FIG. 4. For lipidic cubic phase (LCP) crystallization of the human A2A adenosine receptor in meso, glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr, 60, 1795 (2004)) were filled with 50 nl receptor-cholesterol-monoolein LCP drops overlaid by 0.8 μl of precipitant solution in each well and sealed with a glass coverslip. Lipid:receptor LCP mixture typically contained monoolein:cholesterol (54%:6% (w/w)) and receptor (40% (w/w)). Crystallization set-ups were performed at ambient temperature (22±2° C.). Plates were incubated and imaged at 20° C. using an automated incubator/imager (RockImager 1000, Formulatrix). Data-collection quality crystals ( ̃100 μm×10 μm×5 μm) were obtained in 30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385. The protein crystallized in the primitive monoclinic space group P21 with one molecule per asymmetric unit and an estimated solvent content of 52%.
3. β2AR(E122W) Initial crystals of β2AR(E122W), lacking inserted T4 lysozyme, have also been obtained. The protein was extracted from insect cell membranes using a mixture of 0.5% w/v dodecyl maltoside (DDM), 0.1% w/v cholesteryl hemisuccinate (CHS) and 1 mM timolol. Timolol was maintained at 1 mM throughout the first steps of the purification. The extracted protein was purified by binding overnight to TalonTM immobilized metal affinity resin followed by a standard washing and elution with 200 mM imidazole. Adenosine triphosphate at 5 mM in combination with 10 mM MgCl2 was used to eliminate chaperone protein contamination. Eluted protein was concentrated to 2.5 mL and desalted into a 0 mM imidazole buffer using a PD10 desalting column (GE-Biosciences). Protein was then bound to 100 μL of Ni-sepharose immobilized metal affinity resin in the presence of PNGase (New England Biolabs) to remove glycosylation, and incubated overnight. After incubation the protein was washed on the column and timolol was replaced by carazolol for structure solution. The protein bound to carazolol was eluted from the Ni-Sepharose column, treated with 100 mM Nacitrate pH 7.5, and concentrated to 50 mg/mL.
The protein solution was then mixed with monoolein containing 10% cholesterol at a ration of 40:60% w/w protein to lipid to generate the lipidic cubic phase used in crystallization trials. The LCP lipid containing protein was dispensed onto glass sandwich crystallization plates at a volume of 20 mL to which 1 μL of crystallization solution was added. The entire experiment in 96 well format was then covered by an additional glass plate which was fastened to the first by virtue of double back sticky tape. Initial crystals have been obtained after 24 hours by addition of a solution containing 35% v/v PEG 400, 100 mM NaSO4 100 mM Bis tris propane pH 7 and 8% 2,6 hexanediol.
By way of a prophetic example, optimized crystals of β2AR(E122W) obtained by this method are screened for their ability to diffract at high resolution, e.g., less than 3.5 Å or, more preferably, less than 3 Å. Guidance for optimization is provided by the optimization protocols set forth herein and in the examples. In combination with the teaching provided herein, one skilled in the art will readily identify appropriate beam settings to obtain diffraction patterns from which a detailed molecular structure of the optimally crystallized protein can be solved.
4. CXCR4-T4L CXCR4, also called fusin, is a GPCR protein specific for stromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule endowed with potent chemotactic activity for lymphocytes. This Example teaches prophetically how the methods of the invention may be used to generate diffraction quality crystals of a fusion protein comprising CXCR4 (CXCR4-T4L).
The cDNA encoding CXCR4 is synthesized by outsourcing to DNA2.0 where the DNA was optimized for human codon usage, elimination of transcribed RNA secondary structure, elimination of ribosome binding sites and avoidance of common restrictions sites used in subsequent cloning procedures. Two initial variants are contracted to be synthesized, the first encoding a wild-type full-length receptor and the second a full length receptor with a fusion protein located between transmembrane helix V and helix VI, effectively eliminating the third intracellular loop (3IL) region of the receptor. In the case of CXCR4, T4-lysozyme (T4L) is the fusion protein fused in the place of the 3IL. A set of guidelines is followed for the incorporation of T4L into the fusion protein which minimizes the possibility of structural disruption and concomitant effects on protein expression and stability. Briefly, the 5′ insertion point for the fusion protein takes precedence over the 3′ insertion point and is located 66 nucleotides (22 residues) downstream of the codon for a conserved proline on helix V of the receptor. If the 3IL section of the receptor is large, the 3′ fusion point is set 87 nucleotides (29 residues) upstream from the codon for the family conserved proline on helix VI. However, as is the case for CXCR4, where the 3IL loop is small, cDNA for the fusion protein is inserted directly into the 3IL loop position dictated by spacing from helix V with no resulting excision of intervening nucleotides. Specifically, T4L is inserted into CXCR4 based on spacing between a conserved proline on helix V and a C-terminal truncation was generated based on literature precedence. Each synthesized cDNA is flanked by an out of frame AscI (GGCGCGCCG) restriction site on the 5′ end and an in frame FseI (GGCCGGCC) on the 3′ end for sub-cloning into a set of four expression vectors. Viral DNA is then generated, amplified from these vectors according to standard protocols and titered using flow cytometry to measure the population of cells expressing the virally encoded GP64 protein.
Protein Expression of CXCR4-T4L
With titered virus in hand, small scale expression trials are carried out in a volume of 5 mL/experiment. Expression levels are assessed using flow cytometry to measure the mean fluorescence intensity (MFI) and percentage of cells expressing the FLAG epitope encoded by the expression screening vectors. Expressing cells are tested with and without permeabilization to generate a ratio between protein inserted in the plasma membrane and protein inside the cellular trafficking machinery. A correlation between cell surface expression and overall protein expression is demonstrated, as well as a correlation between stability and the ratio of cell surface expression/total expression. In addition to these assays, small scale purification after solubilization with dodecyl maltoside (DDM) is carried out to determine the quantity of recoverable protein as well as the quality as measured by size exclusion chromatography. Based on these data it was apparent that the T4L fused receptor is expressing and that it is dimerizing in a ligand independent manner, an indicator of C-terminal non-specific interactions in other receptors. Thus, a C-terminal truncation mutant of CXCR4 is generated.
Protein Purification
The C-terminal truncation of T4L fused CXCR4 was scaled up to production sized expression (5-10L of cell culture) and further processed by large scale purification efforts intended for crystallization trials. Briefly 5-10 L of cells culture are centrifuged and washed with PBS followed by freezing at −80° C. The frozen cellular material is then resuspended in 820 mL of lysis buffer (10 mM Hepes pH 7.5, 10 mM MgCl2, 20 mM KCl) supplemented with protease inhibitor (Roche). The cell suspension is lysed by 20 strokes of a dounce homogenizer and centrifuged at 45,000 rpm in Ti45 ultracentrifuge for 30 minutes. The resulted pellet was separated from the supernatant, resuspended and the process repeated six times to ensure complete removal of soluble protein material. On the final resuspension step the membranes were resuspended in 100 mL of lysis buffer containing 40% v/v glycerol, homogenized with 20 strokes of a dounce homogenizer and frozen in 10 mL aliquots at −80° C. for indefinite storage.
For solubilization and purification, each 10 mL aliquot of frozen membranes is resuspended to 25 mL using lysis buffer to which 100 uM AMD070 ligand is added in addition to protease inhibitor at 2× working concentration and 2 mg/mL iodoacetamide. The membranes are allowed to thaw and incubate with ligand at an appropriate temperature for at least 30 minutes. After the incubation the mixture is diluted two-fold with a 2× solubilization buffer containing 100 mM Hepes pH 7.5, 1M NaCl, 2% w/v DDM 0.2% w/v CHS. The solubilization is allowed to proceed with agitation for at least 2 hours at 4° C. after which insoluble material is separated by centrifugation and discarded. The supernatant is isolated and allowed to bind to 0.5 mL of Talon (Clontech) IMAC resin charged with Co2+ in the presence of 20 mM imidizole buffered to 7.5 and 800 mM NaCl. Binding to the Talon IMAC resin is allowed to proceed with agitation at least 4 hours but most commonly overnight. After binding, the slurry is poured into a gravity column and the resin is separated from the supernatant. The resin is then washed with 80 column volumes (CV) of wash buffer (50 mM Hepes pH 7.5, 800 mM NaCl, 20 mM Imidizole, 0.1% w/v DDM, 0.01% w/v CHS and 100 uM AMD070 (or receptor appropriate ligand). After the initial wash the resin is further treated to adjust the NaCl concentration to 500 mM and to increase the ligand concentration to 300 uM. The protein is then eluted from the resin using 200 mM Imidazole and concentrated to 2.5 mL for removal of the excess imidizole with a PD10 desalting column (GE Biosciences). The ligand concentration is increased to 500 uM and the protein is bound to 100 uL of Ni-Sepharose IMAC resin in the presence of 20,000 units of PNGase (NEB) an endoglycosidase capable of removing N-linked glycosylation. The protein is allowed to bind to the resin and deglycosylate for 6 hours after which the resin is washed with imidizole free elution buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 0.05% w/v DDM, 0.01% w/v CHS and 1 mM AMD 070). After the washing step the protein was eluted from the resin using the same buffer but including 200 mM imidizole. After elution the protein is normally concentrated to approximately 50 mg/mL and tested for integrity by SEC. Crystallizable protein should be >90% free of heterogeneity as judged by SDS-PAGE and contain no detectable aggregated species at high protein concentrations as judged by SEC. If the protein remains of high quality it is reconstituted into lipidic cubic phase containing cholesterol. The reconstituted protein is then dispensed onto glass sandwich crystallization plates and tested for crystallization using the screening methodology described in this Example and Example 1. After mixing, the protein-laden lipidid cubic phase mixture will comprise 3.6-7.2% w/w sterol, 56.5-52.8 w/w % Monoolein and 40% w/w protein solution (a 3:2 ratio of lipid mixture to protein). Initial crystallization conditions use PEG 400 between 25-35%, a salt between 50-500 mM, and a pH between 5.0-7.5.
Example 3
High Resolution Crystal Structure of a Human β2-Adrenergic G protein-Coupled Receptor T4 Lysozyme Fusion Protein
The engineering, functional properties, expression and purification of crystallization grade β2AR-T4L protein are described in more detail in Example 4. Briefly, β2AR-T4L was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography. Using the modified lipidic cubic phase (LCP) crystallization procedure described herein, wherein crystals are grown from a cholesterol-doped monoolein cubic phase, β2AR-T4L crystals were obtained that diffract to a resolution of 2.2 Å. The structure was solved at 2.4 Å resolution. Compared to crystallization in detergents, LCP provides a more native, lipid environment for crystallization, as well as a confinement of protein molecules to two-dimensional membrane sheets that may facilitate the crystallization process through the formation of Type I packing interactions (Caffrey, Curr Opin Struct Biol 10, 486 (2000); Deisenhofer, EMBO J. 8, 2149 (1989); Landau et al., Proc Natl Acad Sci USA 93, 14532 (1996)).
Methods
Lipidic cubic phase crystallization. Crystals of engineered human β2AR (β2AR-T4L) grown from bicelles could not be optimized beyond 3.5 Å resolution (FIG. 5). Lipidic cubic phase (LCP) crystallization trials were therefore performed using an in meso crystallization robot (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004)). 96-well glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)) were filled with 25 or 50 mL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature ( ̃21-23° C.). Crystals were obtained in 30-35% (v/v) PEG 400, 0.1-0.2 M sodium sulfate, 0.1 M Bis-tris propane pH 6.5-7.0 and 5-7% (v/v) 1,4-butanediol using 8-10% (w/w) cholesterol in monoolein as the host lipid (FIG. 6A). Addition of cholesterol and 1,4-butanediol dramatically improved crystals size and shape, thereby enabling high-resolution diffraction. In this instance, additions of phospholipids (dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, asolectin) to the main host LCP lipid monoolein (either alone or in combination with cholesterol) failed to improve crystal quality.
Crystal Harvesting
The average size of the harvested crystals was 30×15×5 μm (largest crystal was 40×20×7 μm). Crystals were harvested directly from the glass sandwich plates, even though these plates have been specifically designed for screening and optimization (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)). Crystals were scooped directly from the LCP using 30 or 50 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease unwanted background scattering. Attempts to dissolve the lipids, either by increasing concentration of PEG 400 or using a mineral oil, typically resulted in a decrease in diffraction power of the crystals.
Data Collection
X-ray data were collected on the 23ID-B beamline (GM/CA CAT) at the Advanced Photon Source, Argonne, Ill. using a 10 μm minibeam (wavelength 1.0332 Å) and a MarMosaic 300 CCD detector (FIG. 6B). Several complete datasets were collected from single crystals at resolution between 2.8 and 3.5 Å using 5× attenuated beam, 5 s exposure and 1° oscillation per frame. However, some crystals diffracted to a maximum of 2.2 Å resolution upon 5 s exposure with 1× attenuated beam. Therefore, 10-20° wedges of high-resolution data were collected from more than 40 crystals (some of the crystals were large enough to allow 2-3 translations). 31 of the best datasets from 27 independent crystals were then combined and scaled against the lower resolution full dataset to obtain complete 2.4 Å data.
One of the challenges during data collection was visualization of colorless microcrystals within an opaque frozen lipid phase and aligning them with the 10 μm minibeam. Because the crystals could not be adequately visualized through the inline optics at the beamline, alignment-by-diffraction techniques were employed. The present invention provides, in one aspect, an optimized crystal search algorithm to locate the crystals without the minibeam. First, the area of the loop containing lipid was scanned in the vertical direction with a highly attenuated and slitted 100×25 μm beam. When diffraction was found, the crystal location was further confined by two additional exposures to an area of  ̃50×25 μm. This area was further coarse-scanned with the collimated and 10× attenuated minibeam using 15 μm steps, following by fine-tuning the position using 5 and 2 μm steps. After locating the crystal in one orientation the loop was rotated 90° and the procedure was repeated. Typically during alignment the crystal was exposed  ̃10 times using 10× attenuated beam and 2 s exposures.
Data Processing
A 90% complete, 2-fold redundant monoclinic dataset was processed from one crystal diffracting to 2.8 Å resolution. Initial indexing of lattice parameters in spacegroup C2 and crystal orientation were performed using HKL2000 (Otwinowski, et al, in Methods in Enzymology C. W. J. Carter, R. M. Sweet, Eds. (Academic Press, New York, 1997), vol. 276, pp. 307-326). The refined lattice parameters and space group were implemented in the data processing program XDS for spot integration which models error explicitly for radiation decay, absorption, and rotation (Kabsch, J Appl Crystallogr 26, 795 (1993)). Because data was collected using a 10 μm beam from microcrystals, maintaining the crystal orientation at the beam center during data collection was especially problematic. It appeared that XDS modeled the crystal orientation error upon rotation about the phi axis better than other data processing programs that were tried, resulting in better merging statistics. In addition to rotational error, the radiation decay was also an issue that was partially corrected by the XDS processing program, enabling a more reliable scaling of datasets from different crystals and translations of crystals. The 2.8 Å data was used as a scaling reference for incorporation of additional wedges of data collected at a much higher exposure. Each new dataset was indexed in XDS using the original unit cell parameters as constants which were then refined along with the crystal orientation, beam geometry, and mosaicity parameters. The refinement was generally stable, resulting in very similar unit cell constants which enabled subsequent scaling. All of the integrated wedges of data were then tested individually against the scaling reference set and included in the final scaled dataset if the merging statistics remained acceptable upon incorporation of the data. In total, 31 wedges of data from 27 crystals were combined with the scaling reference dataset, 22 of which diffracted to a resolution of 2.4 Å or better. Each of the higher resolution datasets were exposed to a much larger dose of radiation resulting in a rapid decay in intensity. Typically 10°-20° wedges were collected from each crystal or translation, 5°-7° of which had diffraction data to 2.4 Å. The final merging statistics for the dataset are shown in Table 2. Based on the mean F/σ(F) of reflections near the three crystallographic axes, the effective resolution is estimated to be 2.4 Å along b* and c* and 2.7 Å along a*. The anisotropy results in the high merging R factors in the last few resolution shells despite the significant I/σ(I) values. The anisotropy is either an inherent property of the crystals or the result of a preferential orientation of the crystals within the mounting loop. Thus, the higher resolution shells were filled in anisotropically by incorporation of the additional data at high exposure levels, while the lower resolution shells have a very high redundancy and low anisotropy.







TABLE 2







Data processing statistics from XDS. A comparison is made between data filtered by a sigma cutoff


and the complete set. All of the data was used in structure solution and refinement.
















# Observed
# Unique



I/




Resolution
refl
refl
Redundancy
Completeness
Rsym
SIGMA
R-meas
Rmrgd-F










Signal/noise ≧ −3















10
3352
333
10.1
87.40%
6.60%
23.25
6.90%
2.70%


8
3591
354
10.1
99.40%
7.60%
22.6
8.00%
2.80%


6
10480
1003
10.4
99.60%
9.90%
20.01
10.50%
4.00%


3
126008
11968
10.5
99.80%
13.30%
14.01
14.00%
6.60%


2.8
33158
3130
10.6
100.10%
38.00%
6.42
39.90%
18.70%


2.7
19702
1893
10.4
99.70%
49.50%
4.96
52.10%
24.90%


2.6
23772
2275
10.4
99.90%
60.20%
4.07
63.40%
30.10%


2.5
14108
2558
5.5
99.30%
58.90%
2.69
65.10%
51.80%


2.4
14672
3060
4.8
99.10%
67.80%
2.18
75.70%
62.80%


Total
248843
26574
9.4
99.50%
12.70%
9.62
13.40%
11.40%







Signal/noise ≧ 0















10
3352
333
10.1
87.40%
6.60%
23.25
6.90%
2.70%


8
3591
354
10.1
99.40%
7.60%
22.6
8.00%
2.80%


6
10480
1003
10.4
99.60%
9.90%
20.01
10.50%
4.00%


3
125628
11923
10.5
99.40%
13.30%
14.06
14.00%
6.50%


2.8
32679
3077
10.6
98.40%
37.60%
6.54
39.50%
17.70%


2.7
19346
1849
10.5
97.40%
48.80%
5.08
51.30%
23.40%


2.6
23201
2210
10.5
97.00%
58.90%
4.2
62.00%
28.00%


2.5
13461
2406
5.6
93.40%
56.40%
2.88
62.30%
45.90%


2.4
13833
2827
4.9
91.50%
64.10%
2.4
71.50%
54.30%


Total
245571
25982
9.5
97.30%
12.60%
9.85
13.30%
10.80%





Rsym = Σhkl|I(hkl) − <I(hkl)>|/Σhkl(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl); R-meas = redundancy independent Rsym; Rmrgd-F = indication of amplitude quality. See, e.g., Murshudov, et al, Acta Crystallogr D D53, 240 (1997).






Structure Solution and Refinement
Initial phases for β2AR-T4L were obtained by molecular replacement using both T4-lysozyme (PDB ID Code 2LZM) and a polyalanine model of the rhodopsin seven-transmembrane bundle (PDB ID Code 1U19) as search models. It was necessary to trim the lysozyme search model to remove residues 12-71 as that domain had shifted conformations relative to the larger section. This domain was later reintroduced to the model by fitting into observed density. Molecular replacement was carried out using the program Phaser by first placing the truncated lysozyme (RFZ=3.74; TFZ=3.65) followed by the rhodopsin model (RFZ=5.2; TFZ=7) (McCoy, Acta Crystallogr D Biol Crystallogr 63, 32 (2007)). In order to optimize placement of the receptor each of the seven helices was refined independently by rigid body maximum likelihood refinement as implemented in Refmac (Initial Rwork/Rfree=0.50/0.51) (Murshudov, et al, Acta Crystallogr D D53, 240 (1997)).
Initial rounds of refinement were carried out using restrained parameters in Refmac. Model rebuilding was performed in Coot utilizing 2 Fo−Fc sigma-A weighted maps, as well as density modified maps calculated using Resolve prime-and-switch phasing which reduces model bias introduced by model based phasing methods (Terwilliger, Acta Crystallogr D D56, 965 (2000)). The Resolve calculated maps were superior to the sigma-A weighted ones in that more of the main chain density could be traced. Density for the bound ligand was visible early in the refinement but was not modeled immediately to allow an unbiased assessment of the phase quality through the improvement of the signal/noise of the observed ligand density. The structure quality is excellent (Table 3), with strong electron density in particular observed in the ligand binding site (FIG. 7), cholesterol binding sites (FIG. 8A), and the proline helix kinks (FIG. 8B).







TABLE 3







Data collection and refinement statistics









β2AR-T4L











Data collection (APS GM/CA CAT 23ID-B, 10 μm beam)*








Space group
C2


Cell dimensions


a, b, c (Å)
106.32, 169.24, 40.15


β (°)
105.62


No. of reflections processed
245,571


No. unique reflections
26,574









Resolution (Å)
50-2.4
(2.5-2.4)


Rsym
12.7
(67.8)


Mean I/σ(I)
9.6
(2.2)


Completeness (%)
99.5
(99.1)


Redundancy
9.4
(4.8)







Refinement*









Resolution (Å)
20-2.4
(2.46-2.4)


No. reflections (test set)
25,247
(1,310)








Rwork/Rfree
19.8 (27.0)/23.2 (30.1)


No. atoms
3,805


Protein
3,544


Ions, lipids, ligand and other
213


Water
48


Overall B-values (Å2)
82


β2AR
77


T4-Lysozyme
75


Carazolol
55


Lipid
100


R.m.s deviations


Bond lengths (Å)
0.013


Bond angles (°)
1.5


Ramachandran plot statistics (%) (excl. Gly,


Pro):


Most favored regions
94.8


Additionally allowed regions
5.0


Generously allowed regions
0.2


Disallowed regions
0





*Highest resolution shell is shown in parenthesis. Rsym = Σhkl|I(hkl) − <I(hkl)>|/Σhkl(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl).






Analysis of β2A Receptor Topology
The final model of β2AR-T4L includes 442 amino acids. β2AR-T4L was treated with iodoacetamide during purification to eliminate free thiols. The model includes a palmitic acid covalently bound to Cys341 (GPCRs are frequently post-translationally modified with palmitoylate on cysteine residues at the C-terminal tail) and an acetamide molecule bound to Cys2656.27. Throughout the description, residues are designated by their position within the β2AR sequence and their Ballesteros-Weinstein designation as a superscript where applicable. Ballesteros-Weinstein numbering is used throughout the text as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated x.50 where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position. The model also includes one carazolol molecule, three cholesterol molecules, two sulfate ions and two butanediol molecules that interact with β2AR. There are also four sulfate ions, a putative disaccharide (modeled as maltose) and a molecule of PEG 400 bound to T4L. For β2AR, excellent electron density is observed for residues 29-342, including the ligand carazolol and the two disulfide bonds Cys1063.25-Cys1915.30 and Cys1844.26-Cys1905.29. The palmitic acid at Cys341 is clearly visible in Fo−Fc omit maps; however, the quality of the electron density is lower than for the rest of the receptor. The N-terminus (residues 1 to 28) and the majority of the C-terminus (residues 343 to 365) are disordered and not visible in the structure.
The β2AR has a fold composed of seven transmembrane helices forming a helical bundle (FIG. 9A). The residues that make up the helices (I to VII) in β2AR are as follows: helix 1291.28 to 601.59 helix II 672.38 to 962.67, helix III 1033.22 to 1363.55, helix IV 1474.39 to 1714.63, helix V 1975.36 to 2295.68, helix VI 2676.29 to 2986.60, and helix VII 3057.32 to 3287.55. The residues forming the intracellular loops (ICL) and extracellular loops (ECL) of β2AR are: ICL1.611.60 to 662.37, ECL1.972.68 to 1023.21, ICL2 1373.56 to 1464.38, ECL2 1724.64 to 1965.35, ICL3 2305.69 to 2666.28 (residues 231 to 262 are replaced by T4-lysozyme residues 2 to 161), and ECL3 2996.61 to 3047.31. Helices II, V, VI and VII each have a proline-induced kink at conserved positions along the span of the transmembrane segments. These kinks are thought to enable the structural rearrangements required for activation of G protein effectors (Yohannan et al., Proc Natl Acad Sci USA 101, 959 (2004)). In addition to the seven membrane spanning helices, β2AR possesses two other helical segments: helix VIII, which is believed to be common to all rhodopsin-like GPCRs (Katragadda et al., Biochim Biophys Acta 1663, 74 (2004)), and an unexpected, short helical segment in the middle of ECL2, which is not present in rhodopsin, and was not predicted by computational secondary structure analysis (FIG. 9A).
In the β2AR-T4L construct, T4L is fused to the truncated cytoplasmic ends of helices V and VI. In the crystal structure, the T4L moiety is tilted slightly away from the center axis of β2AR drawn normal to the membrane (FIG. 9B). As a result, interactions between T4L and β2AR are minimal, with only 400 Å2 of surface area buried between them. The intramolecular contacts between T4L and β2AR include salt bridges between the side chains of T4L-Asp159 and the side-chain amine of β2AR-Lys2275.66 (distance 3.4 Å) and between the guanidinium group of T4L-Arg8 with the side-chain carboxyl of β2AR-Glu2686.30 on helix VI (distance 3.2 Å) (FIG. 9C, Table 4). The latter interaction is noteworthy because it differs from rhodopsin where Glu6.30 forms an ionic bond with Arg3.50 of the conserved D(E)RY motif. This interaction is postulated to be important for maintaining rhodopsin in the inactive state, but the charged groups of the two residues [Arg1313.50 (NH1) and Glu2686.30 (OE1)] are 10 Å apart in the β2AR-T4L structure. The remainder of the lysozyme molecule provides important crystal packing interactions, but does not appear to influence significantly the receptor structure.







TABLE 4







Direct contacts between β2AR and T4L











β2AR atom
T4 Lysozyme
Distance (Å)











van der Waals Contacts










Leu2305.69
Trp 158



Lys2636.25
Asp159



Cys2656.27
Ile9



Leu2666.28
Ile9



Leu2666.28
Glu5







Hydrogen Bond and Salt Bridge Contacts











Lys2275.66 (NZ)
Asp159(OD1)
3.4



Gln2295.68 (O)
Asn2 (N)
3.1



Gln2295.68 (O)
Asn2 (ND2)
3.2



Gln2686.30 (OE2)
Arg8 (NH2)
3.2







Covalent bonds










Leu2305.69
Asn2



Lys2636.25
Tyr161










Crystal Packing Interactions

The β2AR-T4L protein is packed in a C-centered monoclinic lattice with one molecule per asymmetric unit (FIG. 10A). Membrane protein generally can form two types of crystal packing: Type I represents stacks of two dimensional crystals ordered in the third dimension via interactions of hydrophilic parts of membrane proteins. Type II crystals are composed of membrane proteins whose hydrophobic part is shielded by a detergent micelle and all crystal contacts are formed through hydrophilic, solvent exposed parts of protein molecules. As observed in all previous lipidic mesophase grown crystals (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)), the β2AR-T4L crystals adopt Type I packing, featuring a multilayered arrangement in accordance with proposed crystallization mechanism (Caffrey, Curr Opin Struct Biol 10, 486 (2000); P. Nollert, et al., FEBS Lett 504, 179 (2001)). Within each layer, protein molecules form arrays of parallel, symmetry-related dimers. There are four distinct crystal-packing interactions within each layer, three of which are mediated by T4L. The fourth interaction in the array is between two receptor molecules related by a crystallographic two-fold rotation axis. This is the sole interaction between symmetry-related receptors, and is mediated primarily by ordered lipids consisting of six cholesterol and two palmitic acid molecules, the latter being covalently attached to Cys341 in the C-terminal portion of the receptor (O'Dowd et al., J Biol Chem 264, 7564 (1989)) (FIG. 10B). These eight lipid molecules form a two-fold symmetric sheet between receptors. The only direct receptor-receptor contact involves a 2.7 Å pair of ionic interactions between the charged amine group of Lys601.59 in helix I and the carboxylate of Glu338 in helix VIII from the symmetry-related receptor. Remarkably, of the 515 Å2 buried at the receptor symmetry interface, 73% of the crystal contact surface area is mediated by ordered lipid, while only 27% is contributed by protein-protein contacts. The stacking interactions between layers are formed between T4L and extracellular loops ECL2 and ECL3 of the receptor (FIG. 10A). It is unlikely that these contacts affect the orientation of these loops due to the small size of ECL3 and the rigid architecture of ECL2.
Lipid Mediated Receptor Association
Many GPCRs including β2AR are thought to exist as dimers in the plasma membrane, although the location of the dimer interface and the functional significance of dimerization is not clear (Milligan, Mol Pharmacol 66, 1 (2004)). The observation of ordered lipids in the helix I and VIII interface between two symmetry related molecules suggests the association is physiologically relevant (Angers, et al., Proc Natl Acad Sci USA 97, 3684 (2000); Javitch, Mol Pharmacol 66, 1077 (2004); Mercier, et al., J Biol Chem 277, 44925 (2002)). Associations between the equivalent regions of rhodopsin have been found in crystal structures (Salom et al., Proc Natl Acad Sci USA 103, 16123 (2006); Schertler, Curr Opin Struct Biol 15, 408 (2005)) (FIG. 10C).
The role of cholesterol in the physiologic function of β2AR is well documented. Depletion of cholesterol from the membranes of neonatal cardiac myocytes alters the signaling behavior of endogenous β2AR (Xiang, et al., J Biol Chem 277, 34280 (2002)). In untreated cells, activation of β2AR results in sequential coupling to the G proteins Gs and Gi, producing a biphasic effect on myocyte contraction rate. Upon depletion of cholesterol, the β2AR couples more strongly to Gs. This effect may be due to a role of cholesterol in regulating interactions between the β2AR and G proteins, or an effect of cholesterol on β2AR dimerization. The β2AR couples efficiently to Gs as a monomer (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), so cholesterol mediated association (dimerization) may reduce the efficiency of β2AR coupling to Gs. The effects of cholesterol depletion on β2AR signaling may also be a secondary effect of altering subcellular signaling compartments. There is evidence that cells may concentrate signaling molecules, such as GPCRs and their cognate G proteins, by way of membrane microdomains or compartments, such as caveolae (Ostrom, et al., Br J Pharmacol 143, 235 (September, 2004)). This compartmentalization may be a major regulator of receptor-effector coupling. Thus, the importance of cholesterol in forming the observed crystallographic association is consistent with its role in β2AR signaling.
Electrostatic Charge Distribution
Electrostatic charge distribution was calculated using APBS (Gether, Endocr Rev 21, 90 (2000)) and mapped onto a molecular surface representation of β2AR. The analysis reveals three polarized areas within the molecule (FIG. 11A). First, the cytoplasmic face of the receptor is involved in G protein interaction and carries a net positive charge even in the absence of ICL3, which also has a predicted overall positive charge (FIG. 11B). The second site is an electrostatically negative region located within the membrane between helices III, IV and V potentially exposed to the lipid alkyl chains, which is unexpected as the burial of charge within the plasma membrane is thermodynamically unfavorable. A glutamate residue at position 1223.41 may partially account for the observed charge distribution. Finally, the binding site cleft is negatively charged and exposed to solvent by an unusual ECL2 architecture and lack of N-terminal interactions. This negative charge may facilitate ligand binding through electrostatic funneling of positively charged catecholamines (FIG. 11B).
Extracellular Region
The ECLs and amino termini of GPCRs, together with the extracellular halves of the transmembrane helices, are believed to define the ligand-binding site of each receptor (Angers et al., Proc Natl Acad Sci USA 97, 3684 (2000)). Therefore, the ECLs play an important role in the overall pharmacology of any particular receptor. In general, small molecule ligands are thought to bind deeper within the space created by the transmembrane domain helices, whereas larger ligands such as peptides bind closer to the membrane surface near the ECLs (Ji, et al., J Biol Chem 273, 17299 (1998); Gether, Endocr Rev 21, 90 (2000)). Mutagenesis studies suggest that the β2AR binds its ligand deep within the transmembrane helix bundle, which may be related to the observation that the extracellular regions have a rather simple structure with short loops connecting transmembrane helices II and III, and VI and VII (FIG. 12A). ECL2, which links helices IV and V, has a somewhat more extensive architecture that is unanticipated. In contrast to the buried, β-sheet structure of this loop in rhodopsin (FIG. 12B), ECL2 in β2AR is more exposed to the solvent and contains an extra helical segment. Additionally, there is an intra-loop disulfide bond between Cys1844.76 and Cys1905.29 that may help stabilize the more exposed ECL2. A second disulfide bond between Cys1915.30 and Cys1063.25 in helix III effectively ties ECL2 to the transmembrane core (Noda, et al., J Biol Chem 269, 6743 (1994)). The distal portion of ECL2 makes close contacts with ECL1 and contains a glycosylation site at Asn1875.26 (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), which may serve to mask a grouping of aromatic residues on ECL1; in this construct, Asn1875.26 has been mutated to glutamate to aid in crystallization.
Electron density corresponding to the N-terminus was not apparent in the maps and, therefore, residues 1-28 are not included in the model. This disorder contrasts with rhodopsin, in which the N-terminus interacts extensively with the ECLs, forming a small four-strand β-sheet in conjunction with ECL2. This sheet structure forms a cap that effectively isolates the retinal binding site in a hydrophobic pocket (FIG. 13B). The lack of interactions between the N-terminus of β2AR and ECL2 further enables diffusible ligand access to the binding site. However a completely disordered N-terminus may be an artifact induced by the presence of the N-terminal Flag tag which carries an overall positive charge and may disrupt N-terminal interactions.
The short helical region on ECL2 adds a rigid structural element that, along with the two disulfide bonds, constrains the loop to a small range of conformations and helps stabilize the receptor by linking three transmembrane helices (FIG. 13A). This rigid conformation may help to stabilize the core of the receptor and lock ECL2 in a conformation that does not hinder access to the binding pocket.
Ligand Binding Site and Comparison to Rhodopsin
Carazolol is a partial inverse agonist that binds with picomolar affinity to β2AR-T4L producing a reduction of the basal activity of the receptor. The crystal structure reveals extensive interactions between the receptor and carazolol that position the carbazole moiety adjacent to Phe2896.51, Phe2906.52, and Trp2866.48 (FIG. 13A, FIG. 7, and Table 5). In contrast, cis-retinal is a full inverse agonist covalently bound to rhodopsin, which suppresses all activity towards transducin (Palczewski, Annu Rev Biochem 75, 743 (2006)). Carazolol and retinal occupy similar spaces in their respective receptors, with significant overlap of the non-aromatic regions of carazolol. However, the β-ionone ring of retinal extends deep into the binding pocket of rhodopsin and contacts residues on helix V and VI, where it is sandwiched between Phe2125.47 and Tyr2686.51, and interacts with the highly conserved Trp2656.48 (FIG. 13B). It has been proposed that changes in the rotamer of Trp2656.48 occur upon activation of rhodopsin and related family members, and constitutes the “toggle switch” for receptor activation (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)). Accordingly, the interactions between c/s-retinal and Trp2656.48 are likely to contribute to the absence of basal activity in rhodopsin. Carazolol does not interact directly with the toggle switch on helix VI, however it lowers the basal activity of the receptor, and may do so by interacting with Phe2896.51 and Phe2906.52, which form an extended aromatic network surrounding the highly conserved Trp2866.48. As a result, Trp2866.48 adopts the rotamer associated with the inactive state. Thus, the steric constraints imposed by Phe2906.52 appear to structurally mimic the interaction of the β-ionone ring of retinal with the conserved Trp2656.48 and Phe2125.47 on rhodopsin (Shi et al., J Biol Chem 277, 40989 (2002)) (FIG. 13C).







TABLE 5







Direct contacts between β2AR and carazolol.











β2AR atom
Carazolol
Distance (Å)











Hydrogen Bond and Salt Bridge Contacts











Asp1133.32 (OD2)
N19
2.9



Asp1133.32 (OD1)
O17
2.6



Ser2035.42 (OG)
N7
3.2



Asn3127.39 (ND2)
O17
2.9



Asn3127.39 (OD1)
N19
2.9



Tyr3167.43 (OH)
N19
3.4







Hydrophobic and Aromatic Interactions


(closest distance for each residue, <4 Å)











Trp1093.28 (CH2)
C21
3.8



Val1143.33 (CG1)
C11
3.9



Val1173.36 (CG1)
C12
4.0



Thr1183.37 (OG1)
C11
3.9



Phe1935.32 (CE2)
C6
3.5



Tyr1995.38 (CE2)
C2
3.9



Ser2075.46 (CB)
C10
3.6



Trp2866.48 (CH2)
O17
3.4



Phe2896.51 (CE2)
O14
3.7



Phe2906.52 (CZ)
C12
3.5



Asn2936.55 (ND2)
C5
3.6



Tyr3087.35 (OH)
C6
3.6










Structural Alignment and Helix Bundle Reorganization

It has long been thought that class A GPCRs share a similar architecture due to their predicted seven transmembrane helical bundles and sequence conservation within the membrane spanning regions (Lefkowitz, Nat Cell Biol 2, E133 (2000)). We aligned the structure of β2AR-T4L to highest resolution structure of rhodopsin (PDB ID Code 1U19) to evaluate the similarities and differences in ligand binding modes. We used difference distance matrices to select non-divergent areas between the two structures that align to reveal the differences in helix orientation between β2AR-T4L and rhodopsin. For the alignment, residues on β2AR were aligned to equivalent residues on Rhodopsin, respectively: 43-59 to 47-63; 67-95 to 71-99; 122-135 to 126-139; 285-296 to 264-275.
Relative to rhodopsin, the following helical shifts are seen in β2AR-T4L: the extracellular portions of helices I and III angle away from the center of the receptor, helix IV is translated away from the center of the receptor, helix V is translated closer to the center of the receptor and helix VI angles away from the receptor on the cytoplasmic end (FIG. 14). The largest difference is in helix I, which lacks a proline-induced kink found in rhodopsin and is comparatively straight. The angle between the rhodopsin and β2AR positions of helix I is approximately 18° with a shift of 7 Å at the apex on the extracellular face. This structural difference may arise from the need for an accessible binding site in β2AR, which is provided in part by a lack of interactions between the N-terminus and extracellular loop segments. In contrast the N-terminal region in rhodopsin occludes the retinal-binding site through extensive interactions with the extracellular loops (FIG. 12B). Helix V of β2AR is closer to the binding pocket by approximately 3.5 Å on average and its lumenal end is angled more towards helix VI. Helix IV of β2AR is further from the binding site, possibly to remove steric clashes resulting from the modified position of helix V (FIG. 14B, 14C). Helix III pivots further from the binding site about a fulcrum located close to the cytoplasmic end (FIG. 14C). The angle formed between rhodopsin helix III and the β2AR helix III is approximately 7°, yielding a 4 Å displacement out of the binding pocket at the cytoplasmic end of the helix. Helix VI is positioned further from the center of the receptor at the cytoplasmic end as compared to rhodopsin, which is caused by a slight difference in the angle about the proline-induced kink in the helix (FIG. 14C).
The ligand-binding pocket is formed by both structurally conserved and divergent helices as compared to rhodopsin (FIG. 14D). Helices III and V are two of the most conformationally shifted helices and contain the canonical catecholamine binding residues associated with activation of adrenergic family of receptors (Strader et al., J Biol Chem 263, 10267 (1988); Strader, et al., J Biol Chem 264, 13572 (1989); Liapakis et al., J Biol Chem 275, 37779 (2000)). The comparison with rhodopsin shows that the structurally conserved helices provide a common core present throughout the class A GPCRs, whereas the variable helices confer binding site plasticity with a resulting architecture capable of binding a large spectrum of ligands.
Comparison to Rhodopsin-Based GPCR Models
Since the determination of the inactive dark-state rhodopsin structure (Palczewski et al., Science 289, 739 (2000)), a number of homology models of other class A GPCRs have been reported (Bissantz, et al., Proteins 50, 5 (2003); Fano, et al., J Chem Inf Model 46, 1223 (2006); Hobrath, et al., J Med Chem 49, 4470 (2006); Nowak, et al., J Med Chem 49, 205 (2006); Zhang, et al., PLoS Comput Biol 2, e13 (2006)). Typically, homology models start by alignment of so-called fingerprint motifs that are common among the family. These fingerprint motifs are extrapolated to assign coordinates for the entire helical bundle. Loop regions are either ignored or modeled based on databases of loop conformations depending on the application (Bissantz, et. al, Proteins 50, 5 (2003)). A number of models exist for β2AR, some of which have been improved upon with supporting biochemical data (Bissantz, et. al, Proteins 50, 5 (2003); Zhang, et al., PLoS Comput Biol 2, e13 (2006); Freddolino et al., Proc Natl Acad Sci USA 101, 2736 (2004); Furse, et al., J Med Chem 46, 4450 (2003); Gouldson et al., Proteins 56, 67 (2004)). When compared to the β2AR structure reported here (according to the methods described above in this Example), however, all of these models were more similar to rhodopsin, as were models for other receptors (e.g. dopamine, muscarinic, and chemokine). This highlights a general shortcoming in homology models generated from a single structural template. The structural divergence between β2AR and rhodopsin would be quite difficult to predict accurately using only rhodopsin as a template.
Example 4
Structural Insights into β2 Adrenergic Receptor Function
Methods
Molecular Biology for Generation of Mammalian and Sf9 Expression Constructs.
The insect cell expression plasmid that was used as a template for modification of the human β2AR gene has been described previously (Yao et al., Nat Chem Biol 2, 417 (2006)): the wild-type coding sequence of the human β2AR (starting at Gly2) was cloned into the pFastbac1 Sf-9 expression vector (Invitrogen) with the HA signal sequence followed by the Flag epitope tag at the amino terminus and the third glycosylation site mutated as N187E. Using this template, a TAA stop codon was placed between Gly365 and Tyr366, terminating translation without the 48 C-terminal residues of the wild-type β2AR (“β2AR365”). A synthetic DNA cassette encoding the T4 Lysozyme (WT*-C54T, C97A) protein was made by overlapping extension PCR of 50-base oligonucleotides. This cassette was amplified and inserted into the β2AR365 construct between Ile2335.72 and Arg2606.22 (FIG. 21A), using the Quickchange Multi protocol (Stratagene). The corresponding mammalian cell expression plasmid was made by amplifying the entire fusion gene and cloning it into pcDNA3 (Invitrogen). Further deletions in the Sf9 and mammalian cell constructs were made using appropriate synthetic oligonucleotides in the Quickchange Multi protocol (Stratagene). All constructs were confirmed by sequencing.
HEK293 Cell Staining and Immunofluorescence Staining.
HEK293 cells were cultured on plastic dishes at 37° C. with 5% CO2 in Dulbecco's modified Eagle's medium (Cellgro) with 5% fetal bovine serum. For an individual expression experiment, cells at confluency were split, and approximately 100,000 cells were used to seed glass cover slips in the same medium. After 2 d, cells were transfected with the addition of 1 μg of a given pcDNA3-receptor plasmid and 3 μl of Fugene 6 reagent (Roche). 48 h after transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, blocked with PBS+2% goat serum, permeabilized with PBS+2% goat serum+0.5% Nonidet P-40 (Sigma), stained with Alexa488-conjugated M1 anti-FLAG antibody (for receptor) plus DAPI (nuclear) in blocking buffer, and washed with blocking buffer. Cover slips were mounted on microscope slides with Vectashield (Vector Labs) and dried overnight. Staining was visualized with an Axioplan 2 fluorescence imaging system, using a 63× objective and either green (Alexa488/FITC) or blue (DAPI/Hoechst) filter sets. A plasmid pcDNA3-β1AR, expressing an N-terminal FLAG-tagged β1 adrenergic receptor, was used as a positive control for cell-surface staining. Empty pcDNA3 was used as a negative control to assess background staining.
Expression and Purification of β2AR-T4L from Baculovirus-Infected Sf9 Cells.
Recombinant baculovirus was made from pFastbac1-β2AR-T4L using the Bac-to-Bac system (Invitrogen), as described previously (Yao et al., Nat Chem Biol 2, 417 (2006)). The β2AR-T4L protein was expressed in Sf9 insect cells infected with this baculovirus, and solubilized according to previously described methods (Kobilka, Anal Biochem 231, 269 (1995)). Dodecylmaltoside-solubilized receptor with the N-terminal FLAG epitope (DYKDDDA) (SEQ ID NO: 1) was purified by M1 antibody affinity chromatography (Sigma), treated with TCEP/iodoacetamide, and further purified by alprenolol-Sepharose chromatography (Kobilka, Anal Biochem 231, 269 (1995)) to isolate only functional GPCR. Eluted alprenolol-bound receptor was re-bound to M1 FLAG resin, and ligand exchange with 30 μM carazolol was performed on the column. β2AR-T4L was eluted from this final column with 0.2 mg/ml FLAG peptide in HLS buffer (0.1% dodecylmaltoside, 20 mM Hepes, 100 mM NaCl, pH 7.5) plus 30 μM carazolol and 5 mM EDTA. N-linked glycolsylations were removed by treatment with PNGaseF (NEB). Protein was concentrated from  ̃5 mg/ml to 50 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator (Vivascience), and dialyzed against HLS buffer plus 10 μM carazolol.
Binding Measurements on Wild-Type β2AR and β2AR-T4L from Membranes.
Membrane preparation from baculovirus-infected Sf9 cells was performed as described previously (Swaminath, et al., Mol Pharmacol 61, 65 (2002)). For each binding reaction, membranes containing 0.7 μg total membrane protein were used. Saturation binding of [3H]-dihydroalprenolol (DHA) was measured by incubating membranes resuspended in 500 μl binding buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 0.4 mg/ml BSA) with 12 different concentrations of [3H]DHA (Perkin Elmer) between 20 pM and 10 nM. After 1 h incubation with shaking at 230 rpm, membranes were filtered from the binding reactions with a Brandel harvester, washed with binding buffer, and measured for bound [3H]DHA with a Beckman LS6000 scintillation counter. Non-specific binding was assessed by performing identical reactions in the presence of 1 μM alprenolol. For competition binding, membranes resuspended in 500 μl binding buffer were incubated with 0.5 nM [3H]DHA plus increasing concentrations of the competing ligand (all compounds were purchased from Sigma). For (−)-isoproterenol and (−)-epinephrine, concentrations were 100 pM-1 mM, each increasing by a factor of 10. For salbutamol, concentrations were 1 nM-10 mM. For ICI-118,551 and formoterol, concentrations were 1 pM-10 μM. Non-specific binding was measured by using 1 μM unlabeled alprenolol as competing ligand. Each data point in the curves in FIGS. 2A and S1 represents the mean of three separate experiments, each done in triplicate. Binding data were analyzed by nonlinear regression analysis using Graphpad Prism. The values for Kd of [3H]DHA and K, of other ligands are shown in Table 6.







TABLE 6







Saturation Binding











[3H]DHA
Kd ± SE (nM)
Bmax (pmol/mg)







β2AR
0.161 ± 0.012
30.0 ± 0.5



β2AR-T4L
0.180 ± 0.016
21.6 ± 0.5











Competition Binding










Ki [S.E. interval]
Ki [S.E. interval]


Ligand
for β2AR (nM)
for β2AR-T4L (nM)





(−)-isoproteronol
 50.6 [48.9-52.3]
15.7 [15.2-16.2]


(−)-epinephrine
175 [163-188]
56.0 [52.8-59.4]


salbutamol
728 [708-750]
307 [291-323] 


ICI-118,551
  0.617 [0.570-0.668]
 0.626 [0.591-0.662]


formoterol
 3.60 [3.39-3.83]
1.68 [1.55-1.81]





Binding affinities of different ligands for the wild-type β2AR and the fusion protein β2AR-T4L. The saturation and competition binding curves shown in FIG. 22 were fit to theoretical saturation and one-site competition binding models, using the program Graphpad Prism. Ki values were calculated using the Cheng-Prusoff equation: Ki = IC50/(1 + [ligand]/Kd).






Bimane Fluorescence Experiments on Purified, Detergent-Solubilized Receptors
β2AR-T4L and β2AR365 were purified as described above, with two important differences. First, prior to iodoacetamide treatment, FLAG-pure receptor at 2.5 μM (measured by soluble [3H]DHA binding) was incubated with 5 μM monobromobimane for 1 h at 4° C. Second, after binding the bimane-labeled alprenolol-Sepharose-purified receptor to M1 antibody resin, the column was washed extensively with ligand-free buffer before elution. Based on previous precedent (Ghanouni, et al., Proc Natl Acad Sci USA 98, 5997 (2001)), this protocol is expected to target primarily Cys2656.27 for fluorophore derivitization. Fluorescence spectroscopy was performed on a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) with photon-counting mode, using an excitation and emission bandpass of 5 nm. All experiments were done at 25° C. For emission scans, we set excitation at 350 nm and measured emission from 417 to 530 nm with an integration time of 1.0 s nm−1. To determine the effect of ligands, spectra were measured after 15 min incubation with different compounds (at saturating concentrations: [(−)-isoproterenol]=100 μM; [ICI-118,551]=10 μM; [salbutamol]=500 μM). Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. The curves shown in FIG. 22B are each the average of triplicate experiments performed in parallel. λmax values and intensity changes for β2AR-T4L and β2AR365, each incubated with different ligands, are tabulated in Table 7.











TABLE 7










λmax ± SD for
λmax ± SD for



Ligand
(β2AR365 (nm)
β2AR-T4L (nm)







none
448 ± 2
447 ± 2



(−)-isoproteronol
453 ± 2
455 ± 2



ICI-118,551
447 ± 1
446 ± 1



salbutamol
449 ± 1
449 ± 1











Intensity at λmaxLigand/Intensity at λmaxnone











Ligand
β2AR365
β2AR-T4L







(−)-isoproteronol
0.758 ± 0.007
0.824 ± 0.006



ICI-118,551
1.013 ± 0.008
1.028 ± 0.008



salbutamol
0.950 ± 0.013
0.928 ± 0.009







Bimane fluorescence responses for unliganded β2AR365 and β2AR-T4L, incubated for 15 min with different ligands. Top panel shows the λmax for fluorescence emission spectra (excitation at 350 nm and emission from 417 to 530 nm) collected after 15 min incubation with ligand. Each value is mean ± standard deviation for triplicate experiments performed in parallel. Bottom panel shows the change in fluorescence intensity after incubation with ligand, represented as the ratio of Intensity at λmax of the ligand to Intensity at λmax of the control no ligand (“none”) response.






Comparing the proteolytic stability of unliganded β2AR and β2AR-T4L.
The limited trypsin proteolysis protocol was adapted from Jiang et al., Biochemistry 44, 1163 (2005). Carazolol-bound β2AR-T4L or wild-type β2AR (each at 30 mg/ml) were diluted 10-fold into HLS buffer (see above) and TPCK-trypsin was added at a 1:1000 ratio (wt:wt). The digests were incubated at room temperature. At various time points, aliquots were removed and flash frozen on dry ice/ethanol. After the last aliquot was removed, all samples were thawed, and an equal volume of 10% SDS/PAGE loading buffer was added to each. Samples were then analyzed by electrophoresis on 12% polyacrylamide gels, followed by staining with Coomassie blue (FIG. 16).
Comparing the Stability of Unliganded β2AR and β2AR-T4L
Unliganded β2AR365 and β2AR-T4L were each purified as described above for the bimane experiments. 200 μl 0.02 mg/ml receptor in HLS buffer was incubated at 37° C. on a heating block. At the time points indicated in FIG. 17, samples were briefly spun and gently vortexed and 16.5 μl was removed and diluted 18.2-fold in HLS (300 μl total). Then 4×5 μl was removed for determination of total binding and 2×5 μl was removed for nonspecific binding. To measure soluble binding, 5 μl diluted receptor was added to 105 μl HLS (400-fold final dilution of receptor) containing 10 nM [3H]DHA±10 μM cold alprenolol. Reactions were incubated 30 min at RT, then on ice until processing. 100 μl of each reaction was applied to a 1 ml G50 column to separate protein from residual unbound [3H]DHA, and receptor was eluted using 1.1 ml ice-cold HLS. Bound [3H]DHA was quantified on a Beckman LS6000 scintillation counter.
Carazolol Dissociation from the “Wild-Type” Receptor NAR365
β2AR365 was purified with carazolol bound, according to the protocol described above for β2ART4L. Carazolol-bound receptor (at approximately 50 μM concentration) was dialyzed in the dark against 1L dialysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 0.1% dodecylmaltoside, 300 micromolar alprenolol) at room temperature with stirring. At indicated time points, two samples were removed from the parafilm-sealed open-ended dialysis chamber, diluted into fresh dialysis buffer, and carazolol emission spectra were obtained on a Spex FluoroMax spectrofluorometer (using excitation at 330 nm and emission from 335 to 400 nm). As internal standards for every time point, samples were removed for determination of protein concentration using the Bio-Rad Protein DC kit (FIG. 19).
CAM and UCM Mutants
The CAMs (constitutively active mutants) described in the literature that are the basis for FIG. 26A and the associated discussion are: L124A (Tao, et al., Mol Endocrinol 14, 1272 (2000)), C116F (Zuscik, et al., J Biol Chem 273, 3401 (1998)), D130A (Rasmussen et al., Mol Pharmacol 56, 175 (1999)), L272C (Jensen et al., J Biol Chem 276, 9279 (2001)), and C285T (Shi et al., J Biol Chem 277, 40989 (2002)). The UCMs (uncoupling mutations) from the literature that were used for FIG. 26C are: D79N (Chung, et al., J Biol Chem 263, 4052 (1988)), F139A (Moro, et al., J Biol Chem 269, 6651 (1994)), T1641 (Green, et al., J Biol Chem 268, 23116 (1993)), N318K (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987)), N322A (Barak, et al., Biochemistry 34, 15407 (1995)), P323A (Barak, et al., Biochemistry 34, 15407 (1995)), Y326A (Barak, et al., Biochemistry 34, 15407 (1995)), L339A (Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)), and L340A (Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)).
Biochemical and Structural Analysis of β2AR-T4L
The β2AR fusion protein in which T4 Lysozyme replaces most of the third intracellular loop of the GPCR (“β2AR-T4L”) retains near-native pharmacologic properties. The β2AR-T4L protein was crystallized in lipidic cubic phase, as described in the Examples above, and the resulting 2.4 Å resolution crystal structure reveals the interface between the receptor and the ligand carazolol, a partial inverse agonist. The efficacy of a ligand describes the effect of the ligand on the functional properties of a GPCR. For purposes of the Examples only, agonists are defined as ligands that fully activate the receptor; partial agonists induce submaximal activation even at saturating concentrations; inverse agonists inhibit basal receptor activity, and antagonists have no effect on basal activity, but competitively block access of other ligands. Carazolol, is defined as a partial inverse agonist because it suppresses only 50% of the basal activity of the β2AR. Analysis of mutagenesis data in light of the structure clarifies the roles of different amino acids in inverse agonist binding, and implies that rearrangement of the binding pocket accompanies agonist binding. In addition, the structure reveals how mutations known to cause constitutive activity or uncoupling of agonist binding and G-protein activation are distributed between the ligand-binding pocket and the cytoplasmic surface of the protein, such that changes in side chains due to interaction with the ligand can be transmitted through the structure to the site of G protein interaction.
Cloning of β2AR-T4L
DNA encoding the T4L protein (C54T, C97A) (Matsumura, et al., Proc Natl Acad Sci USA 86, 6562 (1989)) was initially cloned into the human β2AR gene, guided by comparison of ICL3 length and sequence among class A GPCRs (Horn et al., Nucleic Acids Res 31, 294 (2003)): residues 2345.73-2596.21 of the β2AR were replaced by residues 2-164 of T4L (construct “E3” in FIG. 21A). In addition, the receptor was truncated at position 365, which aligns approximately with the position of the rhodopsin carboxyl terminus. Although these modifications resulted in a receptor that was expressed efficiently in Sf9 cells, further optimization was carried out to reduce the length of the junction between the receptor and the T4L termini, as described in the methods above. Several candidate constructs are illustrated in FIG. 21A, and selected immunofluorescence images of transfected, permeabilized HEK293 cells are shown in FIG. 21B. Relative to the initial construct, we could remove three residues from the cytoplasmic end of helix V, three residues from the C-terminal end of T4L, and three residues from the N terminus of helix VI, all without losing significant cell-surface expression. The final construct used for crystallization trials (“β2AR-T4L”) has residues 231570-2626.24 of the β2AR replaced by amino acids 2-161 of T4L (“1D” in FIG. 21A). Similar reduction of flexibility through minimization of linker length has been important in previous crystallization studies on soluble fusion proteins (Smyth, et al., Protein Sci 12, 1313 (2003)).
Functional Properties of β2AR-T4L
We measured saturation binding of [3H]DHA to the β2AR-T4L, as well as competition binding of the inverse agonist ICI-118,551 and several agonists (FIG. 22A, FIG. 15 and Table 6). The results show that β2AR-T4L has wild-type affinity for the antagonist [3H]DHA and the inverse agonist ICI-118,551, whereas the affinity for both agonists (isoproterenol, epinephrine, formoterol) and a partial agonist (salbutamol) is two to three-fold higher relative to wild-type β2AR. Higher agonist binding affinity is a property associated with constitutively active mutants (CAMs) of GPCRs. CAMs of the β2AR also exhibit elevated basal, agonist-independent activation of Gs, and typically have lower expression levels and reduced stability (Gether et al., J Biol Chem 272, 2587 (1997); Rasmussen et al., Mol Pharmacol 56, 175 (1999)). β2ART4L exhibits binding properties of a CAM, but it expresses at levels exceeding 1 mg per liter of Sf9 cell culture, is more resistant to trypsin proteolysis than the wild-type β2AR (FIG. 16), and retains binding activity in detergent at 37° C. as well as the wild-type receptor (FIG. 17).
β2AR-T4L did not couple to Gs, as expected due to the replacement of ICL3 by T4L. To assess whether the fused protein alters receptor function at the level of its ability to undergo conformational changes, we used a covalently attached fluorescent probe as a reporter for ligand-induced structural changes. Fluorophores attached at Cys2656.27, at the cytoplasmic end of helix VI, detect agonist-induced conformational changes that correlate with the efficacy of the agonist towards G protein activation (Ghanouni et al., J Biol Chem 276, 24433 (2001); Ghanouni, et al., Proc Natl Acad Sci USA 98, 5997 (2001); Swaminath et al., J Biol Chem 279, 686 (2004); Swaminath et al., J Biol Chem 280, 22165 (2005)). Detergent-solubilized β2AR365 (wild-type receptor truncated at 365) and β2AR-T4L were each labeled with monobromobimane. Addition of the agonist isoproterenol to purified β2AR365 induces a decrease in fluorescence intensity and a shift in λmax for the attached bimane probe (FIG. 22B and Table 7). These changes in intensity and λmax are consistent with an agonist-induced increase in polarity around bimane. A smaller change is observed with the partial agonist salbutamol, while the inverse agonist ICI-118,551 had little effect. For the β2AR-T4L, there are subtle differences in the baseline spectrum of the bimane-labeled fusion protein, as might be expected if the environment around Cys2656.27 is altered by T4L. However, the full agonist isoproterenol induces a qualitatively similar decrease in intensity and rightward shift in λmax. Thus the presence of the fused T4L does not prevent agonist-induced conformational changes. The partial agonist salbutamol induced larger responses in β2AR-T4L than were observed in wild-type β2AR, and there was a small increase in fluorescence in response to the inverse agonist ICI-118,551. These are properties observed in CAMs (Gether et al., J Biol Chem 272, 2587 (1997); Samama, et al., J Biol Chem 268, 4625 (1993)) and are consistent with the higher affinities for agonists and partial agonists exhibited by β2AR-T4L. Therefore, we conclude that the T4L fusion induces a partial constitutively active phenotype in the β2AR, likely caused by changes at the cytoplasmic ends of helices V and VI.
Comparison between β2AR-T4L and β2AR-Fab Structures
The β2AR-T4L fusion strategy is validated by comparison of its structure to the structure of wild-type β2AR complexed with a Fab that recognizes a three dimensional epitope consisting of the amino and carboxyl-terminal ends of ICL3, determined at an anisotropic resolution of 3.4 Å/3.7 Å (Rasmussen et al., Nature, 7168:355-6 (2007)). FIG. 23A illustrates the similarity between the fusion and antibody complex approaches to β2AR crystallization, in that both strategies rely on attachment (covalent or non-covalent, respectively) of a soluble protein partner between helices V and VI. A major difference between the two structures is that the extracellular loops and the carazolol ligand could not be modeled in the β2AR-Fab complex, whereas these regions are resolved in the structure of β2AR-T4L. Nonetheless, it is clear that the T4L insertion does not significantly alter the receptor. Superposition of the two structures (FIG. 18) illustrates that the transmembrane helices of the receptor components are very similar (RMSD=0.8 Å for 154 common modeled transmembrane Cα positions, versus 2.3 Å between β2AR-T4L and the 154 equivalent residues in rhodopsin), especially when the modest resolution of the Fab complex is taken into account.
There is one significant difference between the Fab-complex and chimeric receptor structures that can be attributed to the presence of T4L. The cytoplasmic end of helix VI is pulled outward as a result of the fusion to the carboxyl terminus of T4L, which alters the packing of Phe2646.26 at the end of helix VI (FIG. 23B). In the Fab-complex β2AR, interactions between Phe2646.26 and residues in helix V, helix VI, and ICL2 may be important in maintaining the β2AR in the basal state. The loss of these packing interactions in β2AR-T4L could contribute to the higher agonist binding affinity characteristic of a CAM.
An unexpected difference between the structure of rhodopsin and the β2AR-T4L involves the sequence E/DRY found at the cytoplasmic end of helix III in 71% of class A GPCRs. In rhodopsin, Glu1343.49 and Arg1353.50 form a network of hydrogen bond and ionic interactions with Glu2476.30 at the cytoplasmic end of helix VI. These interactions have been referred to as an “ionic lock” that stabilizes the inactive state of rhodopsin and other class A members (Ballesteros et al., J Biol Chem 276, 29171 (2001)). However, the arrangement of the homologous residues is significantly different in β2AR-T4L: Arg1313.50 interacts primarily with Asp1303.49 and a sulfate ion rather than with Glu2686.30, and the distance between helix III and helix VI is greater than in rhodopsin (FIG. 23C). The fact that similar ionic lock structures were obtained using two different approaches suggests that a broken ionic lock is a genuine feature of the carazolol-bound state of the receptor.
Ligand Binding to the β2AR
The β2AR-T4L fusion protein was purified and crystallized in complex with the inverse agonist carazolol. Carazolol stabilizes the β2AR against extremes of pH and temperature, perhaps related to its unusually high binding affinity (Kd<0.1 nM) and slow dissociation kinetics (t1/2 ̃30 h) (FIG. 19). The interactions between carazolol and β2AR-T4L are depicted schematically in FIG. 24. The carbazole ring system is oriented roughly perpendicular to the plane of the membrane, and the alkylamine chain (atoms 15-22 in the model) is nearly parallel to the heterocycle (FIGS. 25A-B). As described in Example 3, above, carazolol was modeled into the electron density as the (S)-(−) isomer due to the higher affinity of this enantiomer, despite the fact that a racemic mixture of the ligand was used in crystallization. Asp1133.32, Tyr3167.43, and Asn3127.39 present a constellation of polar functional groups to the alkylamine and alcohol moieties of the ligand, with Asp1133.32 and Asn3127.39 sidechains forming close contacts (<3 Å) with O17 and N19 atoms of carazolol (FIGS. 24 and 25A-B). Asp1133.32 was one of the first β2AR residues shown to be important for ligand binding; notably the D113N mutation causes complete loss of detectable affinity for antagonists (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987)) and a decrease in the potency of agonists towards cell-based G protein activation by over 4 orders of magnitude (Strader et al., J Biol Chem 263, 10267 (1988)). Likewise, mutations of Asn3127.39 perturb β2AR binding to agonists and antagonists: changes to nonpolar amino acids (Ala or Phe) reduce affinities to undetectable levels, while retention of a polar functionality (Thr or Gln) gives partial affinity (Suryanarayana, et al., Mol Pharmacol 44, 111 (1993)). On the opposite end of the ligand near helix V, N7 of the carbazole heterocycle forms a hydrogen bond with the side chain hydroxyl of Ser2035.42. Interestingly, mutations of Ser2035.42 specifically decrease β2AR affinity towards catecholamine agonists and aryloxyalkylamine ligands with nitrogen-containing heterocycles such as pindolol (Liapakis et al., J Biol Chem 275, 37779 (2000)), and by implication carazolol. Thus, the polar interactions between carazolol and the receptor observed in the crystal structure agree with the known biochemical data. The contribution of Tyr3167.43 to antagonist and agonist affinity remains to be tested; this residue is conserved as tyrosine in all sequenced adrenergic receptor genes (Horn et al., Nucleic Acids Res 31, 294 (2003)).
FIG. 25C shows the tight packing between carazolol and surrounding amino acids that buries 790 Å2 of surface area from solvent; specific contacts are depicted schematically in FIG. 24. Notable among the hydrophobic residues contacting carazolol are Val1143.33, Phe2906.52, and Phe1935.32. The side chain of Val1143.33 from helix III makes multiple contacts with the C8—C13 ring of the carbazole heterocycle, and Phe2906.52 from helix VI forms an edge-to-face aromatic interaction with the same ring. As a result, these two amino acids form a hydrophobic “sandwich” with the portion of the aryl moiety that is common to many adrenergic antagonists. Mutation of Val1143.33 to alanine was shown to decrease β2AR affinity towards the antagonist alprenolol by an order of magnitude, as well as lowering affinity for the agonist epinephrine 300-fold (P. Chelikani et al., Proc Natl Acad Sci USA 104, 7027 (2007)). Phe1935.32 is different from other carazolol contact residues in that it is located on the ECL2, in the path of hormone accessibility to the binding pocket. This amino acid contributes more buried surface area than any other residue to the interface between β2AR-T4L and carazolol (see Table 8). Therefore, Phe1935.32 is likely to contribute significantly to the energy of β2AR-carazolol complex formation, and the position of this residue on the extracellular side of the binding site may allow it to act as a gate that contributes to the unusually slow dissociation of the ligand (FIG. 19).










TABLE 8







β2AR residue
Surface area buried (Å2)



















Trp1093.28
21.4



Thr1103.29
5.7



Asp1133.32
19.3



Val1143.33
25.5



Val1173.36
8.5



Thr1183.37
1.9



Phe1935.32
51.2



Thr1955.34
7.4



Tyr1995.38
7.6



Ala2005.39
10.0



Ser2035.42
9.0



Ser2045.43
4.6



Ser2075.46
6.3



Trp2866.48
3.1



Phe2896.51
20.0



Phe2906.52
19.0



Phe2936.55
18.7



Tyr3087.35
14.4



Asn3127.39
22.5



Tyr3167.43
6.5







Buried surface area contributions at the β2AR-T4L/carazolol interface. Solvent accessible surface area calculations were done with the CNS software package (Brunger et al., Acta Crystallogr D Biol Crystallogr 54, 905 (1998)), using a probe radius of 1.4 Å. Buried surface area contributions of individual residues were determined by calculating solvent-accessible surface area per residue for the full β2ART4L/carazolol model, and subtracting these numbers from the calculated values for the receptor model without carazolol.






Analysis of the binding pocket provides insights into the structural basis for pharmacologic selectivity between the β2AR and closely related adrenergic receptors such as the β1AR. The affinities of these two receptors for certain ligands, such as ICI-118,551, betaxolol and RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)), differ by up to 100-fold. Curiously, all of the amino acids in the carazolol binding pocket are conserved between the β1AR and β2AR (see FIG. 20). The majority of the 94 amino acid differences between the β1AR and β2AR are found in the cytoplasmic and extracellular loops. While residues that differ in the transmembrane segments generally face the lipid bilayer, eight residues lie at the interface between helices and may influence helix packing. The structural basis for pharmacologic differences between β1AR and β2AR must, therefore, arise from amino acid differences in the entrance to the binding pocket or subtle differences in the packing of helices. Evidence for the latter comes from chimeric receptor studies (Frielle, et al., Proc Natl Acad Sci USA 85, 9494 (1988)) in which successive exchange of helices between β1AR and β2ARs led to a gradual change in affinity for the β2AR selective ICI-118,551 and the β1AR selective betaxolol.
As discussed above, β2AR-T4L shows CAM-like properties with respect to agonist binding affinities, suggesting that the unliganded β2AR-T4L may exist in a more active conformation than the wild type-β2AR. Nevertheless, as shown in FIG. 22B, β2AR-T4L can be stabilized in an inactive conformation by an inverse agonist. Since β2AR-T4L was crystallized with bound carazolol, a partial inverse agonist, the structure most likely represents an inactive state. This is consistent with the similarity of the β2AR-T4L and β2AR-Fab5 carazolol-bound structures. To assess whether conformational changes are required to accommodate catecholamines, a model of isoproterenol was placed in the binding site such that common atoms (16-22 in FIG. 24) were superimposed onto the analogous carazolol coordinates in the crystal structure (FIG. 25D). Residues Ser2045.43 and Ser2075.46 are critical for catecholamine binding and activation of the β2AR, with Ser2045.43 hydrogen bonding to the meta-hydroxyl and Ser2075.46 to the para-hydroxyl of the catechol ring, respectively (Strader, et al., J Biol Chem 264, 13572 (1989)). In our model, the catechol hydroxyls of isoproterenol face the appropriate serines on helix V, but the distances are too long for hydrogen bonding (6.8 Å from meta-hydroxyl oxygen to the sidechain oxygen of Ser2045.43, 4.8 Å from the para-hydroxyl oxygen to the sidechain oxygen of Ser2075.46). In addition, Asn2936.55 and Tyr3087.35, two residues expected to form selective interactions with agonists based on the literature (Wieland, et al., Proc Natl Acad Sci USA 93, 9276 (1996); Kikkawa, et al., Mol Pharmacol 53, 128 (1998)), are too distant to form productive polar or hydrophobic contacts with the modeled isoproteronol molecule. These observations suggest that agonist binding requires changes in the binding site relative to the carazolol-bound structure, unless common structural components of agonists and inverse agonists bind in a significantly different manner.
Structural Insights into β2AR Activation
Analysis of mutations that affect β2AR function provides insights into structural rearrangements that are likely to occur during receptor activation. FIG. 26A illustrates the location of amino acids for which mutations lead to elevated basal, agonist-independent activity (constitutively active mutations, CAMs), as well as amino acids for which mutations impair agonist activation (uncoupling mutations, UCMs). Residues for which CAMs have been described are likely to be involved in interactions that maintain the receptor in the inactive conformation. These amino acids are centrally located on helices III and VI. In contrast, positions in which UCMs have been observed are likely to form intramolecular interactions that stabilize the active state. A cluster of UCMs are found at the cytoplasmic end of helix VII. Neither CAMs nor UCMs are directly involved in agonist binding. Although the CAMs and UCMs are not directly connected in sequence, it is evident from the structure that they are linked through packing interactions, such that movements in one will likely affect the packing of others. For example, FIG. 26A (right panel) shows all amino acids with atoms within 4 Å of the two centrally located CAMs, Leu1243.43 (Tao, et al., Mol Endocrinol 14, 1272 (2000)) and Leu2726.34 (Jensen et al., J Biol Chem 276, 9279 (2001)). Several amino acids that pack against these CAMs also interact with one or more UCMs. Trp2866.48 lies at the base of the binding pocket. It has been proposed that agonist binding leads to a change in the rotameric state of Trp2866.48 with subsequent changes in the angle of the helical kink formed by Pro2886.50 (Shi et al., J Biol Chem 277, 40989 (2002)). It is likely that an agonist-induced change in the rotameric state of Trp2866.48 will be linked to changes in sidechains of CAMs and UCMs through packing interactions and propagated to the cytoplasmic ends of the helices and the associated intracellular loops that interact with G proteins and other signaling molecules.
In the structures of both rhodopsin and the β2AR, a cluster of water molecules lies near the most highly conserved class A GPCR residues (FIG. 26B). It has been proposed that these water molecules may play a role in the structural changes involved in receptor activation (Pardo, et al., Chembiochem 8, 19 (2007)). FIG. 26C shows the network of potential hydrogen bonding interactions that link Trp2866.48 with conserved amino acids extending to the cytoplasmic ends of helices. UCMs have been identified for three amino acids linked by this network—N3227.49, P3237.50, and Y3267.53 (Barak et al., Biochemistry 34, 15407 (1995)). This relatively loose-packed, water filled region is likely to be important in allowing conformational transitions, as there will be fewer steric restraints to sidechain repacking.
All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.










APPENDIX 1







HEADER
MEMBRANE PROTEIN/HYDROLASE
05-OCT-07
2RH1








TITLE
HIGH RESOLUTION CRYSTAL STRUCTURE OF HUMAN B2-ADRENERGIC G


TITLE
2 PROTEIN-COUPLED RECEPTOR









COMPND

MOL_ID: 1;


COMPND
2
MOLECULE: BETA-2-ADRENERGIC RECEPTOR/T4-LYSOZYME CHIMERA;


COMPND
3
CHAIN: A;


COMPND
4
SYNONYM: BETA-2 ADRENERGIC RECEPTOR, BETA-2 ADRENOCEPTOR,


COMPND
5
BETA-2 ADRENORECEPTOR/LYSIS PROTEIN, MURAMIDASE,


COMPND
6
ENDOLYSIN;


COMPND
7
ENGINEERED: YES;


COMPND
8
MUTATION: YES


SOURCE

MOL_ID: 1;


SOURCE
2
ORGANISM_SCIENTIFIC: HOMO SAPIENS, ENTEROBACTERIA PHAGE T4;


SOURCE
3
ORGANISM_COMMON: HUMAN,;


SOURCE
4
STRAIN: ,;


SOURCE
5
GENE: ADRB2, ADRB2R, B2AR/E;


SOURCE
6
EXPRESSION_SYSTEM: SPODOPTERA FRUGIPERDA;


SOURCE
7
EXPRESSION_SYSTEM_COMMON: FALL ARMYWORM;


SOURCE
8
EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID;


SOURCE
9
EXPRESSION_SYSTEM_PLASMID: PFASTBAC1;


SOURCE
10
OTHER_DETAILS: THE CONSTRUCT HAS BEEN OBTAINED BY


SOURCE
11
OVERLAPPING EXTENSION PCR


KEYWDS

GPCR, 7TM, ADRENERGIC, FUSION, LIPIDIC CUBIC PHASE, LIPIDIC,


KEYWDS
2
MESOPHASE, CHOLESTEROL, MEMBRANE PROTEIN, MEMBRANE 


KEYWDS
3
PROTEIN/HYDROLASE COMPLEX








EXPDTA
X-RAY DIFFRACTION









AUTHOR

V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. F. RASMUSSEN,


AUTHOR
2
F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, B. K. KOBILKA,


AUTHOR
3
R. C. STEVENS












REVDAT
5
07-OCT-08
2RH1
1
REMARK


REVDAT
4
11-DEC-07
2RH1
1
REMARK


REVDAT
3
04-DEC-07
2RH1
1
JRNL


REVDAT
2
06-NOV-07
2RH1
1
JRNL HELIX SHEET


REVDAT
1
30-OCT-07
2RH1
0









JRNL
AUTH
V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. RASMUSSEN,


JRNL
AUTH 2
F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS,


JRNL
AUTH 3
B. K. KOBILKA, R. C. STEVENS


JRNL
TITL
HIGH-RESOLUTION CRYSTAL STRUCTURE OF AN ENGINEERED


JRNL
TITL 2
HUMAN BETA2-ADRENERGIC G PROTEIN-COUPLED RECEPTOR.










JRNL
REF
SCIENCE
V. 318 1258 2007


JRNL
REFN
ASTM SCIEAS
US ISSN 0036-8075









REMARK
1



REMARK
1
REFERENCE 1










REMARK
1
AUTH
D. M. ROSENBAUM, V. CHEREZOV, M. A. HANSON,


REMARK
1
AUTH 2
S. G. F. RASMUSSEN, F. S. THIAN, T. S. KOBILKA, H. J. CHOI,


REMARK
1
AUTH 3
X. J. YAO, W. I. WEIS, R. C. STEVENS, B. K. KOBILKA


REMARK
1
TITL 
GPCR ENGINEERING YIELDS HIGH-RESOLUTION STRUCTURAL


REMARK
1
TITL 2
INSIGHTS INTO BETA2 ADRENERGIC RECEPTOR FUNCTION.


REMARK
1
REF
TO BE PUBLISHED









REMARK
1
REFN


REMARK
2



REMARK
2
RESOLUTION. 2.40 ANGSTROMS.


REMARK
3



REMARK
3
REFINEMENT.


REMARK
3
PROGRAM: REFMAC 5.2.0019


REMARK
3
AUTHORS: MURSHUDOV, VAGIN, DODSON


REMARK
3



REMARK
3
REFINEMENT TARGET: MAXIMUM LIKELIHOOD


REMARK
3



REMARK
3
DATA USED IN REFINEMENT.










REMARK
3
RESOLUTION RANGE HIGH (ANGSTROMS):
  2.40


REMARK
3
RESOLUTION RANGE LOW (ANGSTROMS):
  19.95


REMARK
3
DATA CUTOFF (SIGMA(F)):
  0.000


REMARK
3
COMPLETENESS FOR RANGE (%):
  99.8


REMARK
3
NUMBER OF REFLECTIONS:
26506









REMARK
3



REMARK
3
FIT TO DATA USED IN REFINEMENT.










REMARK
3
CROSS-VALIDATION METHOD:
THROUGHOUT


REMARK
3
FREE R VALUE TEST SET SELECTION:
RANDOM


REMARK
3
R VALUE (WORKING + TEST SET):
  0.198


REMARK
3
R VALUE (WORKING SET):
  0.196


REMARK
3
FREE R VALUE:
  0.232


REMARK
3
FREE R VALUE TEST SET SIZE (%):
  4.900


REMARK
3
FREE R VALUE TEST SET COUNT:
1310


REMARK
3



REMARK
3
FIT IN THE HIGHEST RESOLUTION BIN.










REMARK
3
TOTAL NUMBER OF BINS USED:
 20


REMARK
3
BIN RESOLUTION RANGE HIGH:
  2.40


REMARK
3
BIN RESOLUTION RANGE LOW:
  2.46


REMARK
3
REFLECTION IN BIN (WORKING SET):
1829


REMARK
3
BIN COMPLETENESS (WORKING + TEST) (%):
 98.76


REMARK
3
BIN R VALUE (WORKING SET):
  0.2700


REMARK
3
BIN FREE R VALUE SET COUNT:
 78


REMARK
3
BIN FREE R VALUE:
  0.3010


REMARK
3










REMARK
3
NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.


REMARK
3
ALL ATOMS: 3804


REMARK
3



REMARK
3
B VALUES.










REMARK
3
FROM WILSON PLOT (A**2):
63.91


REMARK
3
MEAN B VALUE (OVERALL, A**2):
63.91


REMARK
3
OVERALL ANISOTROPIC B VALUE.











REMARK
3
B11 (A**2):
0.43000



REMARK
3
B22 (A**2):
−3.46000


REMARK
3
B33 (A**2):
4.25000


REMARK
3
B12 (A**2):
0.00000


REMARK
3
B13 (A**2):
2.26000


REMARK
3
B23 (A**2):
0.00000


REMARK
3










REMARK
3
ESTIMATED OVERALL COORDINATE ERROR.










REMARK
3
ESU BASED ON R VALUE (A):
 0.295


REMARK
3
ESU BASED ON FREE R VALUE (A):
 0.220


REMARK
3
ESU BASED ON MAXIMUM LIKELIHOOD (A):
 0.203


REMARK
3
ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2):
18.501


REMARK
3


REMARK
3
CORRELATION COEFFICIENTS.










REMARK
3
CORRELATION COEFFICIENT FO-FC:
0.963


REMARK
3
CORRELATION COEFFICIENT FO-FC FREE:
0.944


REMARK
3













REMARK
3
RMS DEVIATIONS FROM IDEAL VALUES
COUNT
RMS
WEIGHT



REMARK
3
BOND LENGTHS REFINED ATOMS (A):
3843;
0.013;
0.022


REMARK
3
BOND LENGTHS OTHERS (A):
2622;
0.000;
0.020


REMARK
3
BOND ANGLES REFINED ATOMS (DEGREES):
5219;
1.500;
2.000


REMARK
3
BOND ANGLES OTHERS (DEGREES):
6377;
4.099;
3.002


REMARK
3
TORSION ANGLES, PERIOD 1 (DEGREES):
441;
3.876;
5.000


REMARK
3
TORSION ANGLES, PERIOD 2 (DEGREES):
154;
31.604;
23.182


REMARK
3
TORSION ANGLES, PERIOD 3 (DEGREES):
627;
11.383;
15.000


REMARK
3
TORSION ANGLES, PERIOD 4 (DEGREES):
22;
12.164;
15.000


REMARK
3
CHIRAL-CENTER RESTRAINTS (A**3):
610;
0.060;
0.200


REMARK
3
GENERAL PLANES REFINED ATOMS (A):
4008;
0.001;
0.020


REMARK
3
GENERAL PLANES OTHERS (A):
804;
0.001;
0.020


REMARK
3
NON-BONDED CONTACTS REFINED ATOMS (A):
926;
0.187;
0.300


REMARK
3
NON-BONDED CONTACTS OTHERS (A):
2443;
0.194;
0.300


REMARK
3
NON-BONDED TORSION REFINED ATOMS (A):
1935;
0.180;
0.500


REMARK
3
NON-BONDED TORSION OTHERS (A):
1580;
0.107;
0.500


REMARK
3
H-BOND (X . . . Y) REFINED ATOMS (A):
194;
0.164;
0.500


REMARK
3
H-BOND (X . . . Y) OTHERS (A):
NULL;
NULL;
NULL


REMARK
3
POTENTIAL METAL-ION REFINED ATOMS (A):
NULL;
NULL;
NULL


REMARK
3
POTENTIAL METAL-ION OTHERS (A):
NULL;
NULL;
NULL


REMARK
3
SYMMETRY VDW REFINED ATOMS (A):
7;
0.155;
0.300


REMARK
3
SYMMETRY VDW OTHERS (A):
29;
0.140;
0.300


REMARK
3
SYMMETRY H-BOND REFINED ATOMS (A):
6;
0.192;
0.500


REMARK
3
SYMMETRY H-BOND OTHERS (A):
NULL;
NULL;
NULL


REMARK
3
SYMMETRY METAL-ION REFINED ATOMS (A):
NULL;
NULL;
NULL


REMARK
3
SYMMETRY METAL-ION OTHERS (A):
NULL;
NULL;
NULL


REMARK
3


REMARK
3
ISOTROPIC THERMAL FACTOR RESTRAINTS.
COUNT
RMS
WEIGHT


REMARK
3
MAIN-CHAIN BOND REFINED ATOMS (A**2):
2884;
2.352;
2.000


REMARK
3
MAIN-CHAIN BOND OTHER ATOMS (A**2):
896;
0.097;
2.000


REMARK
3
MAIN-CHAIN ANGLE REFINED ATOMS (A**2):
3571;
2.767;
2.500


REMARK
3
SIDE-CHAIN BOND REFINED ATOMS (A**2):
1974;
4.582;
2.000


REMARK
3
SIDE-CHAIN ANGLE REFINED ATOMS (A**2):
1648;
5.849;
2.500


REMARK
3


REMARK
3
ANISOTROPIC THERMAL FACTOR RESTRAINTS.
COUNT
RMS
WEIGHT


REMARK
3
RIGID-BOND RESTRAINTS (A**2):
NULL;
NULL;
NULL


REMARK
3
SPHERICITY; FREE ATOMS (A**2):
NULL;
NULL;
NULL


REMARK
3
SPHERICITY; BONDED ATOMS (A**2):
NULL;
NULL;
NULL


REMARK
3


REMARK
3
NCS RESTRAINTS STATISTICS


REMARK
3
NUMBER OF DIFFERENT NCS GROUPS: NULL


REMARK
3


REMARK
3
TLS DETAILS


REMARK
3
NUMBER OF TLS GROUPS: 3


REMARK
3


REMARK
3
TLS GROUP: 1


REMARK
3
NUMBER OF COMPONENTS GROUP: 2













REMARK
3
COMPONENTS
C
SSSEQI TO
C
SSSEQI


REMARK
3
RESIDUE RANGE:
A
1002
A
1011


REMARK
3
RESIDUE RANGE:
A
1062
A
1161









REMARK
3
ORIGIN FOR THE GROUP (A): −23.6840  58.4050  30.4850


REMARK
3
T TENSOR













REMARK
3
T11:
−0.2208
T22:
−0.2598



REMARK
3
T33:
−0.7033
T12:
0.0432


REMARK
3
T13:
−0.0594
T23:
−0.0241









REMARK
3
L TENSOR













REMARK
3
L11:
3.1220
L22:
2.6914



REMARK
3
L33:
5.9787
L12:
0.8643


REMARK
3
L13:
0.6146
L23:
−0.8632









REMARK
3
S TENSOR















REMARK
3
S11:
−0.1453
S12:
−0.2225
S13:
0.2116



REMARK
3
S21:
0.0984
S22:
−0.0656
S23:
−0.0133


REMARK
3
S31:
−0.1849
S32:
−0.1761
S33:
0.2109


REMARK
3









REMARK
3
TLS GROUP: 2


REMARK
3
NUMBER OF COMPONENTS GROUP: 1













REMARK
3
COMPONENTS
C
SSSEQI TO
C
SSSEQI


REMARK
3
RESIDUE RANGE:
A
1012
A
1061









REMARK
3
ORIGIN FOR THE GROUP (A): −35.0580  69.0010  11.9610


REMARK
3
T TENSOR













REMARK
3
T11:
0.0414
T22:
−0.0871



REMARK
3
T33:
−0.4908
T12:
0.0577


REMARK
3
T13:
−0.1559
T23:
−0.0085









REMARK
3
L TENSOR













REMARK
3
L11:
9.6406
L22:
16.6501



REMARK
3
L33:
7.1133
L12:
−6.5961


REMARK
3
L13:
−0.9803
L23:
3.2882









REMARK
3
S TENSOR















REMARK
3
S11:
−0.1628
S12:
−0.1008
S13:
0.4114



REMARK
3
S21:
−0.7585
S22:
−0.1058
S23:
0.7355


REMARK
3
S31:
−0.6590
S32:
−0.6073
S33:
0.2686


REMARK
3









REMARK
3
TLS GROUP: 3


REMARK
3
NUMBER OF COMPONENTS GROUP: 2













REMARK
3
COMPONENTS
C
SSSEQI TO
C
SSSEQI


REMARK
3
RESIDUE RANGE:
A
 29
A
230


REMARK
3
RESIDUE RANGE:
A
263
A
342









REMARK
3
ORIGIN FOR THE GROUP (A): −33.0740  20.0130  7.1220


REMARK
3
T TENSOR













REMARK
3
T11:
−0.0103
T22:
−0.2341



REMARK
3
T33:
−0.5401
T12:
−0.0025


REMARK
3
T13:
−0.0974
T23:
−0.0034









REMARK
3
L TENSOR













REMARK
3
L11:
2.3670
L22:
6.1551



REMARK
3
L33:
1.9314
L12:
2.1068


REMARK
3
L13:
0.8591
L23:
0.7864









REMARK
3
S TENSOR















REMARK
3
S11:
−0.0346
S12:
0.0267
S13:
−0.2068



REMARK
3
S21:
−0.5009
S22:
0.0712
S23:
0.2388


REMARK
3
S31:
0.3208
S32:
0.0002
S33:
−0.0366


REMARK
3









REMARK
3
BULK SOLVENT MODELLING.


REMARK
3
METHOD USED: MASK


REMARK
3
PARAMETERS FOR MASK CALCULATION










REMARK
3
VDW PROBE RADIUS:
1.40


REMARK
3
ION PROBE RADIUS:
0.80


REMARK
3
SHRINKAGE RADIUS:
0.80


REMARK
3









REMARK
3
OTHER REFINEMENT REMARKS: HYDROGENS HAVE BEEN ADDED IN THE


REMARK
3
RIDING POSITIONS. WATER #548 HAS STRONG DIFFERENCE DENSITY BUT


REMARK
3
WEAK 2FO-FC DENSITY.


REMARK
4


REMARK
4
2RH1 COMPLIES WITH FORMAT V. 3.1, 01-AUG-2007


REMARK
100


REMARK
100
THIS ENTRY HAS BEEN PROCESSED BY RCSB.


REMARK
100
THE RCSB ID CODE IS RCSB044849.


REMARK
200


REMARK
200
EXPERIMENTAL DETAILS










REMARK
200
EXPERIMENT TYPE:
X-RAY DIFFRACTION


REMARK
200
DATE OF DATA COLLECTION:
22-JUN-2007; 18-JUL-2007


REMARK
200
TEMPERATURE (KELVIN):
78; 78


REMARK
200
PH:
6.75


REMARK
200
NUMBER OF CRYSTALS USED:
27


REMARK
200


REMARK
200
SYNCHROTRON (Y/N):
Y; Y


REMARK
200
RADIATION SOURCE:
APS; APS


REMARK
200
BEAMLINE:
23-ID-B; 23-ID-B


REMARK
200
X-RAY GENERATOR MODEL:
NULL


REMARK
200
MONOCHROMATIC OR LAUE (M/L):
M


REMARK
200
WAVELENGTH OR RANGE (A):
1.03321; 1.03321


REMARK
200
MONOCHROMATOR:
DOUBLE CRYSTAL


REMARK
200
OPTICS:
MIRRORS; MIRRORS


REMARK
200


REMARK
200
DETECTOR TYPE:
CCD; CCD


REMARK
200
DETECTOR MANUFACTURER:
MARMOSAIC 300 MM CCD;


REMARK
200

MARMOSAIC 300 MM CCD


REMARK
200
INTENSITY-INTEGRATION SOFTWARE:
XDS


REMARK
200
DATA SCALING SOFTWARE:
XDS


REMARK
200










REMARK
200
NUMBER OF UNIQUE REFLECTIONS:
26506


REMARK
200
RESOLUTION RANGE HIGH (A):
  2.400


REMARK
200
RESOLUTION RANGE LOW (A):
  20.000


REMARK
200
REJECTION CRITERIA (SIGMA(I)):
  −3.000


REMARK
200


REMARK
200
OVERALL.


REMARK
200
COMPLETENESS FOR RANGE (%):
99.2


REMARK
200
DATA REDUNDANCY:
NULL


REMARK
200
R MERGE (I):
 0.12700


REMARK
200
R SYM (I):
NULL


REMARK
200
<I/SIGMA(I)> FOR THE DATA SET:
 9.6200


REMARK
200









REMARK
200
IN THE HIGHEST RESOLUTION SHELL.










REMARK
200
HIGHEST RESOLUTION SHELL, RANGE HIGH (A):
 2.40


REMARK
200
HIGHEST RESOLUTION SHELL, RANGE LOW (A):
 2.50


REMARK
200
COMPLETENESS FOR SHELL (%):
99.1


REMARK
200
DATA REDUNDANCY IN SHELL:
NULL


REMARK
200
R MERGE FOR SHELL (I):
 0.67800


REMARK
200
R SYM FOR SHELL (I):
NULL


REMARK
200
<I/SIGMA(I)> FOR SHELL:
 2.200


REMARK
200









REMARK
200
DIFFRACTION PROTOCOL: SINGLE WAVELENGTH


REMARK
200
METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT


REMARK
200
SOFTWARE USED: PHASER


REMARK
200
STARTING MODEL: PDB ENTRIES 1U19, 2LZM


REMARK
200


REMARK
200
REMARK: THIS STRUCTURE IS A PART OF THE ROADMAP/PSI COMMUNITY


REMARK
200
OUTREACH PROGRAM, NOT A SPECIFIC PSI TARGET.


REMARK
280


REMARK
280
CRYSTAL










REMARK
280
SOLVENT CONTENT, VS (%):
59.98


REMARK
280
MATTHEWS COEFFICIENT, VM (ANGSTROMS-3/DA):
 3.07


REMARK
280









REMARK
280
CRYSTALLIZATION CONDITIONS: 30-35% V/V PEG 400, 0.1-0.2M


REMARK
280
NA2SO4, 0.1M BIS-TRIS PROPANE PH 6.5-7.0, 5-7% 1,4-


REMARK
280
BUTANEDIOL, 8-10% CHOLESTEROL, 52-50% MONOOLEIN, PH 6.75,


REMARK
280
LIPIDIC MESOPHASE, TEMPERATURE 293 K


REMARK
290


REMARK
290
CRYSTALLOGRAPHIC SYMMETRY










REMARK
290
SYMMETRY OPERATORS FOR SPACE GROUP:
C 1 2 1


REMARK
290










REMARK
290
SYMOP
SYMMETRY


REMARK
290
NNNMMM
OPERATOR


REMARK
290
1555
X, Y, Z


REMARK
290
2555
−X, Y, −Z


REMARK
290
3555
1/2 + X, 1/2 + Y, Z


REMARK
290
4555
1/2 − X, 1/2 + Y, −Z


REMARK
290









REMARK
290
WHERE NNN -> OPERATOR NUMBER


REMARK
290
MMM -> TRANSLATION VECTOR


REMARK
290


REMARK
290
CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS


REMARK
290
THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM


REMARK
290
RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY


REMARK
290
RELATED MOLECULES.















REMARK
290
SMTRY1
1
1.000000
0.000000
0.000000
0.00000



REMARK
290
SMTRY2
1
0.000000
1.000000
0.000000
0.00000


REMARK
290
SMTRY3
1
0.000000
0.000000
1.000000
0.00000


REMARK
290
SMTRY1
2
−1.000000
0.000000
0.000000
0.00000


REMARK
290
SMTRY2
2
0.000000
1.000000
0.000000
0.00000


REMARK
290
SMTRY3
2
0.000000
0.000000
−1.000000
0.00000


REMARK
290
SMTRY1
3
1.000000
0.000000
0.000000
53.15900


REMARK
290
SMTRY2
3
0.000000
1.000000
0.000000
84.62000


REMARK
290
SMTRY3
3
0.000000
0.000000
1.000000
0.00000


REMARK
290
SMTRY1
4
−1.000000
0.000000
0.000000
53.15900


REMARK
290
SMTRY2
4
0.000000
1.000000
0.000000
84.62000


REMARK
290
SMTRY3
4
0.000000
0.000000
−1.000000
0.00000


REMARK
290









REMARK
290
REMARK: NULL


REMARK
300


REMARK
300
BIOMOLECULE: 1


REMARK
300
SEE REMARK 350 FOR THE AUTHOR PROVIDED AND/OR PROGRAM


REMARK
300
GENERATED ASSEMBLY INFORMATION FOR THE STRUCTURE IN


REMARK
300
THIS ENTRY. THE REMARK MAY ALSO PROVIDE INFORMATION ON


REMARK
300
BURIED SURFACE AREA.


REMARK
300


REMARK
300
REMARK: AUTHORS STATE THAT THE BIOLOGICAL UNIT IS UNKNOWN


REMARK
350


REMARK
350
COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN


REMARK
350
BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE


REMARK
350
MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS


REMARK
350
GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND


REMARK
350
CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN.


REMARK
350


REMARK
350
BIOMOLECULE: 1


REMARK
350
SOFTWARE DETERMINED QUATERNARY STRUCTURE: MONOMERIC


REMARK
350
SOFTWARE USED: PISA


REMARK
350
APPLY THE FOLLOWING TO CHAINS: A














REMARK
350
BIOMT1
1
1.000000
0.000000
0.000000
0.00000


REMARK
350
BIOMT2
1
0.000000
1.000000
0.000000
0.00000


REMARK
350
BIOMT3
1
0.000000
0.000000
1.000000
0.00000


REMARK
375









REMARK
375
SPECIAL POSITION


REMARK
375
THE FOLLOWING ATOMS ARE FOUND TO BE WITHIN 0.15 ANGSTROMS


REMARK
375
OF A SYMMETRY RELATED ATOM AND ARE ASSUMED TO BE ON SPECIAL


REMARK
375
POSITIONS.


REMARK
375











REMARK
375
ATOM RES
CSSEQI



REMARK
375
HOH
A 520
LIES ON A SPECIAL POSITION.


REMARK
465









REMARK
465
MISSING RESIDUES


REMARK
465
THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE


REMARK
465
EXPERIMENT. (M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN


REMARK
465
IDENTIFIER; SSEQ = SEQUENCE NUMBER; I = INSERTION CODE.)


REMARK
465













REMARK
465
M
RES
C
SSEQI



REMARK
465

ASP
A
−6


REMARK
465

TYR
A
−5


REMARK
465

LYS
A
−4


REMARK
465

ASP
A
−3


REMARK
465

ASP
A
−2


REMARK
465

ASP
A
−1


REMARK
465

ALA
A
0


REMARK
465

MET
A
1


REMARK
465

GLY
A
2


REMARK
465

GLN
A
3


REMARK
465

PRO
A
4


REMARK
465

GLY
A
5


REMARK
465

ASN
A
6


REMARK
465

GLY
A
7


REMARK
465

SER
A
8


REMARK
465

ALA
A
9


REMARK
465

PHE
A
10


REMARK
465

LEU
A
11


REMARK
465

LEU
A
12


REMARK
465

ALA
A
13


REMARK
465

PRO
A
14


REMARK
465

ASN
A
15


REMARK
465

ARG
A
16


REMARK
465

SER
A
17


REMARK
465

HIS
A
18


REMARK
465

ALA
A
19


REMARK
465

PRO
A
20


REMARK
465

ASP
A
21


REMARK
465

HIS
A
22


REMARK
465

ASP
A
23


REMARK
465

VAL
A
24


REMARK
465

THR
A
25


REMARK
465

GLN
A
26


REMARK
465

GLN
A
27


REMARK
465

ARG
A
28


REMARK
465

ARG
A
343


REMARK
465

ARG
A
344


REMARK
465

SER
A
345


REMARK
465

SER
A
346


REMARK
465

LEU
A
347


REMARK
465

LYS
A
348


REMARK
465

ALA
A
349


REMARK
465

TYR
A
350


REMARK
465

GLY
A
351


REMARK
465

ASN
A
352


REMARK
465

GLY
A
353


REMARK
465

TYR
A
354


REMARK
465

SER
A
355


REMARK
465

SER
A
356


REMARK
465

ASN
A
357


REMARK
465

GLY
A
358


REMARK
465

ASN
A
359


REMARK
465

THR
A
360


REMARK
465

GLY
A
361


REMARK
465

GLU
A
362


REMARK
465

GLN
A
363


REMARK
465

SER
A
364


REMARK
465

GLY
A
365


REMARK
470









REMARK
470
MISSING ATOM


REMARK
470
THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M = MODEL NUMBER;


REMARK
470
RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER;


REMARK
470
I = INSERTION CODE):












REMARK
470
M
RES
CSSEQI
ATOMS


REMARK
470

ASP
A 29
CG OD1 OD2


REMARK
500









REMARK
500
GEOMETRY AND STEREOCHEMISTRY


REMARK
500
SUBTOPIC: CLOSE CONTACTS IN SAME ASYMMETRIC UNIT


REMARK
500


REMARK
500
THE FOLLOWING ATOMS ARE IN CLOSE CONTACT.


REMARK
500
















REMARK
500
ATM1
RES
C
SSEQI
ATM2
RES
C
SSEQI


REMARK
500
SG
CYS
A
341
O2
PLM
A
415   1.88


REMARK
500









REMARK
500
REMARK: NULL


REMARK
500


REMARK
500
GEOMETRY AND STEREOCHEMISTRY


REMARK
500
SUBTOPIC: TORSION ANGLES


REMARK
500


REMARK
500
TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS:


REMARK
500
(M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN IDENTIFIER;


REMARK
500
SSEQ = SEQUENCE NUMBER; I = INSERTION CODE).


REMARK
500


REMARK
500
STANDARD TABLE:


REMARK
500
FORMAT: (10X, I3, 1X, A3, 1X, A1, I4, A1, 4X, F7.2, 3X, F7.2)


REMARK
500


REMARK
500
EXPECTED VALUES: GJ KLEYWEGT AND TA JONES (1996). PHI/PSI-


REMARK
500
CHOLOGY: RAMACHANDRAN REVISITED. STRUCTURE 4, 1395-1400


REMARK
500














REMARK
500
M
RES
CSSEQI
PSI
PHI



REMARK
500

VAL
A 86
−64.27
−100.05


REMARK
500

TYR
A 141
−22.19
73.12


REMARK
500









REMARK
500
REMARK: NULL


REMARK
600


REMARK
600
HETEROGEN


REMARK
600
THE PALMITIC ACID (PLM) AND ACETAMIDE (ACM) GROUPS ARE


REMARK
600
COVALENTLY LINKED TO THEIR RESPECTIVE CYSTEINE RESIDUES.


REMARK
610


REMARK
610
MISSING HETEROATOM


REMARK
610
THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M = MODEL NUMBER;


REMARK
610
RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER;


REMARK
610
I = INSERTION CODE):












REMARK
610
M
RES
C
SSEQI


REMARK
610

12P
A
416


REMARK
800











REMARK
800
SITE





REMARK
800
SITE_IDENTIFIER: AC1


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
12P
A
416


REMARK
800
SITE_IDENTIFIER: AC2


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
ACM
A
411


REMARK
800
SITE_IDENTIFIER: AC3


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
BU1
A
409


REMARK
800
SITE_IDENTIFIER: AC5


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
CAU
A
408


REMARK
800
SITE_IDENTIFIER: AC6


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
CLR
A
412


REMARK
800
SITE_IDENTIFIER: AC7


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
CLR
A
413


REMARK
800
SITE_IDENTIFIER: AC9


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
MAL
A
401


REMARK
800
SITE_IDENTIFIER: BC1


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
PLM
A
415


REMARK
800
SITE_IDENTIFIER: BC2


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
402


REMARK
800
SITE_IDENTIFIER: BC3


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
403


REMARK
800
SITE_IDENTIFIER: BC4


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
404


REMARK
800
SITE_IDENTIFIER: BC5


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
405


REMARK
800
SITE_IDENTIFIER: BC6


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
406


REMARK
800
SITE_IDENTIFIER: BC7


REMARK
800
SITE_DESCRIPTION: BINDING SITE FOR RESIDUE
SO4
A
407


REMARK
999









REMARK
999
SEQUENCE THE STRUCTURE IS AN INTERNAL FUSION PROTEIN WITH


REMARK
999
LYSOZYME. AN OFFSET 1000 HAS BEEN ADDED TO ORIGINAL


REMARK
999
SEQUENCE DATABASE RESIDUE NUMBERS (2-161) OF THE LYSOZYME


REMARK
999
PART IN COORDINATES TO DISTINGUISH THE LYSOZYME PART IN THE


REMARK
999
CHAIN. THEREFORE THE RESIDUES OF LYSOZYME PART HAVE NUMBERS


REMARK
999
A1002-A1161.
















DBREF
2RH1
A
  1
 230
UNP
P07550
ADRB2_HUMAN
 1
230


DBREF
2RH1
A
1002
1161
UNP
P00720
LYS_BPT4
 2
161


DBREF
2RH1
A
 263
 365
UNP
P07550
ADRB2_HUMAN
263
365














SEQADV
2RH1
ASP
A
−6
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
TYR
A
−5
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
LYS
A
−4
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
ASP
A
−3
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
ASP
A
−2
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
ASP
A
−1
UNP
P07550
EXPRESSION TAG


SEQADV
2RH1
ALA
A
0
UNP
P07550
EXPRESSION TAG
















SEQADV
2RH1
GLU
A
187
UNP
P07550
ASN
187
ENGINEERED


SEQADV
2RH1
THR
A
1054
UNP
P00720
CYS
 54
ENGINEERED


SEQADV
2RH1
ALA
A
1097
UNP
P00720
CYS
 97
ENGINEERED























SEQRES
1
A
500
ASP
TYR
LYS
ASP
ASP
ASP
ALA
MET
GLY
GLN
PRO
GLY
ASN


SEQRES
2
A
500
GLY
SER
ALA
PHE
LEU
LEU
ALA
PRO
ASN
ARG
SER
HIS
ALA


SEQRES
3
A
500
PRO
ASP
HIS
ASP
VAL
THR
GLN
GLN
ARG
ASP
GLU
VAL
TRP


SEQRES
4
A
500
VAL
VAL
GLY
MET
GLY
ILE
VAL
MET
SER
LEU
ILE
VAL
LEU


SEQRES
5
A
500
ALA
ILE
VAL
PHE
GLY
ASN
VAL
LEU
VAL
ILE
THR
ALA
ILE


SEQRES
6
A
500
ALA
LYS
PHE
GLU
ARG
LEU
GLN
THR
VAL
THR
ASN
TYR
PHE


SEQRES
7
A
500
ILE
THR
SER
LEU
ALA
CYS
ALA
ASP
LEU
VAL
MET
GLY
LEU


SEQRES
8
A
500
ALA
VAL
VAL
PRO
PHE
GLY
ALA
ALA
HIS
ILE
LEU
MET
LYS


SEQRES
9
A
500
MET
TRP
THR
PHE
GLY
ASN
PHE
TRP
CYS
GLU
PHE
TRP
THR


SEQRES
10
A
500
SER
ILE
ASP
VAL
LEU
CYS
VAL
THR
ALA
SER
ILE
GLU
THR


SEQRES
11
A
500
LEU
CYS
VAL
ILE
ALA
VAL
ASP
ARG
TYR
PHE
ALA
ILE
THR


SEQRES
12
A
500
SER
PRO
PHE
LYS
TYR
GLN
SER
LEU
LEU
THR
LYS
ASN
LYS


SEQRES
13
A
500
ALA
ARG
VAL
ILE
ILE
LEU
MET
VAL
TRP
ILE
VAL
SER
GLY


SEQRES
14
A
500
LEU
THR
SER
PHE
LEU
PRO
ILE
GLN
MET
HIS
TRP
TYR
ARG


SEQRES
15
A
500
ALA
THR
HIS
GLN
GLU
ALA
ILE
ASN
CYS
TYR
ALA
GLU
GLU


SEQRES
16
A
500
THR
CYS
CYS
ASP
PHE
PHE
THR
ASN
GLN
ALA
TYR
ALA
ILE


SEQRES
17
A
500
ALA
SER
SER
ILE
VAL
SER
PHE
TYR
VAL
PRO
LEU
VAL
ILE


SEQRES
18
A
500
MET
VAL
PHE
VAL
TYR
SER
ARG
VAL
PHE
GLN
GLU
ALA
LYS


SEQRES
19
A
500
ARG
GLN
LEU
ASN
ILE
PHE
GLU
MET
LEU
ARG
ILE
ASP
GLU


SEQRES
20
A
500
GLY
LEU
ARG
LEU
LYS
ILE
TYR
LYS
ASP
THR
GLU
GLY
TYR


SEQRES
21
A
500
TYR
THR
ILE
GLY
ILE
GLY
HIS
LEU
LEU
THR
LYS
SER
PRO


SEQRES
22
A
500
SER
LEU
ASN
ALA
ALA
LYS
SER
GLU
LEU
ASP
LYS
ALA
ILE


SEQRES
23
A
500
GLY
ARG
ASN
THR
ASN
GLY
VAL
ILE
THR
LYS
ASP
GLU
ALA


SEQRES
24
A
500
GLU
LYS
LEU
PHE
ASN
GLN
ASP
VAL
ASP
ALA
ALA
VAL
ARG


SEQRES
25
A
500
GLY
ILE
LEU
ARG
ASN
ALA
LYS
LEU
LYS
PRO
VAL
TYR
ASP


SEQRES
26
A
500
SER
LEU
ASP
ALA
VAL
ARG
ARG
ALA
ALA
LEU
ILE
ASN
MET


SEQRES
27
A
500
VAL
PHE
GLN
MET
GLY
GLU
THR
GLY
VAL
ALA
GLY
PHE
THR


SEQRES
28
A
500
ASN
SER
LEU
ARG
MET
LEU
GLN
GLN
LYS
ARG
TRP
ASP
GLU


SEQRES
29
A
500
ALA
ALA
VAL
ASN
LEU
ALA
LYS
SER
ARG
TRP
TYR
ASN
GLN


SEQRES
30
A
500
THR
PRO
ASN
ARG
ALA
LYS
ARG
VAL
ILE
THR
THR
PHE
ARG


SEQRES
31
A
500
THR
GLY
THR
TRP
ASP
ALA
TYR
LYS
PHE
CYS
LEU
LYS
GLU


SEQRES
32
A
500
HIS
LYS
ALA
LEU
LYS
THR
LEU
GLY
ILE
ILE
MET
GLY
THR


SEQRES
33
A
500
PHE
THR
LEU
CYS
TRP
LEU
PRO
PHE
PHE
ILE
VAL
ASN
ILE


SEQRES
34
A
500
VAL
HIS
VAL
ILE
GLN
ASP
ASN
LEU
ILE
ARG
LYS
GLU
VAL


SEQRES
35
A
500
TYR
ILE
LEU
LEU
ASN
TRP
ILE
GLY
TYR
VAL
ASN
SER
GLY


SEQRES
36
A
500
PHE
ASN
PRO
LEU
ILE
TYR
CYS
ARG
SER
PRO
ASP
PHE
ARG


SEQRES
37
A
500
ILE
ALA
PHE
GLN
GLU
LEU
LEU
CYS
LEU
ARG
ARG
SER
SER


SEQRES
38
A
500
LEU
LYS
ALA
TYR
GLY
ASN
GLY
TYR
SER
SER
ASN
GLY
ASN


SEQRES
39
A
500
THR
GLY
GLU
GLN
SER
GLY











HET
MAL
A
401
23


HET
SO4
A
402
 5


HET
SO4
A
403
 5


HET
SO4
A
404
 5


HET
SO4
A
405
 5


HET
SO4
A
406
 5


HET
SO4
A
407
 5


HET
CAU
A
408
22


HET
BU1
A
409
 6


HET
BU1
A
410
 6


HET
ACM
A
411
 4


HET
CLR
A
412
28


HET
CLR
A
413
28


HET
CLR
A
414
28


HET
PLM
A
415
17


HET
12P
A
416
21










HETNAM

MAL
MALTOSE


HETNAM

SO4
SULFATE ION


HETNAM

CAU
(2S)-1-(9H-CARBAZOL-4-YLOXY)-3-(ISOPROPYLAMINO)PROPAN-


HETNAM
2
CAU
2-OL


HETNAM

BU1
1,4-BUTANEDIOL


HETNAM

ACM
ACETAMIDE


HETNAM

CLR
CHOLESTEROL


HETNAM

PLM
PALMITIC ACID


HETNAM

12P
DODECAETHYLENE GLYCOL


HETSYN

CAU
(S)-CARAZOLOL


HETSYN

12P
POLYETHYLENE GLYCOL PEG400


FORMUL
2
MAL
C12 H22 O11


FORMUL
3
SO4
6(O4 S 2-)


FORMUL
9
CAU
C18 H22 N2 O2


FORMUL
10
BU1
2(C4 H10 O2)


FORMUL
12
ACM
C2 H5 N O


FORMUL
13
CLR
3(C27 H46 O)


FORMUL
16
PLM
C16 H32 O2


FORMUL
17
12P
C24 H50 O13


FORMUL
18
HOH
*48(H2 O)


















HELIX
1
1
ASP
A
29
LYS
A
60
1
32



HELIX
2
2
VAL
A
67
MET
A
96
1
30


HELIX
3
3
ASN
A
103
THR
A
136
1
34


HELIX
4
4
LYS
A
147
MET
A
171
1
25


HELIX
5
5
HIS
A
178
GLU
A
187
1
10


HELIX
6
6
GLN
A
197
GLN
A
229
1
33


HELIX
7
7
LYS
A
267
ILE
A
298
1
32


HELIX
8
8
LYS
A
305
ARG
A
328
1
24


HELIX
9
9
PRO
A
330
CYS
A
341
1
12


HELIX
10
10
ILE
A
1003
GLU
A
1011
1
9


HELIX
11
11
LEU
A
1039
ILE
A
1050
1
12


HELIX
12
12
LYS
A
1060
ARG
A
1080
1
21


HELIX
13
13
ALA
A
1082
SER
A
1090
1
9


HELIX
14
14
ALA
A
1093
MET
A
1106
1
14


HELIX
15
15
GLU
A
1108
GLY
A
1113
1
6


HELIX
16
16
THR
A
1115
GLN
A
1123
1
9


HELIX
17
17
TRP
A
1126
ALA
A
1134
1
9


HELIX
18
18
ARG
A
1137
GLN
A
1141
1
5


HELIX
19
19
PRO
A
1143
THR
A
1155
1
13

















SHEET
1
1
4
LYS
A
1016
ASP
A
1020
0


SHEET
2
1
4
TYR
A
1024
GLY
A
1028
0


SHEET
3
1
4
HIS
A
1031
THR
A
1034
0


SHEET
4
1
4
GLY
A
1056
THR
A
1059
0

















SSBOND
1
CYS
A
106
CYS
A
191
1555
1555
2.05


SSBOND
2
CYS
A
184
CYS
A
190
1555
1555
2.06


















LINK
SG
CYS
A
265
C2
ACM
A
411
1555
1555
1.61


LINK
SG
CYS
A
341
C1
PLM
A
415
1555
1555
1.62























SITE
1
AC1
2
ASP
A
1072
HOH
A
538









SITE
1
AC2
1
CYS
A
265


SITE
1
AC3
4
LYS
A
263
PHE
A
264
HIS
A
269
HOH
A
502


SITE
1
AC5
4
ASP
A
113
PHE
A
193
ASN
A
312
TYR
A
316


SITE
1
AC6
1
ILE
A
112


SITE
1
AC7
1
HOH
A
520


SITE
1
AC9
7
GLU
A
1011
GLY
A
1030
LEU
A
1032
ASP
A
1070


SITE
2
AC9
7
VAL
A
1103
PHE
A
1104
ARG
A
1145


SITE
1
BC1
2
LEU
A
339
CYS
A
341


SITE
1
BC2
5
VAL
A
67
THR
A
68
ARG
A
131
TYR
A
141


SITE
2
BC2
5
SER
A
143


SITE
1
BC3
4
PHE
A
264
LYS
A
270
LYS
A
273
ARG
A
328


SITE
1
BC4
5
PHE
A
1114
THR
A
1115
ASN
A
1116
SER
A
1117


SITE
2
BC4
5
ASN
A
1132


SITE
1
BC5
6
PRO
A
1143
ASN
A
1144
ARG
A
1145
HOH
A
512


SITE
2
BC5
6
HOH
A
526
HOH
A
531


SITE
1
BC6
1
ARG
A
1095


SITE
1
BC7
2
LEU
A
1015
LYS
A
1016














CRYST1
106.318
169.240
40.154
90.00
105.62
90.00
C 1 2 1 4











ORIGX1
1.000000
0.000000
0.000000
0.00000


ORIGX2
0.000000
1.000000
0.000000
0.00000


ORIGX3
0.000000
0.000000
1.000000
0.00000


SCALE1
0.009406
0.000000
0.002630
0.00000


SCALE2
0.000000
0.005909
0.000000
0.00000


SCALE3
0.000000
0.000000
0.025859
0.00000


















ATOM
1
N
ASP
A
29
−52.822
−1.611
23.137
1.00
98.48
N


ATOM
2
CA
ASP
A
29
−51.922
−2.262
22.148
1.00
98.06
C


ATOM
3
C
ASP
A
29
−52.178
−1.713
20.742
1.00
97.74
C


ATOM
4
O
ASP
A
29
−51.291
−1.100
20.143
1.00
96.54
O


ATOM
5
CB
ASP
A
29
−52.106
−3.786
22.184
1.00
97.64
C


ATOM
6
N
GLU
A
30
−53.394
−1.944
20.236
1.00
98.37
N


ATOM
7
CA
GLU
A
30
−53.821
−1.515
18.887
1.00
98.17
C


ATOM
8
C
GLU
A
30
−54.424
−0.104
18.879
1.00
98.57
C


ATOM
9
O
GLU
A
30
−54.197
0.649
17.943
1.00
99.79
O


ATOM
10
CB
GLU
A
30
−54.840
−2.498
18.305
1.00
99.00
C


ATOM
11
CG
GLU
A
30
−54.377
−3.969
18.286
1.00
99.46
C


ATOM
12
CD
GLU
A
30
−55.432
−4.928
17.733
1.00
98.77
C


ATOM
13
OE1
GLU
A
30
−56.228
−4.527
16.853
1.00
97.60
O


ATOM
14
OE2
GLU
A
30
−55.463
−6.092
18.185
1.00
99.75
O


ATOM
15
N
VAL
A
31
−55.190
0.248
19.918
1.00
97.89
N


ATOM
16
CA
VAL
A
31
−55.757
1.618
20.079
1.00
96.48
C


ATOM
17
C
VAL
A
31
−54.643
2.678
20.185
1.00
95.12
C


ATOM
18
O
VAL
A
31
−54.838
3.837
19.803
1.00
93.37
O


ATOM
19
CB
VAL
A
31
−56.729
1.697
21.306
1.00
96.71
C


ATOM
20
CG1
VAL
A
31
−57.185
3.135
21.580
1.00
96.80
C


ATOM
21
CG2
VAL
A
31
−57.948
0.806
21.077
1.00
96.80
C


ATOM
22
N
TRP
A
32
−53.486
2.259
20.709
1.00
95.21
N


ATOM
23
CA
TRP
A
32
−52.267
3.063
20.735
1.00
94.60
C


ATOM
24
C
TRP
A
32
−51.837
3.491
19.319
1.00
93.60
C


ATOM
25
O
TRP
A
32
−51.335
4.603
19.140
1.00
91.48
O


ATOM
26
CB
TRP
A
32
−51.129
2.273
21.427
1.00
96.93
C


ATOM
27
CG
TRP
A
32
−49.770
2.899
21.304
1.00
98.27
C


ATOM
28
CD1
TRP
A
32
−49.169
3.734
22.197
1.00
100.48
C


ATOM
29
CD2
TRP
A
32
−48.844
2.737
20.216
1.00
100.89
C


ATOM
30
NE1
TRP
A
32
−47.924
4.105
21.737
1.00
101.70
N


ATOM
31
CE2
TRP
A
32
−47.700
3.507
20.523
1.00
102.25
C


ATOM
32
CE3
TRP
A
32
−48.874
2.013
19.010
1.00
100.84
C


ATOM
33
CZ2
TRP
A
32
−46.587
3.578
19.667
1.00
101.34
C


ATOM
34
CZ3
TRP
A
32
−47.771
2.082
18.158
1.00
100.68
C


ATOM
35
CH2
TRP
A
32
−46.640
2.860
18.493
1.00
101.20
C


ATOM
36
N
VAL
A
33
−52.036
2.600
18.332
1.00
92.39
N


ATOM
37
CA
VAL
A
33
−51.667
2.852
16.922
1.00
91.10
C


ATOM
38
C
VAL
A
33
−52.435
4.017
16.332
1.00
89.89
C


ATOM
39
O
VAL
A
33
−51.827
4.918
15.748
1.00
88.01
O


ATOM
40
CB
VAL
A
33
−51.920
1.621
16.012
1.00
91.28
C


ATOM
41
CG1
VAL
A
33
−51.625
1.950
14.553
1.00
93.68
C


ATOM
42
CG2
VAL
A
33
−51.080
0.443
16.467
1.00
93.38
C


ATOM
43
N
VAL
A
34
−53.762
3.986
16.490
1.00
88.20
N


ATOM
44
CA
VAL
A
34
−54.638
5.078
16.041
1.00
87.62
C


ATOM
45
C
VAL
A
34
−54.145
6.388
16.649
1.00
87.34
C


ATOM
46
O
VAL
A
34
−53.871
7.334
15.921
1.00
88.42
O


ATOM
47
CB
VAL
A
34
−56.126
4.852
16.422
1.00
87.61
C


ATOM
48
CG1
VAL
A
34
−56.972
6.064
16.046
1.00
87.66
C


ATOM
49
CG2
VAL
A
34
−56.684
3.592
15.755
1.00
85.12
C


ATOM
50
N
GLY
A
35
−54.036
6.413
17.981
1.00
86.61
N


ATOM
51
CA
GLY
A
35
−53.524
7.576
18.738
1.00
86.08
C


ATOM
52
C
GLY
A
35
−52.123
8.048
18.358
1.00
85.30
C


ATOM
53
O
GLY
A
35
−51.837
9.245
18.408
1.00
85.51
O


ATOM
54
N
MET
A
36
−51.257
7.110
17.981
1.00
85.55
N


ATOM
55
CA
MET
A
36
−49.919
7.431
17.486
1.00
84.86
C


ATOM
56
C
MET
A
36
−50.004
7.888
16.029
1.00
85.06
C


ATOM
57
O
MET
A
36
−49.216
8.733
15.590
1.00
86.04
O


ATOM
58
CB
MET
A
36
−49.001
6.214
17.596
1.00
86.69
C


ATOM
59
CG
MET
A
36
−47.509
6.496
17.400
1.00
88.99
C


ATOM
60
SD
MET
A
36
−46.800
7.642
18.613
1.00
102.42
S


ATOM
61
CE
MET
A
36
−47.213
6.857
20.179
1.00
100.39
C


ATOM
62
N
GLY
A
37
−50.959
7.324
15.287
1.00
83.06
N


ATOM
63
CA
GLY
A
37
−51.227
7.713
13.911
1.00
82.58
C


ATOM
64
C
GLY
A
37
−51.710
9.144
13.811
1.00
81.74
C


ATOM
65
O
GLY
A
37
−51.171
9.912
13.024
1.00
83.08
O


ATOM
66
N
ILE
A
38
−52.724
9.494
14.611
1.00
81.34
N


ATOM
67
CA
ILE
A
38
−53.260
10.869
14.659
1.00
80.88
C


ATOM
68
C
ILE
A
38
−52.153
11.874
15.003
1.00
80.13
C


ATOM
69
O
ILE
A
38
−52.121
12.964
14.453
1.00
81.84
O


ATOM
70
CB
ILE
A
38
−54.432
11.032
15.686
1.00
81.07
C


ATOM
71
CG1
ILE
A
38
−55.635
10.132
15.356
1.00
81.29
C


ATOM
72
CG2
ILE
A
38
−54.912
12.479
15.745
1.00
80.45
C


ATOM
73
CD1
ILE
A
38
−56.310
10.413
14.026
1.00
85.05
C


ATOM
74
N
VAL
A
39
−51.255
11.496
15.911
1.00
79.45
N


ATOM
75
CA
VAL
A
39
−50.128
12.346
16.306
1.00
79.67
C


ATOM
76
C
VAL
A
39
−49.166
12.575
15.154
1.00
79.83
C


ATOM
77
O
VAL
A
39
−48.803
13.717
14.863
1.00
81.09
O


ATOM
78
CB
VAL
A
39
−49.352
11.740
17.496
1.00
81.11
C


ATOM
79
CG1
VAL
A
39
−47.996
12.425
17.671
1.00
78.01
C


ATOM
80
CG2
VAL
A
39
−50.185
11.843
18.770
1.00
81.49
C


ATOM
81
N
MET
A
40
−48.754
11.492
14.506
1.00
80.50
N


ATOM
82
CA
MET
A
40
−47.881
11.589
13.320
1.00
80.67
C


ATOM
83
C
MET
A
40
−48.575
12.333
12.155
1.00
80.62
C


ATOM
84
O
MET
A
40
−47.915
13.033
11.390
1.00
78.80
O


ATOM
85
CB
MET
A
40
−47.405
10.197
12.870
1.00
80.79
C


ATOM
86
CG
MET
A
40
−46.294
9.613
13.745
1.00
80.56
C


ATOM
87
SD
MET
A
40
−45.995
7.863
13.419
1.00
83.92
S


ATOM
88
CE
MET
A
40
−44.486
7.588
14.344
1.00
83.67
C


ATOM
89
N
SER
A
41
−49.896
12.177
12.034
1.00
79.41
N


ATOM
90
CA
SER
A
41
−50.672
12.898
11.020
1.00
79.83
C


ATOM
91
C
SER
A
41
−50.651
14.403
11.269
1.00
80.21
C


ATOM
92
O
SER
A
41
−50.521
15.181
10.324
1.00
82.42
O


ATOM
93
CB
SER
A
41
−52.104
12.394
10.976
1.00
79.47
C


ATOM
94
OG
SER
A
41
−52.128
11.044
10.561
1.00
82.68
O


ATOM
95
N
LEU
A
42
−50.778
14.805
12.534
1.00
79.85
N


ATOM
96
CA
LEU
A
42
−50.675
16.219
12.908
1.00
79.83
C


ATOM
97
C
LEU
A
42
−49.256
16.752
12.687
1.00
78.29
C


ATOM
98
O
LEU
A
42
−49.093
17.917
12.355
1.00
80.15
O


ATOM
99
CB
LEU
A
42
−51.106
16.448
14.364
1.00
80.71
C


ATOM
100
CG
LEU
A
42
−52.586
16.242
14.714
1.00
81.76
C


ATOM
101
CD1
LEU
A
42
−52.774
16.400
16.213
1.00
83.86
C


ATOM
102
CD2
LEU
A
42
−53.508
17.191
13.956
1.00
82.80
C


ATOM
103
N
ILE
A
43
−48.243
15.902
12.876
1.00
77.38
N


ATOM
104
CA
ILE
A
43
−46.850
16.274
12.593
1.00
77.38
C


ATOM
105
C
ILE
A
43
−46.683
16.604
11.112
1.00
77.46
C


ATOM
106
O
ILE
A
43
−46.072
17.608
10.771
1.00
77.58
O


ATOM
107
CB
ILE
A
43
−45.845
15.155
12.995
1.00
78.54
C


ATOM
108
CG1
ILE
A
43
−45.698
15.080
14.520
1.00
80.06
C


ATOM
109
CG2
ILE
A
43
−44.473
15.387
12.366
1.00
74.64
C


ATOM
110
CD1
ILE
A
43
−44.923
13.845
15.002
1.00
77.13
C


ATOM
111
N
VAL
A
44
−47.229
15.750
10.247
1.00
77.58
N


ATOM
112
CA
VAL
A
44
−47.195
15.967
8.801
1.00
76.60
C


ATOM
113
C
VAL
A
44
−47.951
17.251
8.416
1.00
78.14
C


ATOM
114
O
VAL
A
44
−47.445
18.053
7.629
1.00
76.84
O


ATOM
115
CB
VAL
A
44
−47.769
14.755
8.041
1.00
76.85
C


ATOM
116
CG1
VAL
A
44
−47.967
15.079
6.567
1.00
75.75
C


ATOM
117
CG2
VAL
A
44
−46.856
13.533
8.219
1.00
70.47
C


ATOM
118
N
LEU
A
45
−49.150
17.434
8.972
1.00
78.66
N


ATOM
119
CA
LEU
A
45
−49.954
18.638
8.719
1.00
78.89
C


ATOM
120
C
LEU
A
45
−49.219
19.901
9.163
1.00
79.68
C


ATOM
121
O
LEU
A
45
−49.246
20.900
8.460
1.00
80.46
O


ATOM
122
CB
LEU
A
45
−51.306
18.555
9.433
1.00
80.42
C


ATOM
123
CG
LEU
A
45
−52.374
19.590
9.062
1.00
83.12
C


ATOM
124
CD1
LEU
A
45
−52.835
19.414
7.616
1.00
86.47
C


ATOM
125
CD2
LEU
A
45
−53.555
19.485
10.008
1.00
82.85
C


ATOM
126
N
ALA
A
46
−48.571
19.830
10.330
1.00
78.89
N


ATOM
127
CA
ALA
A
46
−47.783
20.933
10.893
1.00
76.32
C


ATOM
128
C
ALA
A
46
−46.639
21.352
9.985
1.00
75.76
C


ATOM
129
O
ALA
A
46
−46.453
22.539
9.731
1.00
78.99
O


ATOM
130
CB
ALA
A
46
−47.227
20.535
12.244
1.00
75.15
C


ATOM
131
N
ILE
A
47
−45.885
20.371
9.505
1.00
72.82
N


ATOM
132
CA
ILE
A
47
−44.751
20.613
8.596
1.00
72.71
C


ATOM
133
C
ILE
A
47
−45.206
21.216
7.267
1.00
69.65
C


ATOM
134
O
ILE
A
47
−44.651
22.209
6.815
1.00
68.44
O


ATOM
135
CB
ILE
A
47
−43.984
19.310
8.290
1.00
73.57
C


ATOM
136
CG1
ILE
A
47
−43.276
18.777
9.540
1.00
73.61
C


ATOM
137
CG2
ILE
A
47
−42.961
19.534
7.179
1.00
73.63
C


ATOM
138
CD1
ILE
A
47
−42.811
17.358
9.399
1.00
71.65
C


ATOM
139
N
VAL
A
48
−46.216
20.606
6.659
1.00
68.32
N


ATOM
140
CA
VAL
A
48
−46.721
21.051
5.360
1.00
69.33
C


ATOM
141
C
VAL
A
48
−47.390
22.440
5.463
1.00
71.00
C


ATOM
142
O
VAL
A
48
−47.115
23.305
4.638
1.00
72.48
O


ATOM
143
CB
VAL
A
48
−47.692
20.011
4.722
1.00
69.55
C


ATOM
144
CG1
VAL
A
48
−48.178
20.489
3.364
1.00
64.85
C


ATOM
145
CG2
VAL
A
48
−47.008
18.642
4.585
1.00
66.28
C


ATOM
146
N
PHE
A
49
−48.248
22.640
6.468
1.00
70.84
N


ATOM
147
CA
PHE
A
49
−48.951
23.916
6.654
1.00
69.45
C


ATOM
148
C
PHE
A
49
−47.994
25.077
6.803
1.00
71.34
C


ATOM
149
O
PHE
A
49
−48.039
26.025
6.017
1.00
71.00
O


ATOM
150
CB
PHE
A
49
−49.869
23.866
7.886
1.00
70.88
C


ATOM
151
CG
PHE
A
49
−50.669
25.123
8.103
1.00
71.45
C


ATOM
152
CD1
PHE
A
49
−51.932
25.254
7.554
1.00
72.92
C


ATOM
153
CD2
PHE
A
49
−50.161
26.176
8.855
1.00
75.73
C


ATOM
154
CE1
PHE
A
49
−52.678
26.414
7.751
1.00
73.41
C


ATOM
155
CE2
PHE
A
49
−50.905
27.336
9.052
1.00
74.53
C


ATOM
156
CZ
PHE
A
49
−52.162
27.450
8.498
1.00
69.47
C


ATOM
157
N
GLY
A
50
−47.133
24.985
7.821
1.00
71.43
N


ATOM
158
CA
GLY
A
50
−46.193
26.040
8.158
1.00
67.82
C


ATOM
159
C
GLY
A
50
−45.210
26.409
7.066
1.00
68.17
C


ATOM
160
O
GLY
A
50
−44.952
27.592
6.852
1.00
67.71
O


ATOM
161
N
ASN
A
51
−44.662
25.402
6.381
1.00
66.99
N


ATOM
162
CA
ASN
A
51
−43.713
25.636
5.273
1.00
67.98
C


ATOM
163
C
ASN
A
51
−44.414
26.145
3.998
1.00
71.16
C


ATOM
164
O
ASN
A
51
−43.847
26.968
3.274
1.00
69.41
O


ATOM
165
CB
ASN
A
51
−42.890
24.395
4.997
1.00
68.88
C


ATOM
166
CG
ASN
A
51
−41.927
24.083
6.134
1.00
71.66
C


ATOM
167
OD1
ASN
A
51
−40.914
24.758
6.305
1.00
69.08
O


ATOM
168
ND2
ASN
A
51
−42.241
23.063
6.913
1.00
68.08
N


ATOM
169
N
VAL
A
52
−45.631
25.656
3.730
1.00
67.56
N


ATOM
170
CA
VAL
A
52
−46.453
26.245
2.687
1.00
70.68
C


ATOM
171
C
VAL
A
52
−46.616
27.731
2.994
1.00
71.64
C


ATOM
172
O
VAL
A
52
−46.463
28.558
2.108
1.00
72.88
O


ATOM
173
CB
VAL
A
52
−47.860
25.599
2.575
1.00
69.27
C


ATOM
174
CG1
VAL
A
52
−48.837
26.549
1.903
1.00
71.90
C


ATOM
175
CG2
VAL
A
52
−47.784
24.286
1.829
1.00
67.05
C


ATOM
176
N
LEU
A
53
−46.921
28.049
4.256
1.00
73.15
N


ATOM
177
CA
LEU
A
53
−47.156
29.441
4.688
1.00
72.57
C


ATOM
178
C
LEU
A
53
−45.913
30.335
4.500
1.00
73.72
C


ATOM
179
O
LEU
A
53
−46.043
31.494
4.120
1.00
76.55
O


ATOM
180
CB
LEU
A
53
−47.632
29.482
6.147
1.00
72.34
C


ATOM
181
CG
LEU
A
53
−48.422
30.705
6.593
1.00
74.13
C


ATOM
182
CD1
LEU
A
53
−49.812
30.716
5.973
1.00
73.24
C


ATOM
183
CD2
LEU
A
53
−48.530
30.738
8.109
1.00
76.12
C


ATOM
184
N
VAL
A
54
−44.723
29.789
4.765
1.00
72.68
N


ATOM
185
CA
VAL
A
54
−43.458
30.517
4.563
1.00
70.92
C


ATOM
186
C
VAL
A
54
−43.183
30.795
3.076
1.00
72.46
C


ATOM
187
O
VAL
A
54
−42.786
31.903
2.711
1.00
72.67
O


ATOM
188
CB
VAL
A
54
−42.255
29.741
5.161
1.00
73.28
C


ATOM
189
CG1
VAL
A
54
−40.937
30.380
4.769
1.00
72.16
C


ATOM
190
CG2
VAL
A
54
−42.376
29.647
6.682
1.00
69.63
C


ATOM
191
N
ILE
A
55
−43.395
29.787
2.233
1.00
69.98
N


ATOM
192
CA
ILE
A
55
−43.164
29.906
0.791
1.00
68.58
C


ATOM
193
C
ILE
A
55
−44.076
30.960
0.177
1.00
69.05
C


ATOM
194
O
ILE
A
55
−43.606
31.844
−0.552
1.00
65.40
O


ATOM
195
CB
ILE
A
55
−43.379
28.569
0.090
1.00
68.25
C


ATOM
196
CG1
ILE
A
55
−42.253
27.591
0.454
1.00
71.45
C


ATOM
197
CG2
ILE
A
55
−43.430
28.766
−1.409
1.00
72.11
C


ATOM
198
CD1
ILE
A
55
−42.573
26.152
0.127
1.00
71.51
C


ATOM
199
N
THR
A
56
−45.369
30.840
0.491
1.00
70.28
N


ATOM
200
CA
THR
A
56
−46.403
31.770
0.067
1.00
69.62
C


ATOM
201
C
THR
A
56
−46.078
33.211
0.454
1.00
71.19
C


ATOM
202
O
THR
A
56
−46.188
34.089
−0.372
1.00
72.89
O


ATOM
203
CB
THR
A
56
−47.780
31.399
0.686
1.00
71.36
C


ATOM
204
OG1
THR
A
56
−48.082
30.019
0.434
1.00
65.43
O


ATOM
205
CG2
THR
A
56
−48.896
32.281
0.110
1.00
69.44
C


ATOM
206
N
ALA
A
57
−45.678
33.423
1.714
1.00
71.45
N


ATOM
207
CA
ALA
A
57
−45.368
34.762
2.263
1.00
70.84
C


ATOM
208
C
ALA
A
57
−44.180
35.464
1.584
1.00
71.90
C


ATOM
209
O
ALA
A
57
−44.220
36.665
1.339
1.00
70.99
O


ATOM
210
CB
ALA
A
57
−45.119
34.666
3.767
1.00
67.19
C


ATOM
211
N
ILE
A
58
−43.128
34.712
1.289
1.00
71.62
N


ATOM
212
CA
ILE
A
58
−41.960
35.250
0.586
1.00
71.02
C


ATOM
213
C
ILE
A
58
−42.294
35.462
−0.910
1.00
76.54
C


ATOM
214
O
ILE
A
58
−41.898
36.471
−1.503
1.00
79.19
O


ATOM
215
CB
ILE
A
58
−40.758
34.321
0.745
1.00
69.83
C


ATOM
216
CG1
ILE
A
58
−40.343
34.235
2.227
1.00
69.16
C


ATOM
217
CG2
ILE
A
58
−39.585
34.805
−0.082
1.00
69.14
C


ATOM
218
CD1
ILE
A
58
−39.371
33.129
2.513
1.00
70.35
C


ATOM
219
N
ALA
A
59
−43.016
34.511
−1.505
1.00
74.84
N


ATOM
220
CA
ALA
A
59
−43.420
34.599
−2.913
1.00
75.72
C


ATOM
221
C
ALA
A
59
−44.422
35.722
−3.174
1.00
76.56
C


ATOM
222
O
ALA
A
59
−44.416
36.311
−4.256
1.00
77.62
O


ATOM
223
CB
ALA
A
59
−43.999
33.268
−3.385
1.00
72.21
C


ATOM
224
N
LYS
A
60
−45.271
36.012
−2.184
1.00
76.98
N


ATOM
225
CA
LYS
A
60
−46.336
37.010
−2.323
1.00
76.24
C


ATOM
226
C
LYS
A
60
−45.932
38.438
−1.965
1.00
77.94
C


ATOM
227
O
LYS
A
60
−46.282
39.371
−2.694
1.00
75.40
O


ATOM
228
CB
LYS
A
60
−47.532
36.605
−1.466
1.00
75.25
C


ATOM
229
CG
LYS
A
60
−48.667
37.578
−1.530
1.00
77.75
C


ATOM
230
CD
LYS
A
60
−49.959
36.950
−1.164
1.00
78.52
C


ATOM
231
CE
LYS
A
60
−51.028
37.983
−1.189
1.00
79.74
C


ATOM
232
NZ
LYS
A
60
−52.299
37.333
−1.081
1.00
83.19
N


ATOM
233
N
PHE
A
61
−45.209
38.597
−0.856
1.00
78.18
N


ATOM
234
CA
PHE
A
61
−44.861
39.919
−0.334
1.00
79.67
C


ATOM
235
C
PHE
A
61
−43.457
40.379
−0.743
1.00
81.98
C


ATOM
236
O
PHE
A
61
−42.443
39.855
−0.253
1.00
78.47
O


ATOM
237
CB
PHE
A
61
−45.055
39.943
1.185
1.00
78.50
C


ATOM
238
CG
PHE
A
61
−46.488
39.744
1.589
1.00
77.03
C


ATOM
239
CD1
PHE
A
61
−46.946
38.513
2.013
1.00
74.37
C


ATOM
240
CD2
PHE
A
61
−47.385
40.799
1.536
1.00
79.08
C


ATOM
241
CE1
PHE
A
61
−48.267
38.334
2.380
1.00
77.21
C


ATOM
242
CE2
PHE
A
61
−48.708
40.621
1.903
1.00
77.13
C


ATOM
243
CZ
PHE
A
61
−49.141
39.382
2.326
1.00
75.36
C


ATOM
244
N
GLU
A
62
−43.450
41.369
−1.648
1.00
84.43
N


ATOM
245
CA
GLU
A
62
−42.247
42.031
−2.208
1.00
86.75
C


ATOM
246
C
GLU
A
62
−41.256
42.491
−1.122
1.00
89.37
C


ATOM
247
O
GLU
A
62
−40.040
42.414
−1.313
1.00
90.84
O


ATOM
248
CB
GLU
A
62
−42.699
43.219
−3.080
1.00
86.58
C


ATOM
249
CG
GLU
A
62
−41.641
43.860
−3.967
1.00
87.94
C


ATOM
250
CD
GLU
A
62
−42.234
44.940
−4.911
1.00
90.76
C


ATOM
251
OE1
GLU
A
62
−43.321
44.720
−5.505
1.00
96.67
O


ATOM
252
OE2
GLU
A
62
−41.611
46.016
−5.065
1.00
89.94
O


ATOM
253
N
ARG
A
63
−41.798
42.964
0.003
1.00
90.60
N


ATOM
254
CA
ARG
A
63
−41.036
43.318
1.207
1.00
88.93
C


ATOM
255
C
ARG
A
63
−40.139
42.177
1.705
1.00
88.02
C


ATOM
256
O
ARG
A
63
−39.007
42.409
2.131
1.00
88.49
O


ATOM
257
CB
ARG
A
63
−42.028
43.691
2.309
1.00
91.49
C


ATOM
258
CG
ARG
A
63
−41.443
43.980
3.698
1.00
95.03
C


ATOM
259
CD
ARG
A
63
−42.573
44.340
4.643
1.00
99.84
C


ATOM
260
NE
ARG
A
63
−42.113
44.841
5.939
1.00
103.70
N


ATOM
261
CZ
ARG
A
63
−42.903
45.373
6.882
1.00
105.87
C


ATOM
262
NH1
ARG
A
63
−44.231
45.491
6.708
1.00
107.12
N


ATOM
263
NH2
ARG
A
63
−42.362
45.796
8.028
1.00
109.62
N


ATOM
264
N
LEU
A
64
−40.658
40.955
1.648
1.00
85.74
N


ATOM
265
CA
LEU
A
64
−39.932
39.775
2.097
1.00
82.11
C


ATOM
266
C
LEU
A
64
−39.032
39.168
1.011
1.00
81.55
C


ATOM
267
O
LEU
A
64
−38.378
38.184
1.272
1.00
78.38
O


ATOM
268
CB
LEU
A
64
−40.923
38.712
2.602
1.00
80.82
C


ATOM
269
CG
LEU
A
64
−41.851
39.090
3.769
1.00
79.80
C


ATOM
270
CD1
LEU
A
64
−42.899
37.999
4.007
1.00
70.96
C


ATOM
271
CD2
LEU
A
64
−41.056
39.337
5.038
1.00
73.56
C


ATOM
272
N
GLN
A
65
−38.992
39.745
−0.198
1.00
82.04
N


ATOM
273
CA
GLN
A
65
−38.174
39.189
−1.308
1.00
83.43
C


ATOM
274
C
GLN
A
65
−36.720
39.672
−1.264
1.00
83.31
C


ATOM
275
O
GLN
A
65
−36.305
40.518
−2.060
1.00
86.84
O


ATOM
276
CB
GLN
A
65
−38.820
39.500
−2.662
1.00
82.54
C


ATOM
277
CG
GLN
A
65
−40.120
38.749
−2.856
1.00
84.91
C


ATOM
278
CD
GLN
A
65
−40.789
39.016
−4.182
1.00
88.24
C


ATOM
279
OE1
GLN
A
65
−40.516
40.018
−4.857
1.00
97.81
O


ATOM
280
NE2
GLN
A
65
−41.681
38.114
−4.569
1.00
97.75
N


ATOM
281
N
THR
A
66
−35.973
39.113
−0.320
1.00
80.37
N


ATOM
282
CA
THR
A
66
−34.561
39.413
−0.090
1.00
79.80
C


ATOM
283
C
THR
A
66
−33.747
38.170
−0.355
1.00
79.26
C


ATOM
284
O
THR
A
66
−34.306
37.105
−0.428
1.00
82.12
O


ATOM
285
CB
THR
A
66
−34.334
39.778
1.353
1.00
79.66
C


ATOM
286
OG1
THR
A
66
−34.641
38.639
2.177
1.00
79.38
O


ATOM
287
CG2
THR
A
66
−35.218
40.953
1.753
1.00
75.58
C


ATOM
288
N
VAL
A
67
−32.431
38.303
−0.492
1.00
76.86
N


ATOM
289
CA
VAL
A
67
−31.561
37.138
−0.771
1.00
76.42
C


ATOM
290
C
VAL
A
67
−31.679
36.080
0.342
1.00
76.58
C


ATOM
291
O
VAL
A
67
−31.869
34.888
0.050
1.00
76.26
O


ATOM
292
CB
VAL
A
67
−30.087
37.543
−0.956
1.00
77.31
C


ATOM
293
CG1
VAL
A
67
−29.185
36.325
−0.914
1.00
75.37
C


ATOM
294
CG2
VAL
A
67
−29.900
38.301
−2.270
1.00
72.45
C


ATOM
295
N
THR
A
68
−31.568
36.512
1.597
1.00
71.98
N


ATOM
296
CA
THR
A
68
−31.754
35.609
2.736
1.00
72.07
C


ATOM
297
C
THR
A
68
−33.028
34.797
2.566
1.00
71.33
C


ATOM
298
O
THR
A
68
−33.001
33.564
2.645
1.00
69.01
O


ATOM
299
CB
THR
A
68
−31.810
36.372
4.062
1.00
71.58
C


ATOM
300
OG1
THR
A
68
−30.546
37.009
4.283
1.00
73.28
O


ATOM
301
CG2
THR
A
68
−32.098
35.420
5.213
1.00
71.59
C


ATOM
302
N
ASN
A
69
−34.139
35.488
2.321
1.00
71.44
N


ATOM
303
CA
ASN
A
69
−35.438
34.810
2.146
1.00
70.87
C


ATOM
304
C
ASN
A
69
−35.534
33.842
0.957
1.00
71.14
C


ATOM
305
O
ASN
A
69
−36.425
32.981
0.941
1.00
73.32
O


ATOM
306
CB
ASN
A
69
−36.571
35.835
2.123
1.00
68.01
C


ATOM
307
CG
ASN
A
69
−36.794
36.501
3.503
1.00
72.26
C


ATOM
308
OD1
ASN
A
69
−36.311
36.018
4.539
1.00
66.06
O


ATOM
309
ND2
ASN
A
69
−37.525
37.606
3.513
1.00
72.74
N


ATOM
310
N
TYR
A
70
−34.640
33.969
−0.023
1.00
72.21
N


ATOM
311
CA
TYR
A
70
−34.578
33.002
−1.133
1.00
73.01
C


ATOM
312
C
TYR
A
70
−33.995
31.695
−0.599
1.00
73.34
C


ATOM
313
O
TYR
A
70
−34.479
30.630
−0.942
1.00
77.66
O


ATOM
314
CB
TYR
A
70
−33.781
33.529
−2.341
1.00
74.09
C


ATOM
315
CG
TYR
A
70
−34.536
34.496
−3.245
1.00
75.84
C


ATOM
316
CD1
TYR
A
70
−35.265
35.553
−2.732
1.00
82.81
C


ATOM
317
CD2
TYR
A
70
−34.514
34.348
−4.620
1.00
84.86
C


ATOM
318
CE1
TYR
A
70
−35.957
36.444
−3.555
1.00
84.18
C


ATOM
319
CE2
TYR
A
70
−35.200
35.229
−5.453
1.00
85.94
C


ATOM
320
CZ
TYR
A
70
−35.921
36.278
−4.918
1.00
84.44
C


ATOM
321
OH
TYR
A
70
−36.603
37.149
−5.771
1.00
85.23
O


ATOM
322
N
PHE
A
71
−32.961
31.788
0.238
1.00
75.67
N


ATOM
323
CA
PHE
A
71
−32.384
30.609
0.904
1.00
73.91
C


ATOM
324
C
PHE
A
71
−33.360
30.004
1.903
1.00
75.76
C


ATOM
325
O
PHE
A
71
−33.426
28.779
2.046
1.00
72.03
O


ATOM
326
CB
PHE
A
71
−31.108
30.949
1.666
1.00
74.63
C


ATOM
327
CG
PHE
A
71
−30.024
31.535
0.817
1.00
72.11
C


ATOM
328
CD1
PHE
A
71
−29.584
30.876
−0.311
1.00
69.28
C


ATOM
329
CD2
PHE
A
71
−29.438
32.748
1.158
1.00
70.30
C


ATOM
330
CE1
PHE
A
71
−28.583
31.413
−1.095
1.00
74.96
C


ATOM
331
CE2
PHE
A
71
−28.449
33.285
0.396
1.00
72.37
C


ATOM
332
CZ
PHE
A
71
−28.013
32.613
−0.749
1.00
76.19
C


ATOM
333
N
ILE
A
72
−34.111
30.863
2.595
1.00
71.39
N


ATOM
334
CA
ILE
A
72
−35.160
30.393
3.485
1.00
71.36
C


ATOM
335
C
ILE
A
72
−36.192
29.597
2.692
1.00
73.02
C


ATOM
336
O
ILE
A
72
−36.652
28.546
3.151
1.00
70.62
O


ATOM
337
CB
ILE
A
72
−35.868
31.541
4.197
1.00
68.07
C


ATOM
338
CG1
ILE
A
72
−34.924
32.236
5.203
1.00
79.57
C


ATOM
339
CG2
ILE
A
72
−37.089
31.044
4.929
1.00
67.38
C


ATOM
340
CD1
ILE
A
72
−34.499
31.384
6.367
1.00
74.31
C


ATOM
341
N
THR
A
73
−36.554
30.100
1.506
1.00
70.93
N


ATOM
342
CA
THR
A
73
−37.500
29.397
0.644
1.00
71.88
C


ATOM
343
C
THR
A
73
−36.937
28.040
0.264
1.00
73.34
C


ATOM
344
O
THR
A
73
−37.663
27.045
0.296
1.00
77.13
O


ATOM
345
CB
THR
A
73
−37.840
30.208
−0.618
1.00
73.11
C


ATOM
346
OG1
THR
A
73
−38.491
31.424
−0.228
1.00
71.91
O


ATOM
347
CG2
THR
A
73
−38.747
29.418
−1.548
1.00
72.28
C


ATOM
348
N
SER
A
74
−35.654
28.000
−0.091
1.00
73.09
N


ATOM
349
CA
SER
A
74
−34.981
26.740
−0.409
1.00
74.48
C


ATOM
350
C
SER
A
74
−35.030
25.775
0.800
1.00
74.98
C


ATOM
351
O
SER
A
74
−35.270
24.570
0.638
1.00
78.94
O


ATOM
352
CB
SER
A
74
−33.546
27.014
−0.877
1.00
74.59
C


ATOM
353
OG
SER
A
74
−32.820
25.829
−1.084
1.00
73.96
O


ATOM
354
N
LEU
A
75
−34.810
26.318
1.995
1.00
73.74
N


ATOM
355
CA
LEU
A
75
−34.911
25.567
3.255
1.00
72.08
C


ATOM
356
C
LEU
A
75
−36.351
25.067
3.482
1.00
71.55
C


ATOM
357
O
LEU
A
75
−36.565
23.897
3.819
1.00
71.52
O


ATOM
358
CB
LEU
A
75
−34.437
26.455
4.418
1.00
72.86
C


ATOM
359
CG
LEU
A
75
−34.085
25.884
5.771
1.00
78.35
C


ATOM
360
CD1
LEU
A
75
−33.026
24.797
5.661
1.00
80.50
C


ATOM
361
CD2
LEU
A
75
−33.614
27.061
6.695
1.00
77.36
C


ATOM
362
N
ALA
A
76
−37.326
25.958
3.290
1.00
69.20
N


ATOM
363
CA
ALA
A
76
−38.758
25.610
3.373
1.00
70.43
C


ATOM
364
C
ALA
A
76
−39.206
24.520
2.362
1.00
70.45
C


ATOM
365
O
ALA
A
76
−40.112
23.747
2.658
1.00
73.21
O


ATOM
366
CB
ALA
A
76
−39.613
26.864
3.200
1.00
67.40
C


ATOM
367
N
CYS
A
77
−38.578
24.461
1.184
1.00
72.62
N


ATOM
368
CA
CYS
A
77
−38.911
23.411
0.176
1.00
72.54
C


ATOM
369
C
CYS
A
77
−38.399
22.039
0.601
1.00
71.97
C


ATOM
370
O
CYS
A
77
−39.084
21.047
0.401
1.00
75.84
O


ATOM
371
CB
CYS
A
77
−38.350
23.752
−1.195
1.00
71.94
C


ATOM
372
SG
CYS
A
77
−39.135
25.162
−1.970
1.00
78.85
S


ATOM
373
N
ALA
A
78
−37.197
22.000
1.181
1.00
71.07
N


ATOM
374
CA
ALA
A
78
−36.609
20.774
1.704
1.00
70.88
C


ATOM
375
C
ALA
A
78
−37.457
20.206
2.846
1.00
71.74
C


ATOM
376
O
ALA
A
78
−37.644
18.997
2.947
1.00
72.80
O


ATOM
377
CB
ALA
A
78
−35.174
21.031
2.172
1.00
68.59
C


ATOM
378
N
ASP
A
79
−37.964
21.083
3.698
1.00
71.88
N


ATOM
379
CA
ASP
A
79
−38.859
20.676
4.783
1.00
71.72
C


ATOM
380
C
ASP
A
79
−40.240
20.312
4.244
1.00
70.22
C


ATOM
381
O
ASP
A
79
−40.872
19.384
4.743
1.00
70.23
O


ATOM
382
CB
ASP
A
79
−38.934
21.775
5.847
1.00
68.87
C


ATOM
383
CG
ASP
A
79
−37.609
21.931
6.630
1.00
82.57
C


ATOM
384
OD1
ASP
A
79
−36.914
20.899
6.889
1.00
83.92
O


ATOM
385
OD2
ASP
A
79
−37.258
23.081
6.992
1.00
79.92
O


ATOM
386
N
LEU
A
80
−40.712
21.034
3.237
1.00
67.28
N


ATOM
387
CA
LEU
A
80
−42.008
20.683
2.616
1.00
68.96
C


ATOM
388
C
LEU
A
80
−41.960
19.284
2.009
1.00
65.98
C


ATOM
389
O
LEU
A
80
−42.889
18.505
2.184
1.00
70.25
O


ATOM
390
CB
LEU
A
80
−42.430
21.705
1.562
1.00
63.48
C


ATOM
391
CG
LEU
A
80
−43.828
21.494
0.969
1.00
70.22
C


ATOM
392
CD1
LEU
A
80
−44.896
21.346
2.048
1.00
68.20
C


ATOM
393
CD2
LEU
A
80
−44.193
22.645
−0.015
1.00
74.86
C


ATOM
394
N
VAL
A
81
−40.877
18.972
1.302
1.00
66.65
N


ATOM
395
CA
VAL
A
81
−40.686
17.628
0.713
1.00
68.44
C


ATOM
396
C
VAL
A
81
−40.559
16.537
1.815
1.00
68.93
C


ATOM
397
O
VAL
A
81
−41.066
15.426
1.647
1.00
71.60
O


ATOM
398
CB
VAL
A
81
−39.475
17.608
−0.253
1.00
68.36
C


ATOM
399
CG1
VAL
A
81
−39.202
16.216
−0.740
1.00
65.96
C


ATOM
400
CG2
VAL
A
81
−39.713
18.581
−1.445
1.00
63.74
C


ATOM
401
N
MET
A
82
−39.889
16.859
2.923
1.00
67.67
N


ATOM
402
CA
MET
A
82
−39.862
15.968
4.099
1.00
69.67
C


ATOM
403
C
MET
A
82
−41.282
15.658
4.578
1.00
67.76
C


ATOM
404
O
MET
A
82
−41.632
14.503
4.804
1.00
66.68
O


ATOM
405
CB
MET
A
82
−39.088
16.603
5.245
1.00
70.60
C


ATOM
406
CG
MET
A
82
−37.580
16.550
5.089
1.00
76.37
C


ATOM
407
SD
MET
A
82
−36.812
15.024
5.666
1.00
86.09
S


ATOM
408
CE
MET
A
82
−37.447
15.024
7.316
1.00
88.14
C


ATOM
409
N
GLY
A
83
−42.089
16.703
4.725
1.00
66.27
N


ATOM
410
CA
GLY
A
83
−43.480
16.564
5.170
1.00
68.36
C


ATOM
411
C
GLY
A
83
−44.416
15.795
4.250
1.00
71.02
C


ATOM
412
O
GLY
A
83
−45.350
15.161
4.730
1.00
72.14
O


ATOM
413
N
LEU
A
84
−44.169
15.853
2.939
1.00
71.19
N


ATOM
414
CA
LEU
A
84
−45.010
15.170
1.939
1.00
73.59
C


ATOM
415
C
LEU
A
84
−44.469
13.816
1.460
1.00
73.92
C


ATOM
416
O
LEU
A
84
−45.224
12.859
1.294
1.00
72.70
O


ATOM
417
CB
LEU
A
84
−45.195
16.069
0.702
1.00
72.52
C


ATOM
418
CG
LEU
A
84
−45.901
17.406
0.885
1.00
75.09
C


ATOM
419
CD1
LEU
A
84
−45.933
18.147
−0.445
1.00
78.12
C


ATOM
420
CD2
LEU
A
84
−47.316
17.232
1.430
1.00
77.53
C


ATOM
421
N
ALA
A
85
−43.166
13.751
1.237
1.00
73.21
N


ATOM
422
CA
ALA
A
85
−42.540
12.584
0.628
1.00
73.60
C


ATOM
423
C
ALA
A
85
−41.858
11.673
1.615
1.00
72.66
C


ATOM
424
O
ALA
A
85
−41.825
10.482
1.377
1.00
78.23
O


ATOM
425
CB
ALA
A
85
−41.533
13.043
−0.426
1.00
74.36
C


ATOM
426
N
VAL
A
86
−41.319
12.218
2.710
1.00
74.33
N


ATOM
427
CA
VAL
A
86
−40.544
11.419
3.678
1.00
71.85
C


ATOM
428
C
VAL
A
86
−41.349
11.025
4.910
1.00
71.02
C


ATOM
429
O
VAL
A
86
−41.584
9.860
5.132
1.00
73.11
O


ATOM
430
CB
VAL
A
86
−39.254
12.136
4.110
1.00
71.51
C


ATOM
431
CG1
VAL
A
86
−38.400
11.213
4.969
1.00
68.65
C


ATOM
432
CG2
VAL
A
86
−38.467
12.587
2.885
1.00
70.81
C


ATOM
433
N
VAL
A
87
−41.773
11.995
5.707
1.00
71.04
N


ATOM
434
CA
VAL
A
87
−42.455
11.692
7.002
1.00
71.84
C


ATOM
435
C
VAL
A
87
−43.718
10.799
6.915
1.00
72.51
C


ATOM
436
O
VAL
A
87
−43.894
9.940
7.783
1.00
76.50
O


ATOM
437
CB
VAL
A
87
−42.798
12.999
7.819
1.00
72.70
C


ATOM
438
CG1
VAL
A
87
−43.506
12.655
9.148
1.00
65.72
C


ATOM
439
CG2
VAL
A
87
−41.549
13.826
8.093
1.00
69.80
C


ATOM
440
N
PRO
A
88
−44.588
10.984
5.886
1.00
74.57
N


ATOM
441
CA
PRO
A
88
−45.792
10.131
5.828
1.00
73.89
C


ATOM
442
C
PRO
A
88
−45.514
8.629
5.696
1.00
74.31
C


ATOM
443
O
PRO
A
88
−46.204
7.831
6.322
1.00
72.95
O


ATOM
444
CB
PRO
A
88
−46.529
10.654
4.580
1.00
72.19
C


ATOM
445
CG
PRO
A
88
−46.040
12.026
4.413
1.00
70.91
C


ATOM
446
CD
PRO
A
88
−44.581
11.922
4.748
1.00
75.19
C


ATOM
447
N
PHE
A
89
−44.517
8.266
4.895
1.00
75.27
N


ATOM
448
CA
PHE
A
89
−44.153
6.857
4.707
1.00
76.82
C


ATOM
449
C
PHE
A
89
−43.325
6.300
5.850
1.00
75.58
C


ATOM
450
O
PHE
A
89
−43.466
5.124
6.182
1.00
76.46
O


ATOM
451
CB
PHE
A
89
−43.434
6.665
3.380
1.00
81.35
C


ATOM
452
CG
PHE
A
89
−44.258
7.080
2.177
1.00
87.88
C


ATOM
453
CD1
PHE
A
89
−45.600
6.699
2.046
1.00
93.20
C


ATOM
454
CD2
PHE
A
89
−43.709
7.840
1.186
1.00
87.00
C


ATOM
455
CE1
PHE
A
89
−46.350
7.082
0.940
1.00
93.35
C


ATOM
456
CE2
PHE
A
89
−44.469
8.219
0.076
1.00
92.27
C


ATOM
457
CZ
PHE
A
89
−45.780
7.839
−0.040
1.00
91.97
C


ATOM
458
N
GLY
A
90
−42.468
7.125
6.444
1.00
74.71
N


ATOM
459
CA
GLY
A
90
−41.738
6.731
7.650
1.00
75.06
C


ATOM
460
C
GLY
A
90
−42.728
6.369
8.749
1.00
74.16
C


ATOM
461
O
GLY
A
90
−42.572
5.341
9.402
1.00
77.84
O


ATOM
462
N
ALA
A
91
−43.747
7.223
8.939
1.00
72.72
N


ATOM
463
CA
ALA
A
91
−44.835
6.994
9.912
1.00
72.43
C


ATOM
464
C
ALA
A
91
−45.543
5.664
9.680
1.00
74.22
C


ATOM
465
O
ALA
A
91
−45.668
4.853
10.604
1.00
74.73
O


ATOM
466
CB
ALA
A
91
−45.849
8.129
9.849
1.00
69.78
C


ATOM
467
N
ALA
A
92
−45.999
5.464
8.437
1.00
73.35
N


ATOM
468
CA
ALA
A
92
−46.699
4.241
8.013
1.00
74.78
C


ATOM
469
C
ALA
A
92
−45.850
2.998
8.219
1.00
73.86
C


ATOM
470
O
ALA
A
92
−46.355
1.985
8.671
1.00
76.34
O


ATOM
471
CB
ALA
A
92
−47.131
4.348
6.541
1.00
73.00
C


ATOM
472
N
HIS
A
93
−44.566
3.098
7.881
1.00
75.22
N


ATOM
473
CA
HIS
A
93
−43.592
2.022
8.080
1.00
75.74
C


ATOM
474
C
HIS
A
93
−43.521
1.575
9.543
1.00
76.69
C


ATOM
475
O
HIS
A
93
−43.429
0.383
9.818
1.00
80.31
O


ATOM
476
CB
HIS
A
93
−42.216
2.487
7.579
1.00
77.29
C


ATOM
477
CG
HIS
A
93
−41.151
1.442
7.633
1.00
75.35
C


ATOM
478
ND1
HIS
A
93
−39.816
1.752
7.759
1.00
74.50
N


ATOM
479
CD2
HIS
A
93
−41.220
0.094
7.579
1.00
76.44
C


ATOM
480
CE1
HIS
A
93
−39.110
0.635
7.779
1.00
78.85
C


ATOM
481
NE2
HIS
A
93
−39.939
−0.386
7.671
1.00
74.19
N


ATOM
482
N
ILE
A
94
−43.568
2.533
10.464
1.00
77.24
N


ATOM
483
CA
ILE
A
94
−43.534
2.243
11.901
1.00
77.99
C


ATOM
484
C
ILE
A
94
−44.855
1.668
12.417
1.00
78.57
C


ATOM
485
O
ILE
A
94
−44.853
0.680
13.162
1.00
80.24
O


ATOM
486
CB
ILE
A
94
−43.193
3.506
12.720
1.00
78.41
C


ATOM
487
CG1
ILE
A
94
−41.745
3.911
12.482
1.00
78.28
C


ATOM
488
CG2
ILE
A
94
−43.404
3.262
14.201
1.00
79.28
C


ATOM
489
CD1
ILE
A
94
−41.379
5.210
13.108
1.00
77.39
C


ATOM
490
N
LEU
A
95
−45.967
2.283
12.024
1.00
78.45
N


ATOM
491
CA
LEU
A
95
−47.302
1.855
12.478
1.00
79.09
C


ATOM
492
C
LEU
A
95
−47.729
0.492
11.925
1.00
80.71
C


ATOM
493
O
LEU
A
95
−48.401
−0.271
12.626
1.00
82.74
O


ATOM
494
CB
LEU
A
95
−48.347
2.916
12.119
1.00
79.29
C


ATOM
495
CG
LEU
A
95
−48.202
4.255
12.843
1.00
78.03
C


ATOM
496
CD1
LEU
A
95
−49.033
5.334
12.164
1.00
77.81
C


ATOM
497
CD2
LEU
A
95
−48.587
4.110
14.306
1.00
75.72
C


ATOM
498
N
MET
A
96
−47.346
0.195
10.685
1.00
80.36
N


ATOM
499
CA
MET
A
96
−47.635
−1.103
10.066
1.00
80.57
C


ATOM
500
C
MET
A
96
−46.592
−2.157
10.421
1.00
78.70
C


ATOM
501
O
MET
A
96
−46.849
−3.336
10.242
1.00
75.20
O


ATOM
502
CB
MET
A
96
−47.727
−0.965
8.552
1.00
80.83
C


ATOM
503
CG
MET
A
96
−48.948
−0.212
8.114
1.00
83.20
C


ATOM
504
SD
MET
A
96
−48.947
0.080
6.357
1.00
91.34
S


ATOM
505
CE
MET
A
96
−50.698
0.372
6.060
1.00
92.81
C


ATOM
506
N
LYS
A
97
−45.429
−1.717
10.918
1.00
80.00
N


ATOM
507
CA
LYS
A
97
−44.302
−2.598
11.317
1.00
81.41
C


ATOM
508
C
LYS
A
97
−43.720
−3.385
10.128
1.00
79.08
C


ATOM
509
O
LYS
A
97
−43.216
−4.494
10.294
1.00
78.90
O


ATOM
510
CB
LYS
A
97
−44.705
−3.535
12.483
1.00
82.51
C


ATOM
511
CG
LYS
A
97
−45.084
−2.800
13.750
1.00
85.16
C


ATOM
512
CD
LYS
A
97
−45.946
−3.663
14.664
1.00
86.38
C


ATOM
513
CE
LYS
A
97
−46.262
−2.937
15.971
1.00
90.74
C


ATOM
514
NZ
LYS
A
97
−46.864
−1.569
15.763
1.00
92.55
N


ATOM
515
N
MET
A
98
−43.797
−2.788
8.941
1.00
78.37
N


ATOM
516
CA
MET
A
98
−43.317
−3.387
7.703
1.00
78.62
C


ATOM
517
C
MET
A
98
−43.508
−2.378
6.592
1.00
78.05
C


ATOM
518
O
MET
A
98
−44.326
−1.458
6.720
1.00
80.11
O


ATOM
519
CB
MET
A
98
−44.045
−4.706
7.375
1.00
79.92
C


ATOM
520
CG
MET
A
98
−45.573
−4.628
7.326
1.00
84.42
C


ATOM
521
SD
MET
A
98
−46.226
−4.350
5.681
1.00
96.30
S


ATOM
522
CE
MET
A
98
−45.936
−5.962
4.938
1.00
95.48
C


ATOM
523
N
TRP
A
99
−42.754
−2.552
5.510
1.00
77.49
N


ATOM
524
CA
TRP
A
99
−42.823
−1.681
4.333
1.00
75.69
C


ATOM
525
C
TRP
A
99
−43.856
−2.268
3.372
1.00
73.64
C


ATOM
526
O
TRP
A
99
−43.802
−3.441
3.079
1.00
77.31
O


ATOM
527
CB
TRP
A
99
−41.444
−1.587
3.691
1.00
74.33
C


ATOM
528
CG
TRP
A
99
−41.339
−0.599
2.633
1.00
74.05
C


ATOM
529
CD1
TRP
A
99
−41.293
−0.838
1.306
1.00
70.83
C


ATOM
530
CD2
TRP
A
99
−41.262
0.821
2.788
1.00
73.53
C


ATOM
531
NE1
TRP
A
99
−41.195
0.331
0.624
1.00
73.91
N


ATOM
532
CE2
TRP
A
99
−41.172
1.368
1.505
1.00
73.77
C


ATOM
533
CE3
TRP
A
99
−41.262
1.680
3.887
1.00
74.17
C


ATOM
534
CZ2
TRP
A
99
−41.083
2.737
1.277
1.00
75.99
C


ATOM
535
CZ3
TRP
A
99
−41.172
3.054
3.664
1.00
71.44
C


ATOM
536
CH2
TRP
A
99
−41.083
3.564
2.370
1.00
77.11
C


ATOM
537
N
THR
A
100
−44.795
−1.451
2.892
1.00
73.03
N


ATOM
538
CA
THR
A
100
−45.840
−1.896
1.924
1.00
72.98
C


ATOM
539
C
THR
A
100
−45.766
−1.236
0.546
1.00
71.23
C


ATOM
540
O
THR
A
100
−46.562
−1.571
−0.333
1.00
71.17
O


ATOM
541
CB
THR
A
100
−47.266
−1.597
2.444
1.00
73.76
C


ATOM
542
OG1
THR
A
100
−47.509
−0.178
2.405
1.00
76.78
O


ATOM
543
CG2
THR
A
100
−47.446
−2.076
3.842
1.00
78.02
C


ATOM
544
N
PHE
A
101
−44.829
−0.312
0.355
1.00
70.30
N


ATOM
545
CA
PHE
A
101
−44.748
0.499
−0.870
1.00
72.26
C


ATOM
546
C
PHE
A
101
−43.772
−0.083
−1.920
1.00
74.09
C


ATOM
547
O
PHE
A
101
−43.621
0.485
−3.018
1.00
74.48
O


ATOM
548
CB
PHE
A
101
−44.414
1.947
−0.481
1.00
72.34
C


ATOM
549
CG
PHE
A
101
−45.268
2.457
0.663
1.00
70.71
C


ATOM
550
CD1
PHE
A
101
−44.766
2.495
1.965
1.00
71.75
C


ATOM
551
CD2
PHE
A
101
−46.572
2.887
0.438
1.00
68.75
C


ATOM
552
CE1
PHE
A
101
−45.552
2.961
3.028
1.00
71.54
C


ATOM
553
CE2
PHE
A
101
−47.364
3.354
1.493
1.00
70.01
C


ATOM
554
CZ
PHE
A
101
−46.853
3.391
2.789
1.00
68.99
C


ATOM
555
N
GLY
A
102
−43.122
−1.207
−1.581
1.00
73.14
N


ATOM
556
CA
GLY
A
102
−42.234
−1.922
−2.502
1.00
73.21
C


ATOM
557
C
GLY
A
102
−40.821
−1.391
−2.546
1.00
72.86
C


ATOM
558
O
GLY
A
102
−40.552
−0.299
−2.091
1.00
72.40
O


ATOM
559
N
ASN
A
103
−39.916
−2.184
−3.114
1.00
74.31
N


ATOM
560
CA
ASN
A
103
−38.484
−1.824
−3.165
1.00
74.88
C


ATOM
561
C
ASN
A
103
−38.129
−0.574
−3.956
1.00
74.74
C


ATOM
562
O
ASN
A
103
−37.251
0.175
−3.515
1.00
76.17
O


ATOM
563
CB
ASN
A
103
−37.626
−2.998
−3.690
1.00
74.34
C


ATOM
564
CG
ASN
A
103
−37.420
−4.108
−2.651
1.00
74.79
C


ATOM
565
OD1
ASN
A
103
−37.242
−5.271
−3.012
1.00
76.47
O


ATOM
566
ND2
ASN
A
103
−37.444
−3.753
−1.363
1.00
74.57
N


ATOM
567
N
PHE
A
104
−38.782
−0.339
−5.098
1.00
74.65
N


ATOM
568
CA
PHE
A
104
−38.478
0.860
−5.884
1.00
75.28
C


ATOM
569
C
PHE
A
104
−38.644
2.100
−5.041
1.00
75.71
C


ATOM
570
O
PHE
A
104
−37.710
2.895
−4.906
1.00
77.93
O


ATOM
571
CB
PHE
A
104
−39.367
1.020
−7.116
1.00
78.02
C


ATOM
572
CG
PHE
A
104
−39.279
2.408
−7.727
1.00
79.37
C


ATOM
573
CD1
PHE
A
104
−38.129
2.802
−8.421
1.00
80.42
C


ATOM
574
CD2
PHE
A
104
−40.337
3.321
−7.603
1.00
81.51
C


ATOM
575
CE1
PHE
A
104
−38.031
4.072
−8.984
1.00
79.83
C


ATOM
576
CE2
PHE
A
104
−40.248
4.598
−8.164
1.00
83.42
C


ATOM
577
CZ
PHE
A
104
−39.091
4.975
−8.857
1.00
82.52
C


ATOM
578
N
TRP
A
105
−39.839
2.255
−4.478
1.00
76.21
N


ATOM
579
CA
TRP
A
105
−40.134
3.416
−3.670
1.00
75.91
C


ATOM
580
C
TRP
A
105
−39.295
3.456
−2.357
1.00
76.37
C


ATOM
581
O
TRP
A
105
−38.972
4.534
−1.899
1.00
74.11
O


ATOM
582
CB
TRP
A
105
−41.645
3.562
−3.386
1.00
78.05
C


ATOM
583
CG
TRP
A
105
−41.870
4.799
−2.623
1.00
82.40
C


ATOM
584
CD1
TRP
A
105
−42.157
4.889
−1.318
1.00
83.43
C


ATOM
585
CD2
TRP
A
105
−41.810
6.147
−3.120
1.00
87.60
C


ATOM
586
NE1
TRP
A
105
−42.288
6.182
−0.950
1.00
83.52
N


ATOM
587
CE2
TRP
A
105
−42.082
6.994
−2.033
1.00
84.67
C


ATOM
588
CE3
TRP
A
105
−41.556
6.716
−4.381
1.00
86.42
C


ATOM
589
CZ2
TRP
A
105
−42.109
8.401
−2.155
1.00
81.05
C


ATOM
590
CZ3
TRP
A
105
−41.583
8.115
−4.507
1.00
84.49
C


ATOM
591
CH2
TRP
A
105
−41.860
8.938
−3.391
1.00
85.45
C


ATOM
592
N
CYS
A
106
−38.952
2.302
−1.775
1.00
74.15
N


ATOM
593
CA
CYS
A
106
−38.086
2.278
−0.573
1.00
75.23
C


ATOM
594
C
CYS
A
106
−36.763
2.982
−0.834
1.00
74.63
C


ATOM
595
O
CYS
A
106
−36.332
3.831
−0.045
1.00
72.22
O


ATOM
596
CB
CYS
A
106
−37.827
0.848
−0.097
1.00
75.82
C


ATOM
597
SG
CYS
A
106
−36.530
0.694
1.167
1.00
75.27
S


ATOM
598
N
GLU
A
107
−36.137
2.618
−1.946
1.00
74.18
N


ATOM
599
CA
GLU
A
107
−34.882
3.219
−2.380
1.00
73.95
C


ATOM
600
C
GLU
A
107
−35.054
4.698
−2.773
1.00
73.77
C


ATOM
601
O
GLU
A
107
−34.185
5.521
−2.469
1.00
69.11
O


ATOM
602
CB
GLU
A
107
−34.337
2.411
−3.542
1.00
74.75
C


ATOM
603
CG
GLU
A
107
−33.001
2.841
−4.117
1.00
78.43
C


ATOM
604
CD
GLU
A
107
−32.677
2.072
−5.397
1.00
82.58
C


ATOM
605
OE1
GLU
A
107
−32.915
0.843
−5.452
1.00
87.52
O


ATOM
606
OE2
GLU
A
107
−32.186
2.688
−6.352
1.00
90.13
O


ATOM
607
N
PHE
A
108
−36.166
5.025
−3.445
1.00
72.86
N


ATOM
608
CA
PHE
A
108
−36.458
6.414
−3.816
1.00
70.73
C


ATOM
609
C
PHE
A
108
−36.705
7.236
−2.556
1.00
68.58
C


ATOM
610
O
PHE
A
108
−36.073
8.275
−2.357
1.00
66.92
O


ATOM
611
CB
PHE
A
108
−37.676
6.506
−4.729
1.00
70.86
C


ATOM
612
CG
PHE
A
108
−37.744
7.783
−5.526
1.00
72.80
C


ATOM
613
CD1
PHE
A
108
−37.339
7.799
−6.868
1.00
78.82
C


ATOM
614
CD2
PHE
A
108
−38.205
8.965
−4.961
1.00
74.96
C


ATOM
615
CE1
PHE
A
108
−37.396
8.977
−7.621
1.00
76.80
C


ATOM
616
CE2
PHE
A
108
−38.262
10.149
−5.720
1.00
74.68
C


ATOM
617
CZ
PHE
A
108
−37.858
10.148
−7.043
1.00
76.41
C


ATOM
618
N
TRP
A
109
−37.628
6.752
−1.723
1.00
66.71
N


ATOM
619
CA
TRP
A
109
−37.960
7.358
−0.431
1.00
68.89
C


ATOM
620
C
TRP
A
109
−36.701
7.635
0.361
1.00
69.04
C


ATOM
621
O
TRP
A
109
−36.504
8.752
0.807
1.00
68.71
O


ATOM
622
CB
TRP
A
109
−38.866
6.437
0.371
1.00
71.86
C


ATOM
623
CG
TRP
A
109
−39.102
6.855
1.747
1.00
70.49
C


ATOM
624
CD1
TRP
A
109
−39.862
7.878
2.149
1.00
72.33
C


ATOM
625
CD2
TRP
A
109
−38.569
6.248
2.938
1.00
74.86
C


ATOM
626
NE1
TRP
A
109
−39.847
7.967
3.516
1.00
76.09
N


ATOM
627
CE2
TRP
A
109
−39.062
6.979
4.025
1.00
72.44
C


ATOM
628
CE3
TRP
A
109
−37.720
5.156
3.187
1.00
77.42
C


ATOM
629
CZ2
TRP
A
109
−38.747
6.669
5.347
1.00
75.85
C


ATOM
630
CZ3
TRP
A
109
−37.404
4.849
4.532
1.00
74.21
C


ATOM
631
CH2
TRP
A
109
−37.919
5.602
5.575
1.00
73.66
C


ATOM
632
N
THR
A
110
−35.861
6.607
0.518
1.00
64.06
N


ATOM
633
CA
THR
A
110
−34.570
6.747
1.190
1.00
66.65
C


ATOM
634
C
THR
A
110
−33.707
7.863
0.621
1.00
68.41
C


ATOM
635
O
THR
A
110
−33.116
8.624
1.385
1.00
71.70
O


ATOM
636
CB
THR
A
110
−33.757
5.477
1.102
1.00
66.89
C


ATOM
637
OG1
THR
A
110
−34.491
4.404
1.699
1.00
70.60
O


ATOM
638
CG2
THR
A
110
−32.427
5.645
1.830
1.00
64.09
C


ATOM
639
N
SER
A
111
−33.636
7.959
−0.709
1.00
69.98
N


ATOM
640
CA
SER
A
111
−32.849
9.017
−1.387
1.00
70.65
C


ATOM
641
C
SER
A
111
−33.343
10.443
−1.092
1.00
73.21
C


ATOM
642
O
SER
A
111
−32.524
11.356
−0.899
1.00
76.91
O


ATOM
643
CB
SER
A
111
−32.825
8.802
−2.909
1.00
72.35
C


ATOM
644
OG
SER
A
111
−32.247
7.565
−3.262
1.00
69.46
O


ATOM
645
N
ILE
A
112
−34.669
10.626
−1.063
1.00
72.31
N


ATOM
646
CA
ILE
A
112
−35.290
11.938
−0.786
1.00
70.06
C


ATOM
647
C
ILE
A
112
−35.011
12.365
0.650
1.00
69.36
C


ATOM
648
O
ILE
A
112
−34.706
13.535
0.916
1.00
68.07
O


ATOM
649
CB
ILE
A
112
−36.819
11.916
−1.006
1.00
68.74
C


ATOM
650
CG1
ILE
A
112
−37.174
11.632
−2.476
1.00
75.90
C


ATOM
651
CG2
ILE
A
112
−37.426
13.234
−0.645
1.00
72.66
C


ATOM
652
CD1
ILE
A
112
−38.665
11.518
−2.704
1.00
73.21
C


ATOM
653
N
ASP
A
113
−35.126
11.403
1.557
1.00
68.09
N


ATOM
654
CA
ASP
A
113
−34.823
11.584
2.973
1.00
69.93
C


ATOM
655
C
ASP
A
113
−33.435
12.218
3.102
1.00
68.24
C


ATOM
656
O
ASP
A
113
−33.311
13.328
3.600
1.00
69.08
O


ATOM
657
CB
ASP
A
113
−34.916
10.225
3.681
1.00
71.22
C


ATOM
658
CG
ASP
A
113
−35.001
10.325
5.163
1.00
71.94
C


ATOM
659
OD1
ASP
A
113
−34.453
11.263
5.771
1.00
73.99
O


ATOM
660
OD2
ASP
A
113
−35.644
9.419
5.727
1.00
78.39
O


ATOM
661
N
VAL
A
114
−32.415
11.501
2.640
1.00
69.76
N


ATOM
662
CA
VAL
A
114
−31.016
11.951
2.687
1.00
69.79
C


ATOM
663
C
VAL
A
114
−30.790
13.289
1.934
1.00
70.38
C


ATOM
664
O
VAL
A
114
−30.022
14.132
2.409
1.00
66.61
O


ATOM
665
CB
VAL
A
114
−30.070
10.827
2.158
1.00
71.48
C


ATOM
666
CG1
VAL
A
114
−28.657
11.338
1.891
1.00
71.98
C


ATOM
667
CG2
VAL
A
114
−30.054
9.643
3.147
1.00
68.67
C


ATOM
668
N
LEU
A
115
−31.455
13.467
0.780
1.00
69.66
N


ATOM
669
CA
LEU
A
115
−31.356
14.717
−0.015
1.00
70.60
C


ATOM
670
C
LEU
A
115
−31.817
15.929
0.773
1.00
72.08
C


ATOM
671
O
LEU
A
115
−31.084
16.909
0.895
1.00
71.75
O


ATOM
672
CB
LEU
A
115
−32.193
14.621
−1.314
1.00
73.84
C


ATOM
673
CG
LEU
A
115
−32.357
15.859
−2.238
1.00
73.55
C


ATOM
674
CD1
LEU
A
115
−31.014
16.399
−2.735
1.00
72.28
C


ATOM
675
CD2
LEU
A
115
−33.278
15.542
−3.431
1.00
66.35
C


ATOM
676
N
CYS
A
116
−33.039
15.841
1.297
1.00
72.44
N


ATOM
677
CA
CYS
A
116
−33.670
16.941
2.034
1.00
73.77
C


ATOM
678
C
CYS
A
116
−32.868
17.364
3.251
1.00
74.35
C


ATOM
679
O
CYS
A
116
−32.711
18.570
3.492
1.00
75.14
O


ATOM
680
CB
CYS
A
116
−35.112
16.586
2.437
1.00
72.52
C


ATOM
681
SG
CYS
A
116
−36.268
16.557
1.013
1.00
80.97
S


ATOM
682
N
VAL
A
117
−32.365
16.391
4.010
1.00
72.36
N


ATOM
683
CA
VAL
A
117
−31.509
16.695
5.167
1.00
73.25
C


ATOM
684
C
VAL
A
117
−30.188
17.351
4.725
1.00
75.09
C


ATOM
685
O
VAL
A
117
−29.729
18.311
5.369
1.00
76.15
O


ATOM
686
CB
VAL
A
117
−31.217
15.439
6.020
1.00
73.55
C


ATOM
687
CG1
VAL
A
117
−30.199
15.737
7.098
1.00
69.89
C


ATOM
688
CG2
VAL
A
117
−32.503
14.916
6.641
1.00
73.99
C


ATOM
689
N
THR
A
118
−29.592
16.837
3.635
1.00
71.84
N


ATOM
690
CA
THR
A
118
−28.329
17.366
3.096
1.00
71.32
C


ATOM
691
C
THR
A
118
−28.501
18.788
2.575
1.00
73.72
C


ATOM
692
O
THR
A
118
−27.710
19.656
2.916
1.00
78.08
O


ATOM
693
CB
THR
A
118
−27.767
16.482
1.949
1.00
72.20
C


ATOM
694
OG1
THR
A
118
−27.512
15.161
2.433
1.00
75.03
O


ATOM
695
CG2
THR
A
118
−26.471
17.066
1.389
1.00
65.55
C


ATOM
696
N
ALA
A
119
−29.534
19.013
1.752
1.00
72.84
N


ATOM
697
CA
ALA
A
119
−29.857
20.351
1.220
1.00
71.95
C


ATOM
698
C
ALA
A
119
−30.142
21.361
2.334
1.00
71.48
C


ATOM
699
O
ALA
A
119
−29.750
22.521
2.226
1.00
73.66
O


ATOM
700
CB
ALA
A
119
−31.054
20.284
0.243
1.00
69.50
C


ATOM
701
N
SER
A
120
−30.818
20.920
3.394
1.00
71.79
N


ATOM
702
CA
SER
A
120
−31.124
21.803
4.531
1.00
71.94
C


ATOM
703
C
SER
A
120
−29.861
22.374
5.153
1.00
73.64
C


ATOM
704
O
SER
A
120
−29.724
23.598
5.252
1.00
74.21
O


ATOM
705
CB
SER
A
120
−31.927
21.071
5.584
1.00
71.95
C


ATOM
706
OG
SER
A
120
−33.202
20.758
5.077
1.00
75.41
O


ATOM
707
N
ILE
A
121
−28.945
21.500
5.562
1.00
74.66
N


ATOM
708
CA
ILE
A
121
−27.694
21.946
6.223
1.00
72.09
C


ATOM
709
C
ILE
A
121
−26.767
22.684
5.253
1.00
74.47
C


ATOM
710
O
ILE
A
121
−26.066
23.606
5.668
1.00
72.06
O


ATOM
711
CB
ILE
A
121
−26.922
20.782
6.903
1.00
72.33
C


ATOM
712
CG1
ILE
A
121
−25.776
21.297
7.823
1.00
75.07
C


ATOM
713
CG2
ILE
A
121
−26.349
19.826
5.858
1.00
69.34
C


ATOM
714
CD1
ILE
A
121
−26.149
22.421
8.816
1.00
75.41
C


ATOM
715
N
GLU
A
122
−26.758
22.289
3.977
1.00
71.61
N


ATOM
716
CA
GLU
A
122
−25.957
23.013
2.976
1.00
73.61
C


ATOM
717
C
GLU
A
122
−26.477
24.430
2.815
1.00
71.97
C


ATOM
718
O
GLU
A
122
−25.684
25.357
2.719
1.00
73.76
O


ATOM
719
CB
GLU
A
122
−25.936
22.298
1.619
1.00
71.23
C


ATOM
720
CG
GLU
A
122
−25.042
21.050
1.616
1.00
78.82
C


ATOM
721
CD
GLU
A
122
−24.898
20.393
0.249
1.00
76.68
C


ATOM
722
OE1
GLU
A
122
−25.210
21.034
−0.777
1.00
81.34
O


ATOM
723
OE2
GLU
A
122
−24.464
19.221
0.208
1.00
82.37
O


ATOM
724
N
THR
A
123
−27.804
24.585
2.788
1.00
71.94
N


ATOM
725
CA
THR
A
123
−28.437
25.916
2.693
1.00
71.90
C


ATOM
726
C
THR
A
123
−28.141
26.762
3.909
1.00
71.32
C


ATOM
727
O
THR
A
123
−27.722
27.903
3.768
1.00
70.52
O


ATOM
728
CB
THR
A
123
−29.940
25.819
2.596
1.00
69.91
C


ATOM
729
OG1
THR
A
123
−30.275
24.924
1.534
1.00
70.66
O


ATOM
730
CG2
THR
A
123
−30.539
27.214
2.354
1.00
68.19
C


ATOM
731
N
LEU
A
124
−28.364
26.197
5.099
1.00
69.53
N


ATOM
732
CA
LEU
A
124
−28.044
26.877
6.330
1.00
70.49
C


ATOM
733
C
LEU
A
124
−26.624
27.353
6.294
1.00
69.98
C


ATOM
734
O
LEU
A
124
−26.349
28.465
6.684
1.00
72.08
O


ATOM
735
CB
LEU
A
124
−28.230
25.963
7.561
1.00
72.66
C


ATOM
736
CG
LEU
A
124
−29.654
25.655
7.991
1.00
71.44
C


ATOM
737
CD1
LEU
A
124
−29.643
24.635
9.128
1.00
74.54
C


ATOM
738
CD2
LEU
A
124
−30.358
26.930
8.384
1.00
68.04
C


ATOM
739
N
CYS
A
125
−25.726
26.509
5.822
1.00
72.50
N


ATOM
740
CA
CYS
A
125
−24.319
26.880
5.727
1.00
75.56
C


ATOM
741
C
CYS
A
125
−24.087
28.030
4.746
1.00
73.80
C


ATOM
742
O
CYS
A
125
−23.222
28.868
4.985
1.00
74.28
O


ATOM
743
CB
CYS
A
125
−23.474
25.682
5.322
1.00
76.67
C


ATOM
744
SG
CYS
A
125
−21.781
25.957
5.620
1.00
84.21
S


ATOM
745
N
VAL
A
126
−24.860
28.059
3.652
1.00
76.58
N


ATOM
746
CA
VAL
A
126
−24.803
29.161
2.654
1.00
74.32
C


ATOM
747
C
VAL
A
126
−25.327
30.458
3.269
1.00
74.06
C


ATOM
748
O
VAL
A
126
−24.820
31.529
2.959
1.00
74.99
O


ATOM
749
CB
VAL
A
126
−25.612
28.848
1.381
1.00
73.89
C


ATOM
750
CG1
VAL
A
126
−25.614
30.050
0.435
1.00
78.42
C


ATOM
751
CG2
VAL
A
126
−25.056
27.612
0.682
1.00
75.52
C


ATOM
752
N
ILE
A
127
−26.341
30.352
4.138
1.00
73.19
N


ATOM
753
CA
ILE
A
127
−26.902
31.527
4.840
1.00
71.30
C


ATOM
754
C
ILE
A
127
−25.846
32.137
5.724
1.00
69.71
C


ATOM
755
O
ILE
A
127
−25.706
33.342
5.750
1.00
73.08
O


ATOM
756
CB
ILE
A
127
−28.150
31.200
5.671
1.00
70.82
C


ATOM
757
CG1
ILE
A
127
−29.301
30.720
4.782
1.00
74.13
C


ATOM
758
CG2
ILE
A
127
−28.636
32.429
6.410
1.00
72.22
C


ATOM
759
CD1
ILE
A
127
−30.450
30.161
5.540
1.00
73.97
C


ATOM
760
N
ALA
A
128
−25.105
31.298
6.440
1.00
70.03
N


ATOM
761
CA
ALA
A
128
−24.031
31.756
7.323
1.00
70.78
C


ATOM
762
C
ALA
A
128
−22.929
32.481
6.577
1.00
70.23
C


ATOM
763
O
ALA
A
128
−22.493
33.544
7.004
1.00
70.16
O


ATOM
764
CB
ALA
A
128
−23.443
30.585
8.092
1.00
72.00
C


ATOM
765
N
VAL
A
129
−22.480
31.905
5.463
1.00
73.67
N


ATOM
766
CA
VAL
A
129
−21.415
32.529
4.657
1.00
70.68
C


ATOM
767
C
VAL
A
129
−21.923
33.853
4.062
1.00
70.47
C


ATOM
768
O
VAL
A
129
−21.212
34.853
4.092
1.00
73.60
O


ATOM
769
CB
VAL
A
129
−20.878
31.595
3.556
1.00
70.74
C


ATOM
770
CG1
VAL
A
129
−19.835
32.328
2.689
1.00
65.41
C


ATOM
771
CG2
VAL
A
129
−20.285
30.331
4.172
1.00
66.69
C


ATOM
772
N
ASP
A
130
−23.149
33.836
3.536
1.00
72.22
N


ATOM
773
CA
ASP
A
130
−23.812
35.034
2.993
1.00
69.02
C


ATOM
774
C
ASP
A
130
−23.965
36.125
4.036
1.00
68.79
C


ATOM
775
O
ASP
A
130
−23.668
37.270
3.758
1.00
66.69
O


ATOM
776
CB
ASP
A
130
−25.195
34.686
2.460
1.00
71.35
C


ATOM
777
CG
ASP
A
130
−25.925
35.902
1.916
1.00
78.10
C


ATOM
778
OD1
ASP
A
130
−25.369
36.570
1.008
1.00
76.18
O


ATOM
779
OD2
ASP
A
130
−27.049
36.181
2.399
1.00
90.58
O


ATOM
780
N
ARG
A
131
−24.434
35.770
5.243
1.00
69.55
N


ATOM
781
CA
ARG
A
131
−24.520
36.762
6.342
1.00
67.36
C


ATOM
782
C
ARG
A
131
−23.143
37.268
6.778
1.00
67.63
C


ATOM
783
O
ARG
A
131
−23.021
38.445
7.169
1.00
65.43
O


ATOM
784
CB
ARG
A
131
−25.291
36.222
7.544
1.00
67.67
C


ATOM
785
CG
ARG
A
131
−26.791
35.942
7.317
1.00
70.59
C


ATOM
786
CD
ARG
A
131
−27.581
37.208
7.051
1.00
78.70
C


ATOM
787
NE
ARG
A
131
−27.386
37.707
5.685
1.00
80.63
N


ATOM
788
CZ
ARG
A
131
−27.690
38.934
5.244
1.00
84.09
C


ATOM
789
NH1
ARG
A
131
−28.227
39.861
6.051
1.00
88.30
N


ATOM
790
NH2
ARG
A
131
−27.454
39.237
3.969
1.00
81.21
N


ATOM
791
N
TYR
A
132
−22.112
36.414
6.716
1.00
66.25
N


ATOM
792
CA
TYR
A
132
−20.772
36.872
7.091
1.00
66.91
C


ATOM
793
C
TYR
A
132
−20.300
37.951
6.123
1.00
69.24
C


ATOM
794
O
TYR
A
132
−19.816
38.995
6.553
1.00
74.15
O


ATOM
795
CB
TYR
A
132
−19.744
35.746
7.153
1.00
64.41
C


ATOM
796
CG
TYR
A
132
−18.362
36.288
7.466
1.00
65.53
C


ATOM
797
CD1
TYR
A
132
−17.989
36.601
8.780
1.00
73.29
C


ATOM
798
CD2
TYR
A
132
−17.433
36.492
6.460
1.00
66.44
C


ATOM
799
CE1
TYR
A
132
−16.716
37.102
9.068
1.00
69.45
C


ATOM
800
CE2
TYR
A
132
−16.170
36.989
6.731
1.00
66.23
C


ATOM
801
CZ
TYR
A
132
−15.809
37.293
8.025
1.00
71.49
C


ATOM
802
OH
TYR
A
132
−14.542
37.787
8.266
1.00
69.82
O


ATOM
803
N
PHE
A
133
−20.435
37.715
4.827
1.00
69.93
N


ATOM
804
CA
PHE
A
133
−20.010
38.759
3.860
1.00
70.80
C


ATOM
805
C
PHE
A
133
−20.920
39.992
3.920
1.00
69.98
C


ATOM
806
O
PHE
A
133
−20.427
41.103
3.794
1.00
70.63
O


ATOM
807
CB
PHE
A
133
−19.846
38.184
2.456
1.00
67.77
C


ATOM
808
CG
PHE
A
133
−18.650
37.278
2.342
1.00
69.98
C


ATOM
809
CD1
PHE
A
133
−17.352
37.802
2.445
1.00
72.82
C


ATOM
810
CD2
PHE
A
133
−18.799
35.914
2.140
1.00
69.69
C


ATOM
811
CE1
PHE
A
133
−16.228
36.981
2.345
1.00
70.61
C


ATOM
812
CE2
PHE
A
133
−17.667
35.081
2.039
1.00
71.02
C


ATOM
813
CZ
PHE
A
133
−16.385
35.620
2.143
1.00
68.76
C


ATOM
814
N
ALA
A
134
−22.221
39.790
4.120
1.00
69.44
N


ATOM
815
CA
ALA
A
134
−23.141
40.911
4.273
1.00
68.56
C


ATOM
816
C
ALA
A
134
−22.724
41.816
5.416
1.00
68.68
C


ATOM
817
O
ALA
A
134
−22.633
43.022
5.229
1.00
71.94
O


ATOM
818
CB
ALA
A
134
−24.562
40.422
4.491
1.00
67.16
C


ATOM
819
N
ILE
A
135
−22.465
41.241
6.589
1.00
68.84
N


ATOM
820
CA
ILE
A
135
−22.145
42.039
7.792
1.00
70.46
C


ATOM
821
C
ILE
A
135
−20.732
42.639
7.824
1.00
73.76
C


ATOM
822
O
ILE
A
135
−20.483
43.567
8.604
1.00
77.85
O


ATOM
823
CB
ILE
A
135
−22.361
41.205
9.107
1.00
68.09
C


ATOM
824
CG1
ILE
A
135
−22.694
42.122
10.290
1.00
70.69
C


ATOM
825
CG2
ILE
A
135
−21.151
40.315
9.387
1.00
54.90
C


ATOM
826
CD1
ILE
A
135
−23.130
41.371
11.531
1.00
69.39
C


ATOM
827
N
THR
A
136
−19.819
42.123
6.998
1.00
72.13
N


ATOM
828
CA
THR
A
136
−18.450
42.658
6.911
1.00
72.48
C


ATOM
829
C
THR
A
136
−18.272
43.602
5.722
1.00
73.00
C


ATOM
830
O
THR
A
136
−17.205
44.194
5.560
1.00
76.38
O


ATOM
831
CB
THR
A
136
−17.408
41.523
6.791
1.00
71.33
C


ATOM
832
OG1
THR
A
136
−17.656
40.755
5.612
1.00
69.78
O


ATOM
833
CG2
THR
A
136
−17.478
40.622
7.980
1.00
76.96
C


ATOM
834
N
SER
A
137
−19.312
43.743
4.890
1.00
73.57
N


ATOM
835
CA
SER
A
137
−19.224
44.546
3.673
1.00
71.50
C


ATOM
836
C
SER
A
137
−19.370
46.004
4.020
1.00
70.30
C


ATOM
837
O
SER
A
137
−20.079
46.302
4.923
1.00
72.29
O


ATOM
838
CB
SER
A
137
−20.327
44.159
2.678
1.00
70.57
C


ATOM
839
OG
SER
A
137
−20.402
45.088
1.612
1.00
73.10
O


ATOM
840
N
PRO
A
138
−18.693
46.924
3.292
1.00
73.40
N


ATOM
841
CA
PRO
A
138
−18.908
48.362
3.552
1.00
71.10
C


ATOM
842
C
PRO
A
138
−20.342
48.823
3.196
1.00
72.24
C


ATOM
843
O
PRO
A
138
−20.791
49.845
3.689
1.00
73.27
O


ATOM
844
CB
PRO
A
138
−17.875
49.042
2.656
1.00
72.76
C


ATOM
845
CG
PRO
A
138
−16.952
47.966
2.220
1.00
75.11
C


ATOM
846
CD
PRO
A
138
−17.713
46.719
2.207
1.00
70.99
C


ATOM
847
N
PHE
A
139
−21.030
48.053
2.343
1.00
75.02
N


ATOM
848
CA
PHE
A
139
−22.436
48.228
2.012
1.00
75.94
C


ATOM
849
C
PHE
A
139
−23.097
47.194
2.924
1.00
81.11
C


ATOM
850
O
PHE
A
139
−23.703
46.209
2.451
1.00
82.84
O


ATOM
851
CB
PHE
A
139
−22.686
47.948
0.517
1.00
70.64
C


ATOM
852
CG
PHE
A
139
−21.537
48.366
−0.376
1.00
68.22
C


ATOM
853
CD1
PHE
A
139
−20.705
47.417
−0.943
1.00
64.99
C


ATOM
854
CD2
PHE
A
139
−21.291
49.686
−0.634
1.00
54.92
C


ATOM
855
CE1
PHE
A
139
−19.676
47.789
−1.736
1.00
53.34
C


ATOM
856
CE2
PHE
A
139
−20.261
50.061
−1.427
1.00
55.02
C


ATOM
857
CZ
PHE
A
139
−19.448
49.112
−1.982
1.00
59.66
C


ATOM
858
N
LYS
A
140
−22.970
47.430
4.239
1.00
82.78
N


ATOM
859
CA
LYS
A
140
−23.326
46.437
5.288
1.00
86.29
C


ATOM
860
C
LYS
A
140
−24.732
45.890
5.161
1.00
84.99
C


ATOM
861
O
LYS
A
140
−25.668
46.622
4.835
1.00
84.00
O


ATOM
862
CB
LYS
A
140
−23.144
47.003
6.710
1.00
86.05
C


ATOM
863
CG
LYS
A
140
−21.716
47.263
7.104
1.00
93.56
C


ATOM
864
CD
LYS
A
140
−21.533
47.754
8.540
1.00
92.32
C


ATOM
865
CE
LYS
A
140
−20.068
48.178
8.748
1.00
94.63
C


ATOM
866
NZ
LYS
A
140
−19.803
48.731
10.115
1.00
103.84
N


ATOM
867
N
TYR
A
141
−24.836
44.589
5.424
1.00
85.69
N


ATOM
868
CA
TYR
A
141
−26.070
43.783
5.383
1.00
87.34
C


ATOM
869
C
TYR
A
141
−26.642
43.451
3.984
1.00
87.45
C


ATOM
870
O
TYR
A
141
−27.393
42.477
3.859
1.00
87.44
O


ATOM
871
CB
TYR
A
141
−27.154
44.369
6.294
1.00
89.48
C


ATOM
872
CG
TYR
A
141
−26.773
44.254
7.729
1.00
86.02
C


ATOM
873
CD1
TYR
A
141
−27.055
43.095
8.434
1.00
82.79
C


ATOM
874
CD2
TYR
A
141
−26.132
45.293
8.380
1.00
87.79
C


ATOM
875
CE1
TYR
A
141
−26.717
42.968
9.747
1.00
95.42
C


ATOM
876
CE2
TYR
A
141
−25.777
45.189
9.716
1.00
94.16
C


ATOM
877
CZ
TYR
A
141
−26.071
44.020
10.400
1.00
97.24
C


ATOM
878
OH
TYR
A
141
−25.728
43.897
11.720
1.00
91.49
O


ATOM
879
N
GLN
A
142
−26.313
44.220
2.958
1.00
83.88
N


ATOM
880
CA
GLN
A
142
−26.686
43.826
1.640
1.00
87.53
C


ATOM
881
C
GLN
A
142
−25.823
42.632
1.368
1.00
85.35
C


ATOM
882
O
GLN
A
142
−24.602
42.682
1.609
1.00
85.83
O


ATOM
883
CB
GLN
A
142
−26.375
44.883
0.590
1.00
86.73
C


ATOM
884
CG
GLN
A
142
−27.186
46.134
0.674
1.00
87.65
C


ATOM
885
CD
GLN
A
142
−26.769
47.145
−0.388
1.00
91.57
C


ATOM
886
OE1
GLN
A
142
−26.382
46.780
−1.508
1.00
90.57
O


ATOM
887
NE2
GLN
A
142
−26.847
48.425
−0.040
1.00
99.82
N


ATOM
888
N
SER
A
143
−26.446
41.562
0.879
1.00
81.37
N


ATOM
889
CA
SER
A
143
−25.716
40.420
0.415
1.00
78.53
C


ATOM
890
C
SER
A
143
−24.884
40.866
−0.772
1.00
77.76
C


ATOM
891
O
SER
A
143
−25.320
41.731
−1.554
1.00
76.92
O


ATOM
892
CB
SER
A
143
−26.662
39.315
−0.030
1.00
79.11
C


ATOM
893
OG
SER
A
143
−25.960
38.321
−0.743
1.00
79.05
O


ATOM
894
N
LEU
A
144
−23.695
40.284
−0.913
1.00
75.59
N


ATOM
895
CA
LEU
A
144
−22.854
40.541
−2.082
1.00
78.96
C


ATOM
896
C
LEU
A
144
−23.461
39.937
−3.371
1.00
77.36
C


ATOM
897
O
LEU
A
144
−23.012
40.278
−4.477
1.00
79.84
O


ATOM
898
CB
LEU
A
144
−21.433
40.007
−1.867
1.00
80.08
C


ATOM
899
CG
LEU
A
144
−20.660
40.517
−0.639
1.00
82.77
C


ATOM
900
CD1
LEU
A
144
−19.218
40.045
−0.740
1.00
84.51
C


ATOM
901
CD2
LEU
A
144
−20.714
42.055
−0.474
1.00
77.04
C


ATOM
902
N
LEU
A
145
−24.466
39.056
−3.210
1.00
74.06
N


ATOM
903
CA
LEU
A
145
−25.162
38.394
−4.293
1.00
75.14
C


ATOM
904
C
LEU
A
145
−26.417
39.116
−4.759
1.00
74.57
C


ATOM
905
O
LEU
A
145
−27.158
39.675
−3.951
1.00
76.73
O


ATOM
906
CB
LEU
A
145
−25.610
36.999
−3.836
1.00
74.59
C


ATOM
907
CG
LEU
A
145
−24.572
36.016
−3.326
1.00
73.05
C


ATOM
908
CD1
LEU
A
145
−25.249
34.715
−2.973
1.00
75.64
C


ATOM
909
CD2
LEU
A
145
−23.502
35.785
−4.364
1.00
79.94
C


ATOM
910
N
THR
A
146
−26.645
39.095
−6.070
1.00
74.52
N


ATOM
911
CA
THR
A
146
−27.901
39.539
−6.654
1.00
76.04
C


ATOM
912
C
THR
A
146
−28.928
38.413
−6.482
1.00
75.70
C


ATOM
913
O
THR
A
146
−28.574
37.282
−6.104
1.00
75.21
O


ATOM
914
CB
THR
A
146
−27.772
39.850
−8.156
1.00
76.46
C


ATOM
915
OG1
THR
A
146
−27.440
38.650
−8.860
1.00
79.82
O


ATOM
916
CG2
THR
A
146
−26.702
40.917
−8.423
1.00
71.60
C


ATOM
917
N
LYS
A
147
−30.193
38.719
−6.760
1.00
77.16
N


ATOM
918
CA
LYS
A
147
−31.279
37.718
−6.687
1.00
77.11
C


ATOM
919
C
LYS
A
147
−30.999
36.539
−7.633
1.00
76.19
C


ATOM
920
O
LYS
A
147
−31.219
35.383
−7.267
1.00
73.96
O


ATOM
921
CB
LYS
A
147
−32.646
38.315
−7.052
1.00
78.39
C


ATOM
922
CG
LYS
A
147
−33.107
39.543
−6.264
1.00
81.83
C


ATOM
923
CD
LYS
A
147
−33.377
39.263
−4.815
1.00
87.01
C


ATOM
924
CE
LYS
A
147
−33.758
40.559
−4.072
1.00
89.91
C


ATOM
925
NZ
LYS
A
147
−35.048
41.155
−4.556
1.00
91.41
N


ATOM
926
N
ASN
A
148
−30.512
36.843
−8.843
1.00
76.47
N


ATOM
927
CA
ASN
A
148
−30.199
35.809
−9.849
1.00
77.14
C


ATOM
928
C
ASN
A
148
−29.023
34.912
−9.476
1.00
76.20
C


ATOM
929
O
ASN
A
148
−29.113
33.689
−9.647
1.00
74.96
O


ATOM
930
CB
ASN
A
148
−29.958
36.431
−11.244
1.00
79.53
C


ATOM
931
CG
ASN
A
148
−31.254
36.824
−11.956
1.00
84.32
C


ATOM
932
OD1
ASN
A
148
−32.355
36.395
−11.586
1.00
89.19
O


ATOM
933
ND2
ASN
A
148
−31.121
37.648
−12.993
1.00
85.88
N


ATOM
934
N
LYS
A
149
−27.933
35.503
−8.976
1.00
73.79
N


ATOM
935
CA
LYS
A
149
−26.801
34.706
−8.474
1.00
76.59
C


ATOM
936
C
LYS
A
149
−27.254
33.809
−7.319
1.00
76.35
C


ATOM
937
O
LYS
A
149
−26.822
32.672
−7.232
1.00
81.05
O


ATOM
938
CB
LYS
A
149
−25.662
35.562
−7.950
1.00
79.46
C


ATOM
939
CG
LYS
A
149
−25.007
36.526
−8.914
1.00
82.58
C


ATOM
940
CD
LYS
A
149
−24.001
37.409
−8.129
1.00
83.85
C


ATOM
941
CE
LYS
A
149
−23.690
38.747
−8.790
1.00
89.02
C


ATOM
942
NZ
LYS
A
149
−23.014
39.645
−7.773
1.00
88.17
N


ATOM
943
N
ALA
A
150
−28.120
34.331
−6.441
1.00
73.36
N


ATOM
944
CA
ALA
A
150
−28.684
33.545
−5.325
1.00
74.46
C


ATOM
945
C
ALA
A
150
−29.457
32.309
−5.827
1.00
75.42
C


ATOM
946
O
ALA
A
150
−29.337
31.239
−5.245
1.00
79.11
O


ATOM
947
CB
ALA
A
150
−29.572
34.416
−4.440
1.00
68.50
C


ATOM
948
N
ARG
A
151
−30.243
32.465
−6.900
1.00
77.55
N


ATOM
949
CA
ARG
A
151
−30.945
31.314
−7.511
1.00
77.51
C


ATOM
950
C
ARG
A
151
−29.983
30.249
−8.022
1.00
77.61
C


ATOM
951
O
ARG
A
151
−30.272
29.057
−7.913
1.00
77.15
O


ATOM
952
CB
ARG
A
151
−31.836
31.709
−8.690
1.00
83.00
C


ATOM
953
CG
ARG
A
151
−33.284
32.095
−8.376
1.00
89.58
C


ATOM
954
CD
ARG
A
151
−33.576
33.556
−8.598
1.00
95.56
C


ATOM
955
NE
ARG
A
151
−34.990
33.794
−8.884
1.00
93.67
N


ATOM
956
CZ
ARG
A
151
−35.510
34.981
−9.220
1.00
98.85
C


ATOM
957
NH1
ARG
A
151
−34.743
36.084
−9.325
1.00
97.06
N


ATOM
958
NH2
ARG
A
151
−36.820
35.073
−9.458
1.00
102.25
N


ATOM
959
N
VAL
A
152
−28.852
30.684
−8.579
1.00
77.11
N


ATOM
960
CA
VAL
A
152
−27.825
29.767
−9.081
1.00
77.01
C


ATOM
961
C
VAL
A
152
−27.144
29.030
−7.919
1.00
76.31
C


ATOM
962
O
VAL
A
152
−26.878
27.845
−8.023
1.00
73.81
O


ATOM
963
CB
VAL
A
152
−26.806
30.502
−9.987
1.00
78.53
C


ATOM
964
CG1
VAL
A
152
−25.612
29.617
−10.311
1.00
85.33
C


ATOM
965
CG2
VAL
A
152
−27.486
30.934
−11.274
1.00
75.42
C


ATOM
966
N
ILE
A
153
−26.874
29.734
−6.824
1.00
76.01
N


ATOM
967
CA
ILE
A
153
−26.278
29.102
−5.632
1.00
76.47
C


ATOM
968
C
ILE
A
153
−27.235
28.053
−5.074
1.00
75.66
C


ATOM
969
O
ILE
A
153
−26.812
26.945
−4.733
1.00
74.99
O


ATOM
970
CB
ILE
A
153
−25.954
30.101
−4.523
1.00
75.93
C


ATOM
971
CG1
ILE
A
153
−24.899
31.127
−4.966
1.00
78.94
C


ATOM
972
CG2
ILE
A
153
−25.402
29.373
−3.320
1.00
75.34
C


ATOM
973
CD1
ILE
A
153
−23.488
30.564
−5.063
1.00
84.91
C


ATOM
974
N
ILE
A
154
−28.517
28.416
−4.990
1.00
75.41
N


ATOM
975
CA
ILE
A
154
−29.570
27.497
−4.545
1.00
75.93
C


ATOM
976
C
ILE
A
154
−29.645
26.286
−5.461
1.00
72.61
C


ATOM
977
O
ILE
A
154
−29.565
25.159
−5.001
1.00
70.17
O


ATOM
978
CB
ILE
A
154
−30.943
28.192
−4.490
1.00
75.92
C


ATOM
979
CG1
ILE
A
154
−30.975
29.183
−3.328
1.00
79.46
C


ATOM
980
CG2
ILE
A
154
−32.072
27.170
−4.312
1.00
73.88
C


ATOM
981
CD1
ILE
A
154
−32.130
30.133
−3.371
1.00
81.26
C


ATOM
982
N
LEU
A
155
−29.793
26.521
−6.754
1.00
75.14
N


ATOM
983
CA
LEU
A
155
−29.826
25.411
−7.725
1.00
75.92
C


ATOM
984
C
LEU
A
155
−28.604
24.497
−7.543
1.00
75.22
C


ATOM
985
O
LEU
A
155
−28.744
23.279
−7.543
1.00
73.02
O


ATOM
986
CB
LEU
A
155
−29.912
25.930
−9.158
1.00
71.84
C


ATOM
987
CG
LEU
A
155
−29.837
24.903
−10.297
1.00
79.20
C


ATOM
988
CD1
LEU
A
155
−30.845
23.769
−10.095
1.00
82.92
C


ATOM
989
CD2
LEU
A
155
−30.038
25.585
−11.666
1.00
75.89
C


ATOM
990
N
MET
A
156
−27.423
25.100
−7.384
1.00
76.89
N


ATOM
991
CA
MET
A
156
−26.186
24.339
−7.184
1.00
77.43
C


ATOM
992
C
MET
A
156
−26.181
23.568
−5.871
1.00
76.37
C


ATOM
993
O
MET
A
156
−25.571
22.504
−5.801
1.00
78.59
O


ATOM
994
CB
MET
A
156
−24.945
25.247
−7.281
1.00
81.89
C


ATOM
995
CG
MET
A
156
−24.609
25.744
−8.716
1.00
83.04
C


ATOM
996
SD
MET
A
156
−24.284
24.442
−9.942
1.00
100.97
S


ATOM
997
CE
MET
A
156
−24.128
25.398
−11.458
1.00
89.68
C


ATOM
998
N
VAL
A
157
−26.851
24.094
−4.841
1.00
76.00
N


ATOM
999
CA
VAL
A
157
−27.000
23.370
−3.572
1.00
74.39
C


ATOM
1000
C
VAL
A
157
−27.835
22.098
−3.784
1.00
74.09
C


ATOM
1001
O
VAL
A
157
−27.447
21.022
−3.327
1.00
74.06
O


ATOM
1002
CB
VAL
A
157
−27.624
24.250
−2.448
1.00
73.43
C


ATOM
1003
CG1
VAL
A
157
−28.152
23.382
−1.321
1.00
69.33
C


ATOM
1004
CG2
VAL
A
157
−26.613
25.269
−1.942
1.00
68.63
C


ATOM
1005
N
TRP
A
158
−28.969
22.227
−4.472
1.00
74.10
N


ATOM
1006
CA
TRP
A
158
−29.817
21.046
−4.777
1.00
74.40
C


ATOM
1007
C
TRP
A
158
−29.131
20.024
−5.683
1.00
75.32
C


ATOM
1008
O
TRP
A
158
−29.313
18.824
−5.482
1.00
78.30
O


ATOM
1009
CB
TRP
A
158
−31.188
21.469
−5.310
1.00
70.20
C


ATOM
1010
CG
TRP
A
158
−31.961
21.950
−4.183
1.00
74.02
C


ATOM
1011
CD1
TRP
A
158
−32.015
23.217
−3.719
1.00
80.00
C


ATOM
1012
CD2
TRP
A
158
−32.803
21.173
−3.333
1.00
72.24
C


ATOM
1013
NE1
TRP
A
158
−32.842
23.290
−2.632
1.00
79.53
N


ATOM
1014
CE2
TRP
A
158
−33.342
22.050
−2.371
1.00
79.45
C


ATOM
1015
CE3
TRP
A
158
−33.157
19.822
−3.292
1.00
73.80
C


ATOM
1016
CZ2
TRP
A
158
−34.220
21.625
−1.374
1.00
71.01
C


ATOM
1017
CZ3
TRP
A
158
−34.026
19.396
−2.307
1.00
73.95
C


ATOM
1018
CH2
TRP
A
158
−34.549
20.299
−1.358
1.00
74.98
C


ATOM
1019
N
ILE
A
159
−28.354
20.495
−6.663
1.00
76.28
N


ATOM
1020
CA
ILE
A
159
−27.572
19.595
−7.526
1.00
75.87
C


ATOM
1021
C
ILE
A
159
−26.509
18.840
−6.713
1.00
76.85
C


ATOM
1022
O
ILE
A
159
−26.416
17.626
−6.830
1.00
76.71
O


ATOM
1023
CB
ILE
A
159
−26.892
20.332
−8.696
1.00
73.88
C


ATOM
1024
CG1
ILE
A
159
−27.933
20.757
−9.735
1.00
75.94
C


ATOM
1025
CG2
ILE
A
159
−25.861
19.431
−9.354
1.00
76.05
C


ATOM
1026
CD1
ILE
A
159
−27.359
21.529
−10.923
1.00
71.99
C


ATOM
1027
N
VAL
A
160
−25.725
19.564
−5.903
1.00
74.32
N


ATOM
1028
CA
VAL
A
160
−24.672
18.947
−5.073
1.00
74.37
C


ATOM
1029
C
VAL
A
160
−25.257
17.970
−4.047
1.00
76.12
C


ATOM
1030
O
VAL
A
160
−24.738
16.864
−3.877
1.00
72.86
O


ATOM
1031
CB
VAL
A
160
−23.805
20.011
−4.357
1.00
75.98
C


ATOM
1032
CG1
VAL
A
160
−22.910
19.378
−3.297
1.00
70.80
C


ATOM
1033
CG2
VAL
A
160
−22.962
20.777
−5.377
1.00
72.65
C


ATOM
1034
N
SER
A
161
−26.332
18.388
−3.376
1.00
76.74
N


ATOM
1035
CA
SER
A
161
−27.052
17.522
−2.436
1.00
76.33
C


ATOM
1036
C
SER
A
161
−27.588
16.253
−3.127
1.00
80.12
C


ATOM
1037
O
SER
A
161
−27.565
15.175
−2.540
1.00
81.12
O


ATOM
1038
CB
SER
A
161
−28.203
18.277
−1.783
1.00
77.19
C


ATOM
1039
OG
SER
A
161
−27.721
19.391
−1.043
1.00
76.25
O


ATOM
1040
N
GLY
A
162
−28.067
16.399
−4.367
1.00
78.97
N


ATOM
1041
CA
GLY
A
162
−28.559
15.268
−5.165
1.00
77.45
C


ATOM
1042
C
GLY
A
162
−27.418
14.346
−5.559
1.00
75.17
C


ATOM
1043
O
GLY
A
162
−27.536
13.134
−5.495
1.00
72.75
O


ATOM
1044
N
LEU
A
163
−26.313
14.958
−5.965
1.00
76.45
N


ATOM
1045
CA
LEU
A
163
−25.098
14.253
−6.355
1.00
78.44
C


ATOM
1046
C
LEU
A
163
−24.517
13.473
−5.171
1.00
79.50
C


ATOM
1047
O
LEU
A
163
−23.962
12.407
−5.356
1.00
80.46
O


ATOM
1048
CB
LEU
A
163
−24.044
15.248
−6.908
1.00
79.15
C


ATOM
1049
CG
LEU
A
163
−23.153
14.848
−8.081
1.00
79.51
C


ATOM
1050
CD1
LEU
A
163
−23.978
14.520
−9.308
1.00
75.74
C


ATOM
1051
CD2
LEU
A
163
−22.183
15.984
−8.386
1.00
78.31
C


ATOM
1052
N
THR
A
164
−24.650
14.007
−3.958
1.00
82.19
N


ATOM
1053
CA
THR
A
164
−24.150
13.328
−2.751
1.00
84.31
C


ATOM
1054
C
THR
A
164
−25.196
12.399
−2.061
1.00
85.47
C


ATOM
1055
O
THR
A
164
−24.814
11.585
−1.225
1.00
83.60
O


ATOM
1056
CB
THR
A
164
−23.560
14.350
−1.758
1.00
84.63
C


ATOM
1057
OG1
THR
A
164
−24.500
15.405
−1.514
1.00
86.96
O


ATOM
1058
CG2
THR
A
164
−22.291
14.950
−2.337
1.00
82.27
C


ATOM
1059
N
SER
A
165
−26.485
12.525
−2.415
1.00
87.63
N


ATOM
1060
CA
SER
A
165
−27.562
11.705
−1.832
1.00
88.01
C


ATOM
1061
C
SER
A
165
−28.057
10.621
−2.778
1.00
90.10
C


ATOM
1062
O
SER
A
165
−28.029
9.438
−2.432
1.00
94.95
O


ATOM
1063
CB
SER
A
165
−28.745
12.583
−1.454
1.00
92.00
C


ATOM
1064
OG
SER
A
165
−29.324
13.166
−2.614
1.00
96.93
O


ATOM
1065
N
PHE
A
166
−28.512
11.027
−3.967
1.00
87.60
N


ATOM
1066
CA
PHE
A
166
−29.078
10.078
−4.951
1.00
85.74
C


ATOM
1067
C
PHE
A
166
−28.042
9.150
−5.573
1.00
85.31
C


ATOM
1068
O
PHE
A
166
−28.313
7.967
−5.704
1.00
85.33
O


ATOM
1069
CB
PHE
A
166
−29.884
10.789
−6.073
1.00
85.68
C


ATOM
1070
CG
PHE
A
166
−31.350
10.954
−5.753
1.00
84.00
C


ATOM
1071
CD1
PHE
A
166
−32.307
10.148
−6.362
1.00
82.71
C


ATOM
1072
CD2
PHE
A
166
−31.773
11.910
−4.847
1.00
78.83
C


ATOM
1073
CE1
PHE
A
166
−33.659
10.302
−6.066
1.00
84.17
C


ATOM
1074
CE2
PHE
A
166
−33.124
12.065
−4.551
1.00
79.25
C


ATOM
1075
CZ
PHE
A
166
−34.062
11.265
−5.155
1.00
80.39
C


ATOM
1076
N
LEU
A
167
−26.870
9.673
−5.954
1.00
83.84
N


ATOM
1077
CA
LEU
A
167
−25.839
8.813
−6.579
1.00
83.08
C


ATOM
1078
C
LEU
A
167
−25.510
7.568
−5.738
1.00
83.34
C


ATOM
1079
O
LEU
A
167
−25.725
6.477
−6.235
1.00
84.70
O


ATOM
1080
CB
LEU
A
167
−24.543
9.568
−6.947
1.00
82.52
C


ATOM
1081
CG
LEU
A
167
−24.431
10.342
−8.250
1.00
81.28
C


ATOM
1082
CD1
LEU
A
167
−23.003
10.847
−8.415
1.00
76.32
C


ATOM
1083
CD2
LEU
A
167
−24.815
9.492
−9.427
1.00
75.15
C


ATOM
1084
N
PRO
A
168
−25.003
7.727
−4.480
1.00
82.18
N


ATOM
1085
CA
PRO
A
168
−24.657
6.540
−3.663
1.00
81.22
C


ATOM
1086
C
PRO
A
168
−25.803
5.561
−3.419
1.00
80.15
C


ATOM
1087
O
PRO
A
168
−25.578
4.352
−3.439
1.00
79.49
O


ATOM
1088
CB
PRO
A
168
−24.226
7.145
−2.321
1.00
80.06
C


ATOM
1089
CG
PRO
A
168
−23.858
8.515
−2.614
1.00
82.53
C


ATOM
1090
CD
PRO
A
168
−24.720
8.968
−3.734
1.00
83.35
C


ATOM
1091
N
ILE
A
169
−27.007
6.086
−3.191
1.00
80.34
N


ATOM
1092
CA
ILE
A
169
−28.181
5.246
−2.931
1.00
81.83
C


ATOM
1093
C
ILE
A
169
−28.670
4.542
−4.207
1.00
83.09
C


ATOM
1094
O
ILE
A
169
−28.974
3.347
−4.167
1.00
85.69
O


ATOM
1095
CB
ILE
A
169
−29.316
6.029
−2.245
1.00
80.40
C


ATOM
1096
CG1
ILE
A
169
−28.845
6.522
−0.876
1.00
82.52
C


ATOM
1097
CG2
ILE
A
169
−30.538
5.150
−2.069
1.00
80.48
C


ATOM
1098
CD1
ILE
A
169
−29.913
7.192
−0.059
1.00
83.50
C


ATOM
1099
N
GLN
A
170
−28.743
5.274
−5.322
1.00
83.76
N


ATOM
1100
CA
GLN
A
170
−29.161
4.691
−6.617
1.00
83.63
C


ATOM
1101
C
GLN
A
170
−28.056
3.817
−7.243
1.00
83.39
C


ATOM
1102
O
GLN
A
170
−28.374
2.864
−7.960
1.00
86.11
O


ATOM
1103
CB
GLN
A
170
−29.606
5.780
−7.616
1.00
84.12
C


ATOM
1104
CG
GLN
A
170
−30.766
6.740
−7.123
1.00
84.07
C


ATOM
1105
CD
GLN
A
170
−32.159
6.134
−7.119
1.00
80.98
C


ATOM
1106
OE1
GLN
A
170
−32.370
5.018
−7.557
1.00
77.62
O


ATOM
1107
NE2
GLN
A
170
−33.123
6.895
−6.614
1.00
76.26
N


ATOM
1108
N
MET
A
171
−26.777
4.134
−6.981
1.00
82.82
N


ATOM
1109
CA
MET
A
171
−25.636
3.286
−7.436
1.00
83.78
C


ATOM
1110
C
MET
A
171
−25.340
2.113
−6.493
1.00
83.40
C


ATOM
1111
O
MET
A
171
−24.487
1.279
−6.816
1.00
79.60
O


ATOM
1112
CB
MET
A
171
−24.336
4.104
−7.631
1.00
84.08
C


ATOM
1113
CG
MET
A
171
−24.315
5.014
−8.854
1.00
84.82
C


ATOM
1114
SD
MET
A
171
−24.372
4.141
−10.445
1.00
93.60
S


ATOM
1115
CE
MET
A
171
−22.962
3.018
−10.356
1.00
86.88
C


ATOM
1116
N
HIS
A
172
−26.032
2.056
−5.346
1.00
83.18
N


ATOM
1117
CA
HIS
A
172
−25.909
0.954
−4.373
1.00
83.41
C


ATOM
1118
C
HIS
A
172
−24.544
0.943
−3.621
1.00
83.06
C


ATOM
1119
O
HIS
A
172
−24.082
−0.107
−3.168
1.00
83.35
O


ATOM
1120
CB
HIS
A
172
−26.218
−0.415
−5.050
1.00
82.96
C


ATOM
1121
CG
HIS
A
172
−27.640
−0.563
−5.505
1.00
83.78
C


ATOM
1122
ND1
HIS
A
172
−28.273
−1.780
−5.517
1.00
79.73
N


ATOM
1123
CD2
HIS
A
172
−28.552
0.332
−5.957
1.00
82.46
C


ATOM
1124
CE1
HIS
A
172
−29.509
−1.639
−5.954
1.00
77.50
C


ATOM
1125
NE2
HIS
A
172
−29.705
−0.364
−6.229
1.00
81.47
N


ATOM
1126
N
TRP
A
173
−23.915
2.116
−3.495
1.00
83.44
N


ATOM
1127
CA
TRP
A
173
−22.647
2.253
−2.758
1.00
83.86
C


ATOM
1128
C
TRP
A
173
−22.823
2.178
−1.244
1.00
83.60
C


ATOM
1129
O
TRP
A
173
−21.888
1.833
−0.520
1.00
83.83
O


ATOM
1130
CB
TRP
A
173
−21.953
3.576
−3.084
1.00
83.86
C


ATOM
1131
CG
TRP
A
173
−21.492
3.720
−4.491
1.00
84.03
C


ATOM
1132
CD1
TRP
A
173
−21.496
2.766
−5.475
1.00
84.91
C


ATOM
1133
CD2
TRP
A
173
−20.939
4.893
−5.079
1.00
82.79
C


ATOM
1134
NE1
TRP
A
173
−20.988
3.281
−6.631
1.00
84.59
N


ATOM
1135
CE2
TRP
A
173
−20.636
4.585
−6.419
1.00
81.34
C


ATOM
1136
CE3
TRP
A
173
−20.671
6.184
−4.602
1.00
84.75
C


ATOM
1137
CZ2
TRP
A
173
−20.079
5.517
−7.291
1.00
85.31
C


ATOM
1138
CZ3
TRP
A
173
−20.115
7.115
−5.470
1.00
84.09
C


ATOM
1139
CH2
TRP
A
173
−19.825
6.777
−6.799
1.00
86.15
C


ATOM
1140
N
TYR
A
174
−24.018
2.505
−0.777
1.00
81.87
N


ATOM
1141
CA
TYR
A
174
−24.330
2.478
0.643
1.00
82.95
C


ATOM
1142
C
TYR
A
174
−24.461
1.103
1.261
1.00
84.39
C


ATOM
1143
O
TYR
A
174
−24.328
0.993
2.451
1.00
85.16
O


ATOM
1144
CB
TYR
A
174
−25.646
3.224
0.916
1.00
82.89
C


ATOM
1145
CG
TYR
A
174
−26.913
2.473
0.522
1.00
80.98
C


ATOM
1146
CD1
TYR
A
174
−27.679
1.794
1.475
1.00
79.89
C


ATOM
1147
CD2
TYR
A
174
−27.340
2.442
−0.798
1.00
79.78
C


ATOM
1148
CE1
TYR
A
174
−28.838
1.110
1.111
1.00
81.50
C


ATOM
1149
CE2
TYR
A
174
−28.489
1.767
−1.174
1.00
80.48
C


ATOM
1150
CZ
TYR
A
174
−29.234
1.104
−0.221
1.00
81.26
C


ATOM
1151
OH
TYR
A
174
−30.365
0.439
−0.601
1.00
79.71
O


ATOM
1152
N
ARG
A
175
−24.709
0.070
0.457
1.00
82.91
N


ATOM
1153
CA
ARG
A
175
−25.158
−1.227
0.984
1.00
81.71
C


ATOM
1154
C
ARG
A
175
−24.093
−2.047
1.678
1.00
80.96
C


ATOM
1155
O
ARG
A
175
−22.922
−1.998
1.313
1.00
80.38
O


ATOM
1156
CB
ARG
A
175
−25.819
−2.057
−0.136
1.00
82.53
C


ATOM
1157
CG
ARG
A
175
−26.861
−1.242
−0.863
1.00
85.26
C


ATOM
1158
CD
ARG
A
175
−28.068
−1.948
−1.318
1.00
83.28
C


ATOM
1159
NE
ARG
A
175
−27.867
−2.850
−2.413
1.00
82.86
N


ATOM
1160
CZ
ARG
A
175
−28.850
−3.346
−3.172
1.00
86.74
C


ATOM
1161
NH1
ARG
A
175
−30.142
−3.019
−2.963
1.00
84.23
N


ATOM
1162
NH2
ARG
A
175
−28.541
−4.184
−4.169
1.00
81.76
N


ATOM
1163
N
ALA
A
176
−24.539
−2.793
2.689
1.00
81.02
N


ATOM
1164
CA
ALA
A
176
−23.696
−3.687
3.454
1.00
81.16
C


ATOM
1165
C
ALA
A
176
−23.664
−5.020
2.737
1.00
81.72
C


ATOM
1166
O
ALA
A
176
−24.541
−5.308
1.920
1.00
81.33
O


ATOM
1167
CB
ALA
A
176
−24.231
−3.853
4.854
1.00
81.01
C


ATOM
1168
N
THR
A
177
−22.651
−5.821
3.056
1.00
81.43
N


ATOM
1169
CA
THR
A
177
−22.421
−7.128
2.422
1.00
80.81
C


ATOM
1170
C
THR
A
177
−22.741
−8.348
3.319
1.00
81.25
C


ATOM
1171
O
THR
A
177
−22.430
−9.481
2.947
1.00
81.14
O


ATOM
1172
CB
THR
A
177
−20.959
−7.215
1.944
1.00
80.88
C


ATOM
1173
OG1
THR
A
177
−20.080
−7.111
3.071
1.00
80.59
O


ATOM
1174
CG2
THR
A
177
−20.656
−6.090
0.961
1.00
80.78
C


ATOM
1175
N
HIS
A
178
−23.358
−8.123
4.482
1.00
80.75
N


ATOM
1176
CA
HIS
A
178
−23.719
−9.227
5.397
1.00
80.59
C


ATOM
1177
C
HIS
A
178
−25.129
−9.723
5.047
1.00
79.62
C


ATOM
1178
O
HIS
A
178
−25.924
−8.985
4.473
1.00
80.66
O


ATOM
1179
CB
HIS
A
178
−23.574
−8.824
6.878
1.00
80.59
C


ATOM
1180
CG
HIS
A
178
−24.512
−7.752
7.320
1.00
80.24
C


ATOM
1181
ND1
HIS
A
178
−24.229
−6.410
7.189
1.00
85.34
N


ATOM
1182
CD2
HIS
A
178
−25.731
−7.825
7.895
1.00
81.58
C


ATOM
1183
CE1
HIS
A
178
−25.238
−5.702
7.664
1.00
86.20
C


ATOM
1184
NE2
HIS
A
178
−26.163
−6.537
8.098
1.00
88.13
N


ATOM
1185
N
GLN
A
179
−25.413
−10.973
5.405
1.00
79.51
N


ATOM
1186
CA
GLN
A
179
−26.652
−11.670
4.994
1.00
79.05
C


ATOM
1187
C
GLN
A
179
−27.968
−11.024
5.453
1.00
77.54
C


ATOM
1188
O
GLN
A
179
−28.903
−10.937
4.667
1.00
76.07
O


ATOM
1189
CB
GLN
A
179
−26.624
−13.140
5.467
1.00
79.94
C


ATOM
1190
CG
GLN
A
179
−27.540
−14.079
4.668
1.00
82.48
C


ATOM
1191
CD
GLN
A
179
−27.127
−14.218
3.201
1.00
85.58
C


ATOM
1192
OE1
GLN
A
179
−25.963
−14.006
2.838
1.00
84.07
O


ATOM
1193
NE2
GLN
A
179
−28.082
−14.578
2.355
1.00
88.48
N


ATOM
1194
N
GLU
A
180
−28.026
−10.587
6.713
1.00
76.40
N


ATOM
1195
CA
GLU
A
180
−29.224
−9.915
7.278
1.00
76.08
C


ATOM
1196
C
GLU
A
180
−29.656
−8.694
6.441
1.00
74.97
C


ATOM
1197
O
GLU
A
180
−30.854
−8.437
6.268
1.00
72.77
O


ATOM
1198
CB
GLU
A
180
−28.957
−9.518
8.738
1.00
77.18
C


ATOM
1199
CG
GLU
A
180
−30.098
−8.829
9.499
1.00
78.62
C


ATOM
1200
CD
GLU
A
180
−29.685
−8.430
10.920
1.00
78.93
C


ATOM
1201
OE1
GLU
A
180
−30.024
−7.303
11.336
1.00
77.22
O


ATOM
1202
OE2
GLU
A
180
−29.022
−9.239
11.618
1.00
82.04
O


ATOM
1203
N
ALA
A
181
−28.666
−7.962
5.929
1.00
73.84
N


ATOM
1204
CA
ALA
A
181
−28.887
−6.814
5.061
1.00
74.58
C


ATOM
1205
C
ALA
A
181
−29.359
−7.291
3.699
1.00
74.81
C


ATOM
1206
O
ALA
A
181
−30.389
−6.828
3.201
1.00
74.62
O


ATOM
1207
CB
ALA
A
181
−27.610
−6.005
4.921
1.00
72.34
C


ATOM
1208
N
ILE
A
182
−28.592
−8.218
3.110
1.00
76.12
N


ATOM
1209
CA
ILE
A
182
−28.905
−8.843
1.800
1.00
76.87
C


ATOM
1210
C
ILE
A
182
−30.356
−9.336
1.743
1.00
77.84
C


ATOM
1211
O
ILE
A
182
−31.047
−9.109
0.745
1.00
78.53
O


ATOM
1212
CB
ILE
A
182
−27.924
−10.003
1.453
1.00
76.51
C


ATOM
1213
CG1
ILE
A
182
−26.515
−9.466
1.192
1.00
75.02
C


ATOM
1214
CG2
ILE
A
182
−28.387
−10.756
0.226
1.00
76.30
C


ATOM
1215
CD1
ILE
A
182
−25.478
−10.540
0.959
1.00
75.60
C


ATOM
1216
N
ASN
A
183
−30.796
−10.000
2.817
1.00
79.14
N


ATOM
1217
CA
ASN
A
183
−32.185
−10.442
2.958
1.00
79.97
C


ATOM
1218
C
ASN
A
183
−33.152
−9.263
2.924
1.00
81.18
C


ATOM
1219
O
ASN
A
183
−34.160
−9.323
2.232
1.00
81.85
O


ATOM
1220
CB
ASN
A
183
−32.392
−11.225
4.260
1.00
79.70
C


ATOM
1221
CG
ASN
A
183
−31.725
−12.586
4.246
1.00
78.69
C


ATOM
1222
OD1
ASN
A
183
−30.711
−12.800
3.586
1.00
76.53
O


ATOM
1223
ND2
ASN
A
183
−32.301
−13.522
4.984
1.00
78.92
N


ATOM
1224
N
CYS
A
184
−32.835
−8.200
3.671
1.00
81.90
N


ATOM
1225
CA
CYS
A
184
−33.675
−6.990
3.698
1.00
81.53
C


ATOM
1226
C
CYS
A
184
−33.742
−6.278
2.348
1.00
80.40
C


ATOM
1227
O
CYS
A
184
−34.791
−5.743
1.982
1.00
80.62
O


ATOM
1228
CB
CYS
A
184
−33.189
−5.995
4.751
1.00
82.03
C


ATOM
1229
SG
CYS
A
184
−34.281
−4.556
4.896
1.00
86.09
S


ATOM
1230
N
TYR
A
185
−32.630
−6.270
1.614
1.00
80.51
N


ATOM
1231
CA
TYR
A
185
−32.588
−5.626
0.289
1.00
80.30
C


ATOM
1232
C
TYR
A
185
−33.500
−6.328
−0.693
1.00
80.59
C


ATOM
1233
O
TYR
A
185
−34.322
−5.683
−1.354
1.00
82.14
O


ATOM
1234
CB
TYR
A
185
−31.165
−5.597
−0.278
1.00
79.80
C


ATOM
1235
CG
TYR
A
185
−30.177
−4.798
0.552
1.00
80.24
C


ATOM
1236
CD1
TYR
A
185
−30.497
−3.522
1.018
1.00
76.86
C


ATOM
1237
CD2
TYR
A
185
−28.923
−5.313
0.866
1.00
79.73
C


ATOM
1238
CE1
TYR
A
185
−29.604
−2.797
1.769
1.00
78.10
C


ATOM
1239
CE2
TYR
A
185
−28.021
−4.589
1.618
1.00
79.03
C


ATOM
1240
CZ
TYR
A
185
−28.365
−3.334
2.066
1.00
78.33
C


ATOM
1241
OH
TYR
A
185
−27.471
−2.621
2.811
1.00
79.23
O


ATOM
1242
N
ALA
A
186
−33.344
−7.645
−0.776
1.00
79.03
N


ATOM
1243
CA
ALA
A
186
−34.189
−8.489
−1.622
1.00
78.43
C


ATOM
1244
C
ALA
A
186
−35.659
−8.474
−1.178
1.00
78.71
C


ATOM
1245
O
ALA
A
186
−36.557
−8.506
−2.023
1.00
79.49
O


ATOM
1246
CB
ALA
A
186
−33.661
−9.914
−1.627
1.00
76.05
C


ATOM
1247
N
GLU
A
187
−35.887
−8.422
0.140
1.00
79.25
N


ATOM
1248
CA
GLU
A
187
−37.237
−8.399
0.713
1.00
79.62
C


ATOM
1249
C
GLU
A
187
−37.976
−7.085
0.400
1.00
79.69
C


ATOM
1250
O
GLU
A
187
−37.549
−5.998
0.814
1.00
80.47
O


ATOM
1251
CB
GLU
A
187
−37.181
−8.643
2.234
1.00
79.95
C


ATOM
1252
CG
GLU
A
187
−38.523
−8.617
2.992
1.00
82.26
C


ATOM
1253
CD
GLU
A
187
−39.521
−9.671
2.533
1.00
86.24
C


ATOM
1254
OE1
GLU
A
187
−39.137
−10.643
1.845
1.00
92.89
O


ATOM
1255
OE2
GLU
A
187
−40.711
−9.530
2.863
1.00
88.95
O


ATOM
1256
N
GLU
A
188
−39.081
−7.216
−0.334
1.00
79.07
N


ATOM
1257
CA
GLU
A
188
−39.930
−6.085
−0.751
1.00
79.55
C


ATOM
1258
C
GLU
A
188
−40.619
−5.361
0.404
1.00
79.89
C


ATOM
1259
O
GLU
A
188
−40.897
−4.161
0.292
1.00
79.15
O


ATOM
1260
CB
GLU
A
188
−40.983
−6.562
−1.747
1.00
79.62
C


ATOM
1261
CG
GLU
A
188
−42.048
−7.458
−1.121
1.00
83.66
C


ATOM
1262
CD
GLU
A
188
−42.614
−8.463
−2.084
1.00
84.99
C


ATOM
1263
OE1
GLU
A
188
−42.812
−8.122
−3.268
1.00
95.21
O


ATOM
1264
OE2
GLU
A
188
−42.866
−9.605
−1.652
1.00
89.59
O


ATOM
1265
N
THR
A
189
−40.891
−6.089
1.496
1.00
77.96
N


ATOM
1266
CA
THR
A
189
−41.533
−5.512
2.676
1.00
75.91
C


ATOM
1267
C
THR
A
189
−40.541
−5.063
3.755
1.00
76.43
C


ATOM
1268
O
THR
A
189
−40.961
−4.656
4.836
1.00
76.97
O


ATOM
1269
CB
THR
A
189
−42.562
−6.481
3.300
1.00
75.78
C


ATOM
1270
OG1
THR
A
189
−41.883
−7.617
3.843
1.00
74.95
O


ATOM
1271
CG2
THR
A
189
−43.580
−6.923
2.252
1.00
70.98
C


ATOM
1272
N
CYS
A
190
−39.242
−5.131
3.464
1.00
78.00
N


ATOM
1273
CA
CYS
A
190
−38.209
−4.690
4.377
1.00
77.37
C


ATOM
1274
C
CYS
A
190
−37.588
−3.443
3.799
1.00
74.95
C


ATOM
1275
O
CYS
A
190
−37.131
−3.464
2.658
1.00
75.26
O


ATOM
1276
CB
CYS
A
190
−37.136
−5.755
4.545
1.00
79.47
C


ATOM
1277
SG
CYS
A
190
−35.924
−5.347
5.847
1.00
85.35
S


ATOM
1278
N
CYS
A
191
−37.574
−2.368
4.582
1.00
75.05
N


ATOM
1279
CA
CYS
A
191
−36.955
−1.110
4.175
1.00
77.17
C


ATOM
1280
C
CYS
A
191
−36.140
−0.537
5.329
1.00
77.29
C


ATOM
1281
O
CYS
A
191
−36.450
0.523
5.856
1.00
81.42
O


ATOM
1282
CB
CYS
A
191
−38.024
−0.135
3.680
1.00
77.85
C


ATOM
1283
SG
CYS
A
191
−37.383
1.370
2.899
1.00
80.61
S


ATOM
1284
N
ASP
A
192
−35.091
−1.270
5.703
1.00
78.03
N


ATOM
1285
CA
ASP
A
192
−34.141
−0.851
6.741
1.00
76.97
C


ATOM
1286
C
ASP
A
192
−32.909
−0.347
6.054
1.00
75.94
C


ATOM
1287
O
ASP
A
192
−32.428
−0.979
5.114
1.00
77.63
O


ATOM
1288
CB
ASP
A
192
−33.762
−2.005
7.667
1.00
77.44
C


ATOM
1289
CG
ASP
A
192
−34.889
−2.419
8.586
1.00
80.00
C


ATOM
1290
OD1
ASP
A
192
−35.724
−1.561
8.959
1.00
82.18
O


ATOM
1291
OD2
ASP
A
192
−34.941
−3.619
8.943
1.00
80.09
O


ATOM
1292
N
PHE
A
193
−32.388
0.785
6.517
1.00
75.48
N


ATOM
1293
CA
PHE
A
193
−31.237
1.416
5.890
1.00
75.22
C


ATOM
1294
C
PHE
A
193
−29.909
0.865
6.443
1.00
75.43
C


ATOM
1295
O
PHE
A
193
−29.160
1.573
7.133
1.00
73.54
O


ATOM
1296
CB
PHE
A
193
−31.351
2.947
6.043
1.00
74.11
C


ATOM
1297
CG
PHE
A
193
−30.399
3.730
5.175
1.00
76.00
C


ATOM
1298
CD1
PHE
A
193
−30.300
3.471
3.793
1.00
76.25
C


ATOM
1299
CD2
PHE
A
193
−29.600
4.736
5.724
1.00
76.96
C


ATOM
1300
CE1
PHE
A
193
−29.428
4.189
2.984
1.00
73.80
C


ATOM
1301
CE2
PHE
A
193
−28.718
5.465
4.905
1.00
79.72
C


ATOM
1302
CZ
PHE
A
193
−28.638
5.183
3.528
1.00
75.51
C


ATOM
1303
N
PHE
A
194
−29.641
−0.409
6.127
1.00
74.51
N


ATOM
1304
CA
PHE
A
194
−28.376
−1.061
6.455
1.00
74.06
C


ATOM
1305
C
PHE
A
194
−27.340
−0.493
5.527
1.00
74.74
C


ATOM
1306
O
PHE
A
194
−27.542
−0.522
4.308
1.00
73.83
O


ATOM
1307
CB
PHE
A
194
−28.423
−2.577
6.235
1.00
73.04
C


ATOM
1308
CG
PHE
A
194
−29.226
−3.318
7.256
1.00
73.26
C


ATOM
1309
CD1
PHE
A
194
−28.674
−3.630
8.499
1.00
75.81
C


ATOM
1310
CD2
PHE
A
194
−30.534
−3.712
6.984
1.00
74.89
C


ATOM
1311
CE1
PHE
A
194
−29.412
−4.320
9.457
1.00
74.15
C


ATOM
1312
CE2
PHE
A
194
−31.282
−4.405
7.936
1.00
75.40
C


ATOM
1313
CZ
PHE
A
194
−30.720
−4.709
9.176
1.00
74.86
C


ATOM
1314
N
THR
A
195
−26.241
0.022
6.087
1.00
73.91
N


ATOM
1315
CA
THR
A
195
−25.157
0.559
5.278
1.00
72.89
C


ATOM
1316
C
THR
A
195
−23.811
−0.039
5.644
1.00
71.51
C


ATOM
1317
O
THR
A
195
−23.636
−0.565
6.748
1.00
70.29
O


ATOM
1318
CB
THR
A
195
−25.034
2.092
5.403
1.00
73.36
C


ATOM
1319
OG1
THR
A
195
−24.316
2.431
6.598
1.00
75.79
O


ATOM
1320
CG2
THR
A
195
−26.415
2.762
5.409
1.00
75.39
C


ATOM
1321
N
ASN
A
196
−22.863
0.047
4.709
1.00
69.76
N


ATOM
1322
CA
ASN
A
196
−21.483
−0.332
4.996
1.00
70.25
C


ATOM
1323
C
ASN
A
196
−20.870
0.823
5.783
1.00
70.56
C


ATOM
1324
O
ASN
A
196
−21.350
1.967
5.694
1.00
73.34
O


ATOM
1325
CB
ASN
A
196
−20.676
−0.721
3.732
1.00
70.71
C


ATOM
1326
CG
ASN
A
196
−20.478
0.426
2.743
1.00
71.43
C


ATOM
1327
OD1
ASN
A
196
−19.940
1.481
3.071
1.00
74.37
O


ATOM
1328
ND2
ASN
A
196
−20.920
0.205
1.515
1.00
72.46
N


ATOM
1329
N
GLN
A
197
−19.824
0.524
6.544
1.00
69.71
N


ATOM
1330
CA
GLN
A
197
−19.195
1.507
7.446
1.00
69.84
C


ATOM
1331
C
GLN
A
197
−18.570
2.687
6.711
1.00
68.45
C


ATOM
1332
O
GLN
A
197
−18.745
3.832
7.137
1.00
68.34
O


ATOM
1333
CB
GLN
A
197
−18.141
0.829
8.319
1.00
70.49
C


ATOM
1334
CG
GLN
A
197
−18.723
−0.221
9.264
1.00
73.78
C


ATOM
1335
CD
GLN
A
197
−17.662
−1.017
9.977
1.00
75.55
C


ATOM
1336
OE1
GLN
A
197
−16.556
−0.528
10.223
1.00
81.19
O


ATOM
1337
NE2
GLN
A
197
−17.988
−2.262
10.319
1.00
83.68
N


ATOM
1338
N
ALA
A
198
−17.855
2.400
5.617
1.00
67.03
N


ATOM
1339
CA
ALA
A
198
−17.222
3.430
4.772
1.00
67.06
C


ATOM
1340
C
ALA
A
198
−18.229
4.480
4.309
1.00
68.00
C


ATOM
1341
O
ALA
A
198
−17.926
5.672
4.310
1.00
67.52
O


ATOM
1342
CB
ALA
A
198
−16.540
2.796
3.576
1.00
67.06
C


ATOM
1343
N
TYR
A
199
−19.420
4.027
3.916
1.00
67.41
N


ATOM
1344
CA
TYR
A
199
−20.503
4.925
3.544
1.00
68.01
C


ATOM
1345
C
TYR
A
199
−21.001
5.678
4.753
1.00
66.60
C


ATOM
1346
O
TYR
A
199
−21.162
6.889
4.697
1.00
64.41
O


ATOM
1347
CB
TYR
A
199
−21.685
4.168
2.935
1.00
69.88
C


ATOM
1348
CG
TYR
A
199
−22.897
5.054
2.714
1.00
67.04
C


ATOM
1349
CD1
TYR
A
199
−23.009
5.831
1.566
1.00
70.50
C


ATOM
1350
CD2
TYR
A
199
−23.932
5.113
3.655
1.00
69.18
C


ATOM
1351
CE1
TYR
A
199
−24.128
6.657
1.352
1.00
71.51
C


ATOM
1352
CE2
TYR
A
199
−25.051
5.933
3.453
1.00
72.40
C


ATOM
1353
CZ
TYR
A
199
−25.141
6.702
2.298
1.00
71.80
C


ATOM
1354
OH
TYR
A
199
−26.236
7.509
2.088
1.00
73.79
O


ATOM
1355
N
ALA
A
200
−21.245
4.942
5.840
1.00
67.80
N


ATOM
1356
CA
ALA
A
200
−21.723
5.531
7.100
1.00
66.98
C


ATOM
1357
C
ALA
A
200
−20.840
6.697
7.533
1.00
65.35
C


ATOM
1358
O
ALA
A
200
−21.354
7.739
7.890
1.00
62.59
O


ATOM
1359
CB
ALA
A
200
−21.789
4.486
8.183
1.00
67.92
C


ATOM
1360
N
ILE
A
201
−19.520
6.505
7.489
1.00
66.75
N


ATOM
1361
CA
ILE
A
201
−18.559
7.565
7.830
1.00
69.39
C


ATOM
1362
C
ILE
A
201
−18.514
8.680
6.785
1.00
70.99
C


ATOM
1363
O
ILE
A
201
−18.768
9.836
7.107
1.00
71.46
O


ATOM
1364
CB
ILE
A
201
−17.126
7.027
7.971
1.00
68.97
C


ATOM
1365
CG1
ILE
A
201
−16.998
6.142
9.210
1.00
70.83
C


ATOM
1366
CG2
ILE
A
201
−16.142
8.181
8.064
1.00
68.40
C


ATOM
1367
CD1
ILE
A
201
−15.610
5.530
9.383
1.00
71.18
C


ATOM
1368
N
ALA
A
202
−18.191
8.311
5.542
1.00
71.53
N


ATOM
1369
CA
ALA
A
202
−18.001
9.273
4.438
1.00
70.39
C


ATOM
1370
C
ALA
A
202
−19.228
10.112
4.136
1.00
71.21
C


ATOM
1371
O
ALA
A
202
−19.112
11.331
3.979
1.00
72.58
O


ATOM
1372
CB
ALA
A
202
−17.558
8.555
3.177
1.00
69.73
C


ATOM
1373
N
SER
A
203
−20.395
9.474
4.054
1.00
71.88
N


ATOM
1374
CA
SER
A
203
−21.633
10.202
3.748
1.00
72.73
C


ATOM
1375
C
SER
A
203
−22.030
11.160
4.861
1.00
75.38
C


ATOM
1376
O
SER
A
203
−22.457
12.274
4.579
1.00
76.69
O


ATOM
1377
CB
SER
A
203
−22.798
9.261
3.492
1.00
73.42
C


ATOM
1378
OG
SER
A
203
−23.944
10.010
3.118
1.00
77.91
O


ATOM
1379
N
SER
A
204
−21.890
10.730
6.114
1.00
72.53
N


ATOM
1380
CA
SER
A
204
−22.266
11.570
7.250
1.00
71.77
C


ATOM
1381
C
SER
A
204
−21.283
12.742
7.442
1.00
73.36
C


ATOM
1382
O
SER
A
204
−21.695
13.822
7.863
1.00
73.43
O


ATOM
1383
CB
SER
A
204
−22.428
10.728
8.525
1.00
69.52
C


ATOM
1384
OG
SER
A
204
−21.292
9.941
8.776
1.00
76.74
O


ATOM
1385
N
ILE
A
205
−19.998
12.537
7.137
1.00
73.22
N


ATOM
1386
CA
ILE
A
205
−19.020
13.638
7.213
1.00
73.31
C


ATOM
1387
C
ILE
A
205
−19.284
14.674
6.118
1.00
76.06
C


ATOM
1388
O
ILE
A
205
−19.399
15.872
6.405
1.00
76.12
O


ATOM
1389
CB
ILE
A
205
−17.569
13.147
7.126
1.00
72.39
C


ATOM
1390
CG1
ILE
A
205
−17.173
12.458
8.435
1.00
71.69
C


ATOM
1391
CG2
ILE
A
205
−16.625
14.303
6.876
1.00
70.67
C


ATOM
1392
CD1
ILE
A
205
−15.812
11.794
8.390
1.00
72.10
C


ATOM
1393
N
VAL
A
206
−19.380
14.201
4.877
1.00
74.65
N


ATOM
1394
CA
VAL
A
206
−19.591
15.081
3.713
1.00
77.11
C


ATOM
1395
C
VAL
A
206
−20.972
15.757
3.665
1.00
75.89
C


ATOM
1396
O
VAL
A
206
−21.071
16.933
3.317
1.00
75.71
O


ATOM
1397
CB
VAL
A
206
−19.356
14.321
2.388
1.00
75.98
C


ATOM
1398
CG1
VAL
A
206
−19.647
15.210
1.194
1.00
77.29
C


ATOM
1399
CG2
VAL
A
206
−17.920
13.819
2.324
1.00
80.65
C


ATOM
1400
N
SER
A
207
−22.023
15.025
4.009
1.00
76.66
N


ATOM
1401
CA
SER
A
207
−23.377
15.585
3.972
1.00
77.79
C


ATOM
1402
C
SER
A
207
−23.720
16.457
5.165
1.00
78.97
C


ATOM
1403
O
SER
A
207
−24.437
17.437
4.995
1.00
83.06
O


ATOM
1404
CB
SER
A
207
−24.420
14.488
3.922
1.00
78.06
C


ATOM
1405
OG
SER
A
207
−24.231
13.633
2.814
1.00
83.89
O


ATOM
1406
N
PHE
A
208
−23.216
16.103
6.356
1.00
79.98
N


ATOM
1407
CA
PHE
A
208
−23.601
16.771
7.609
1.00
77.07
C


ATOM
1408
C
PHE
A
208
−22.463
17.477
8.319
1.00
78.35
C


ATOM
1409
O
PHE
A
208
−22.511
18.695
8.470
1.00
79.76
O


ATOM
1410
CB
PHE
A
208
−24.254
15.753
8.552
1.00
76.98
C


ATOM
1411
CG
PHE
A
208
−24.676
16.321
9.888
1.00
78.18
C


ATOM
1412
CD1
PHE
A
208
−25.792
17.141
9.984
1.00
77.60
C


ATOM
1413
CD2
PHE
A
208
−23.957
16.025
11.051
1.00
75.04
C


ATOM
1414
CE1
PHE
A
208
−26.190
17.668
11.216
1.00
77.26
C


ATOM
1415
CE2
PHE
A
208
−24.350
16.545
12.278
1.00
80.26
C


ATOM
1416
CZ
PHE
A
208
−25.475
17.373
12.357
1.00
77.27
C


ATOM
1417
N
TYR
A
209
−21.450
16.739
8.756
1.00
78.29
N


ATOM
1418
CA
TYR
A
209
−20.386
17.351
9.593
1.00
78.96
C


ATOM
1419
C
TYR
A
209
−19.583
18.463
8.941
1.00
78.89
C


ATOM
1420
O
TYR
A
209
−19.222
19.422
9.625
1.00
79.13
O


ATOM
1421
CB
TYR
A
209
−19.425
16.295
10.131
1.00
80.47
C


ATOM
1422
CG
TYR
A
209
−20.047
15.425
11.181
1.00
82.05
C


ATOM
1423
CD1
TYR
A
209
−20.330
14.088
10.937
1.00
80.35
C


ATOM
1424
CD2
TYR
A
209
−20.360
15.948
12.440
1.00
84.46
C


ATOM
1425
CE1
TYR
A
209
−20.906
13.292
11.913
1.00
83.44
C


ATOM
1426
CE2
TYR
A
209
−20.936
15.163
13.424
1.00
85.84
C


ATOM
1427
CZ
TYR
A
209
−21.208
13.835
13.159
1.00
85.25
C


ATOM
1428
OH
TYR
A
209
−21.776
13.073
14.145
1.00
89.70
O


ATOM
1429
N
VAL
A
210
−19.304
18.351
7.645
1.00
78.18
N


ATOM
1430
CA
VAL
A
210
−18.557
19.404
6.944
1.00
77.41
C


ATOM
1431
C
VAL
A
210
−19.350
20.740
6.944
1.00
77.38
C


ATOM
1432
O
VAL
A
210
−18.853
21.716
7.495
1.00
80.02
O


ATOM
1433
CB
VAL
A
210
−18.108
18.976
5.515
1.00
78.51
C


ATOM
1434
CG1
VAL
A
210
−17.758
20.202
4.653
1.00
73.94
C


ATOM
1435
CG2
VAL
A
210
−16.932
18.022
5.609
1.00
76.43
C


ATOM
1436
N
PRO
A
211
−20.557
20.780
6.339
1.00
75.78
N


ATOM
1437
CA
PRO
A
211
−21.304
22.033
6.379
1.00
76.70
C


ATOM
1438
C
PRO
A
211
−21.721
22.509
7.793
1.00
78.77
C


ATOM
1439
O
PRO
A
211
−21.917
23.707
7.967
1.00
81.95
O


ATOM
1440
CB
PRO
A
211
−22.538
21.738
5.506
1.00
78.00
C


ATOM
1441
CG
PRO
A
211
−22.687
20.307
5.514
1.00
77.13
C


ATOM
1442
CD
PRO
A
211
−21.305
19.746
5.601
1.00
80.23
C


ATOM
1443
N
LEU
A
212
−21.848
21.598
8.767
1.00
77.02
N


ATOM
1444
CA
LEU
A
212
−22.178
21.965
10.159
1.00
75.25
C


ATOM
1445
C
LEU
A
212
−21.034
22.723
10.815
1.00
75.40
C


ATOM
1446
O
LEU
A
212
−21.248
23.765
11.404
1.00
76.71
O


ATOM
1447
CB
LEU
A
212
−22.492
20.727
11.017
1.00
73.79
C


ATOM
1448
CG
LEU
A
212
−22.759
20.906
12.537
1.00
77.60
C


ATOM
1449
CD1
LEU
A
212
−24.102
21.571
12.759
1.00
79.25
C


ATOM
1450
CD2
LEU
A
212
−22.691
19.558
13.306
1.00
70.72
C


ATOM
1451
N
VAL
A
213
−19.826
22.183
10.711
1.00
76.14
N


ATOM
1452
CA
VAL
A
213
−18.636
22.808
11.311
1.00
76.70
C


ATOM
1453
C
VAL
A
213
−18.372
24.190
10.713
1.00
77.08
C


ATOM
1454
O
VAL
A
213
−18.039
25.132
11.437
1.00
76.69
O


ATOM
1455
CB
VAL
A
213
−17.380
21.915
11.146
1.00
79.00
C


ATOM
1456
CG1
VAL
A
213
−16.111
22.656
11.562
1.00
78.68
C


ATOM
1457
CG2
VAL
A
213
−17.534
20.627
11.955
1.00
77.70
C


ATOM
1458
N
ILE
A
214
−18.528
24.295
9.396
1.00
74.65
N


ATOM
1459
CA
ILE
A
214
−18.372
25.561
8.690
1.00
74.83
C


ATOM
1460
C
ILE
A
214
−19.423
26.574
9.162
1.00
72.99
C


ATOM
1461
O
ILE
A
214
−19.075
27.692
9.502
1.00
72.81
O


ATOM
1462
CB
ILE
A
214
−18.480
25.384
7.144
1.00
73.17
C


ATOM
1463
CG1
ILE
A
214
−17.318
24.547
6.603
1.00
76.80
C


ATOM
1464
CG2
ILE
A
214
−18.501
26.748
6.450
1.00
71.35
C


ATOM
1465
CD1
ILE
A
214
−17.443
24.202
5.126
1.00
72.96
C


ATOM
1466
N
MET
A
215
−20.692
26.159
9.171
1.00
73.55
N


ATOM
1467
CA
MET
A
215
−21.824
26.990
9.611
1.00
75.44
C


ATOM
1468
C
MET
A
215
−21.589
27.580
10.979
1.00
77.02
C


ATOM
1469
O
MET
A
215
−21.690
28.787
11.150
1.00
79.77
O


ATOM
1470
CB
MET
A
215
−23.099
26.153
9.675
1.00
74.74
C


ATOM
1471
CG
MET
A
215
−24.374
26.874
10.124
1.00
73.35
C


ATOM
1472
SD
MET
A
215
−25.653
25.672
10.545
1.00
82.79
S


ATOM
1473
CE
MET
A
215
−25.102
25.185
12.188
1.00
78.98
C


ATOM
1474
N
VAL
A
216
−21.281
26.709
11.938
1.00
73.57
N


ATOM
1475
CA
VAL
A
216
−21.029
27.111
13.319
1.00
77.98
C


ATOM
1476
C
VAL
A
216
−19.847
28.077
13.424
1.00
78.25
C


ATOM
1477
O
VAL
A
216
−19.960
29.124
14.061
1.00
82.48
O


ATOM
1478
CB
VAL
A
216
−20.760
25.888
14.249
1.00
74.95
C


ATOM
1479
CG1
VAL
A
216
−20.358
26.350
15.613
1.00
73.89
C


ATOM
1480
CG2
VAL
A
216
−21.995
24.992
14.334
1.00
72.42
C


ATOM
1481
N
PHE
A
217
−18.724
27.724
12.802
1.00
78.36
N


ATOM
1482
CA
PHE
A
217
−17.559
28.595
12.820
1.00
77.83
C


ATOM
1483
C
PHE
A
217
−17.880
29.962
12.227
1.00
74.98
C


ATOM
1484
O
PHE
A
217
−17.661
30.981
12.862
1.00
74.40
O


ATOM
1485
CB
PHE
A
217
−16.394
27.992
12.054
1.00
80.26
C


ATOM
1486
CG
PHE
A
217
−15.236
28.930
11.918
1.00
83.15
C


ATOM
1487
CD1
PHE
A
217
−14.381
29.152
12.994
1.00
87.33
C


ATOM
1488
CD2
PHE
A
217
−14.993
29.600
10.717
1.00
87.93
C


ATOM
1489
CE1
PHE
A
217
−13.296
30.029
12.881
1.00
85.31
C


ATOM
1490
CE2
PHE
A
217
−13.912
30.478
10.592
1.00
87.26
C


ATOM
1491
CZ
PHE
A
217
−13.063
30.692
11.677
1.00
86.57
C


ATOM
1492
N
VAL
A
218
−18.400
29.958
11.009
1.00
72.95
N


ATOM
1493
CA
VAL
A
218
−18.693
31.202
10.299
1.00
72.35
C


ATOM
1494
C
VAL
A
218
−19.739
32.039
11.007
1.00
70.18
C


ATOM
1495
O
VAL
A
218
−19.547
33.238
11.123
1.00
72.08
O


ATOM
1496
CB
VAL
A
218
−19.126
30.965
8.828
1.00
69.97
C


ATOM
1497
CG1
VAL
A
218
−19.608
32.264
8.189
1.00
63.51
C


ATOM
1498
CG2
VAL
A
218
−17.973
30.393
8.039
1.00
69.43
C


ATOM
1499
N
TYR
A
219
−20.822
31.428
11.479
1.00
69.91
N


ATOM
1500
CA
TYR
A
219
−21.867
32.204
12.154
1.00
70.79
C


ATOM
1501
C
TYR
A
219
−21.482
32.646
13.561
1.00
72.41
C


ATOM
1502
O
TYR
A
219
−22.135
33.532
14.094
1.00
73.85
O


ATOM
1503
CB
TYR
A
219
−23.216
31.482
12.201
1.00
70.68
C


ATOM
1504
CG
TYR
A
219
−24.373
32.468
12.149
1.00
72.35
C


ATOM
1505
CD1
TYR
A
219
−24.698
33.112
10.968
1.00
75.81
C


ATOM
1506
CD2
TYR
A
219
−25.136
32.755
13.263
1.00
77.93
C


ATOM
1507
CE1
TYR
A
219
−25.757
34.025
10.892
1.00
74.63
C


ATOM
1508
CE2
TYR
A
219
−26.203
33.673
13.190
1.00
70.80
C


ATOM
1509
CZ
TYR
A
219
−26.499
34.292
12.013
1.00
79.33
C


ATOM
1510
OH
TYR
A
219
−27.544
35.187
11.958
1.00
75.98
O


ATOM
1511
N
SER
A
220
−20.451
32.053
14.170
1.00
71.26
N


ATOM
1512
CA
SER
A
220
−19.982
32.579
15.456
1.00
72.46
C


ATOM
1513
C
SER
A
220
−19.265
33.886
15.124
1.00
73.33
C


ATOM
1514
O
SER
A
220
−19.384
34.833
15.876
1.00
71.17
O


ATOM
1515
CB
SER
A
220
−19.110
31.593
16.240
1.00
71.03
C


ATOM
1516
OG
SER
A
220
−17.988
31.169
15.514
1.00
78.76
O


ATOM
1517
N
ARG
A
221
−18.539
33.924
13.990
1.00
71.00
N


ATOM
1518
CA
ARG
A
221
−17.878
35.164
13.507
1.00
71.92
C


ATOM
1519
C
ARG
A
221
−18.847
36.268
13.102
1.00
71.03
C


ATOM
1520
O
ARG
A
221
−18.485
37.440
13.144
1.00
75.74
O


ATOM
1521
CB
ARG
A
221
−16.940
34.887
12.319
1.00
73.28
C


ATOM
1522
CG
ARG
A
221
−15.789
33.967
12.607
1.00
81.79
C


ATOM
1523
CD
ARG
A
221
−14.737
34.643
13.460
1.00
91.32
C


ATOM
1524
NE
ARG
A
221
−13.695
33.711
13.900
1.00
99.40
N


ATOM
1525
CZ
ARG
A
221
−12.642
34.037
14.669
1.00
104.74
C


ATOM
1526
NH1
ARG
A
221
−12.463
35.296
15.107
1.00
108.58
N


ATOM
1527
NH2
ARG
A
221
−11.754
33.095
15.009
1.00
102.27
N


ATOM
1528
N
VAL
A
222
−20.065
35.898
12.705
1.00
72.12
N


ATOM
1529
CA
VAL
A
222
−21.114
36.862
12.390
1.00
70.08
C


ATOM
1530
C
VAL
A
222
−21.544
37.555
13.680
1.00
72.52
C


ATOM
1531
O
VAL
A
222
−21.666
38.787
13.704
1.00
74.66
O


ATOM
1532
CB
VAL
A
222
−22.314
36.199
11.688
1.00
69.66
C


ATOM
1533
CG1
VAL
A
222
−23.527
37.099
11.713
1.00
70.73
C


ATOM
1534
CG2
VAL
A
222
−21.940
35.844
10.245
1.00
74.61
C


ATOM
1535
N
PHE
A
223
−21.771
36.782
14.752
1.00
72.29
N


ATOM
1536
CA
PHE
A
223
−22.115
37.388
16.054
1.00
71.42
C


ATOM
1537
C
PHE
A
223
−21.020
38.303
16.562
1.00
69.83
C


ATOM
1538
O
PHE
A
223
−21.324
39.411
16.970
1.00
71.03
O


ATOM
1539
CB
PHE
A
223
−22.461
36.334
17.105
1.00
72.71
C


ATOM
1540
CG
PHE
A
223
−23.766
35.640
16.857
1.00
69.91
C


ATOM
1541
CD1
PHE
A
223
−24.927
36.370
16.563
1.00
71.96
C


ATOM
1542
CD2
PHE
A
223
−23.850
34.266
16.920
1.00
73.93
C


ATOM
1543
CE1
PHE
A
223
−26.121
35.741
16.338
1.00
73.93
C


ATOM
1544
CE2
PHE
A
223
−25.055
33.624
16.693
1.00
76.95
C


ATOM
1545
CZ
PHE
A
223
−26.190
34.367
16.404
1.00
78.80
C


ATOM
1546
N
GLN
A
224
−19.766
37.854
16.534
1.00
70.23
N


ATOM
1547
CA
GLN
A
224
−18.632
38.724
16.916
1.00
71.28
C


ATOM
1548
C
GLN
A
224
−18.655
40.034
16.155
1.00
72.34
C


ATOM
1549
O
GLN
A
224
−18.462
41.077
16.742
1.00
76.18
O


ATOM
1550
CB
GLN
A
224
−17.293
38.077
16.639
1.00
71.04
C


ATOM
1551
CG
GLN
A
224
−17.007
36.807
17.433
1.00
82.15
C


ATOM
1552
CD
GLN
A
224
−15.565
36.308
17.276
1.00
85.53
C


ATOM
1553
OE1
GLN
A
224
−14.706
36.986
16.673
1.00
85.57
O


ATOM
1554
NE2
GLN
A
224
−15.290
35.107
17.824
1.00
87.83
N


ATOM
1555
N
GLU
A
225
−18.892
39.969
14.839
1.00
74.23
N


ATOM
1556
CA
GLU
A
225
−18.986
41.187
14.026
1.00
73.90
C


ATOM
1557
C
GLU
A
225
−20.161
42.043
14.442
1.00
72.00
C


ATOM
1558
O
GLU
A
225
−19.987
43.221
14.670
1.00
69.39
O


ATOM
1559
CB
GLU
A
225
−19.073
40.880
12.525
1.00
75.78
C


ATOM
1560
CG
GLU
A
225
−17.713
40.806
11.881
1.00
85.48
C


ATOM
1561
CD
GLU
A
225
−16.990
42.134
11.934
1.00
92.43
C


ATOM
1562
OE1
GLU
A
225
−17.670
43.174
11.759
1.00
86.05
O


ATOM
1563
OE2
GLU
A
225
−15.755
42.133
12.152
1.00
98.53
O


ATOM
1564
N
ALA
A
226
−21.336
41.439
14.537
1.00
70.43
N


ATOM
1565
CA
ALA
A
226
−22.521
42.145
14.988
1.00
71.14
C


ATOM
1566
C
ALA
A
226
−22.238
42.861
16.326
1.00
70.13
C


ATOM
1567
O
ALA
A
226
−22.513
44.040
16.455
1.00
69.11
O


ATOM
1568
CB
ALA
A
226
−23.683
41.195
15.114
1.00
69.59
C


ATOM
1569
N
LYS
A
227
−21.686
42.139
17.298
1.00
70.92
N


ATOM
1570
CA
LYS
A
227
−21.345
42.716
18.619
1.00
72.35
C


ATOM
1571
C
LYS
A
227
−20.374
43.880
18.510
1.00
70.68
C


ATOM
1572
O
LYS
A
227
−20.498
44.860
19.238
1.00
71.56
O


ATOM
1573
CB
LYS
A
227
−20.728
41.660
19.540
1.00
72.29
C


ATOM
1574
CG
LYS
A
227
−20.673
42.077
21.027
1.00
77.11
C


ATOM
1575
CD
LYS
A
227
−20.272
40.913
21.948
1.00
79.33
C


ATOM
1576
CE
LYS
A
227
−20.366
41.295
23.457
1.00
89.68
C


ATOM
1577
NZ
LYS
A
227
−19.211
42.084
23.975
1.00
91.63
N


ATOM
1578
N
ARG
A
228
−19.419
43.749
17.591
1.00
71.06
N


ATOM
1579
CA
ARG
A
228
−18.380
44.749
17.349
1.00
71.67
C


ATOM
1580
C
ARG
A
228
−18.909
46.028
16.696
1.00
66.45
C


ATOM
1581
O
ARG
A
228
−18.234
47.031
16.714
1.00
70.70
O


ATOM
1582
CB
ARG
A
228
−17.271
44.142
16.486
1.00
73.73
C


ATOM
1583
CG
ARG
A
228
−15.959
44.946
16.395
1.00
80.33
C


ATOM
1584
CD
ARG
A
228
−15.060
44.487
15.239
1.00
85.20
C


ATOM
1585
NE
ARG
A
228
−15.691
44.699
13.931
1.00
95.73
N


ATOM
1586
CZ
ARG
A
228
−15.736
45.853
13.239
1.00
99.68
C


ATOM
1587
NH1
ARG
A
228
−15.178
46.979
13.702
1.00
98.15
N


ATOM
1588
NH2
ARG
A
228
−16.353
45.889
12.052
1.00
97.65
N


ATOM
1589
N
GLN
A
229
−20.105
46.002
16.127
1.00
67.44
N


ATOM
1590
CA
GLN
A
229
−20.718
47.217
15.562
1.00
69.07
C


ATOM
1591
C
GLN
A
229
−21.324
48.107
16.630
1.00
68.99
C


ATOM
1592
O
GLN
A
229
−21.550
49.299
16.388
1.00
73.07
O


ATOM
1593
CB
GLN
A
229
−21.817
46.858
14.603
1.00
67.27
C


ATOM
1594
CG
GLN
A
229
−21.328
46.085
13.403
1.00
79.11
C


ATOM
1595
CD
GLN
A
229
−22.412
45.836
12.358
1.00
81.84
C


ATOM
1596
OE1
GLN
A
229
−22.097
45.437
11.233
1.00
98.22
O


ATOM
1597
NE2
GLN
A
229
−23.703
46.069
12.724
1.00
80.39
N


ATOM
1598
N
LEU
A
230
−21.594
47.546
17.804
1.00
65.76
N


ATOM
1599
CA
LEU
A
230
−22.180
48.318
18.887
1.00
66.37
C


ATOM
1600
C
LEU
A
230
−21.228
49.424
19.268
1.00
62.49
C


ATOM
1601
O
LEU
A
230
−20.068
49.184
19.517
1.00
63.24
O


ATOM
1602
CB
LEU
A
230
−22.492
47.429
20.078
1.00
63.29
C


ATOM
1603
CG
LEU
A
230
−23.526
46.346
19.805
1.00
63.55
C


ATOM
1604
CD1
LEU
A
230
−23.659
45.401
21.017
1.00
56.14
C


ATOM
1605
CD2
LEU
A
230
−24.867
46.998
19.398
1.00
63.01
C


ATOM
1606
N
ASN
A
1002
−21.745
50.645
19.298
1.00
65.96
N


ATOM
1607
CA
ASN
A
1002
−20.959
51.846
19.632
1.00
68.64
C


ATOM
1608
C
ASN
A
1002
−21.911
52.946
20.059
1.00
68.56
C


ATOM
1609
O
ASN
A
1002
−23.125
52.706
20.134
1.00
70.46
O


ATOM
1610
CB
ASN
A
1002
−20.114
52.260
18.442
1.00
68.39
C


ATOM
1611
CG
ASN
A
1002
−20.935
52.517
17.224
1.00
74.98
C


ATOM
1612
OD1
ASN
A
1002
−22.100
52.934
17.313
1.00
65.74
O


ATOM
1613
ND2
ASN
A
1002
−20.342
52.273
16.058
1.00
67.18
N


ATOM
1614
N
ILE
A
1003
−21.390
54.139
20.330
1.00
69.49
N


ATOM
1615
CA
ILE
A
1003
−22.224
55.203
20.901
1.00
70.87
C


ATOM
1616
C
ILE
A
1003
−23.404
55.572
20.013
1.00
70.78
C


ATOM
1617
O
ILE
A
1003
−24.472
55.886
20.521
1.00
74.04
O


ATOM
1618
CB
ILE
A
1003
−21.362
56.453
21.310
1.00
74.80
C


ATOM
1619
CG1
ILE
A
1003
−22.172
57.390
22.229
1.00
76.63
C


ATOM
1620
CG2
ILE
A
1003
−20.788
57.153
20.075
1.00
68.47
C


ATOM
1621
CD1
ILE
A
1003
−21.370
58.560
22.737
1.00
73.42
C


ATOM
1622
N
PHE
A
1004
−23.204
55.527
18.695
1.00
73.63
N


ATOM
1623
CA
PHE
A
1004
−24.261
55.801
17.719
1.00
72.72
C


ATOM
1624
C
PHE
A
1004
−25.409
54.841
17.835
1.00
73.43
C


ATOM
1625
O
PHE
A
1004
−26.574
55.255
17.872
1.00
71.77
O


ATOM
1626
CB
PHE
A
1004
−23.679
55.727
16.309
1.00
75.62
C


ATOM
1627
CG
PHE
A
1004
−24.689
55.716
15.236
1.00
71.84
C


ATOM
1628
CD1
PHE
A
1004
−25.453
56.848
14.979
1.00
82.28
C


ATOM
1629
CD2
PHE
A
1004
−24.892
54.570
14.452
1.00
82.90
C


ATOM
1630
CE1
PHE
A
1004
−26.423
56.843
13.943
1.00
77.44
C


ATOM
1631
CE2
PHE
A
1004
−25.865
54.564
13.410
1.00
71.41
C


ATOM
1632
CZ
PHE
A
1004
−26.620
55.697
13.166
1.00
70.16
C


ATOM
1633
N
GLU
A
1005
−25.074
53.559
17.889
1.00
72.98
N


ATOM
1634
CA
GLU
A
1005
−26.063
52.517
18.064
1.00
72.41
C


ATOM
1635
C
GLU
A
1005
−26.733
52.619
19.416
1.00
74.97
C


ATOM
1636
O
GLU
A
1005
−27.949
52.434
19.517
1.00
74.19
O


ATOM
1637
CB
GLU
A
1005
−25.418
51.130
17.931
1.00
72.97
C


ATOM
1638
CG
GLU
A
1005
−24.759
50.844
16.561
1.00
75.62
C


ATOM
1639
CD
GLU
A
1005
−25.713
51.008
15.396
1.00
84.27
C


ATOM
1640
OE1
GLU
A
1005
−26.925
50.899
15.640
1.00
91.81
O


ATOM
1641
OE2
GLU
A
1005
−25.261
51.243
14.247
1.00
80.28
O


ATOM
1642
N
MET
A
1006
−25.939
52.913
20.453
1.00
71.72
N


ATOM
1643
CA
MET
A
1006
−26.467
53.101
21.783
1.00
72.95
C


ATOM
1644
C
MET
A
1006
−27.562
54.182
21.796
1.00
73.35
C


ATOM
1645
O
MET
A
1006
−28.639
53.963
22.356
1.00
73.69
O


ATOM
1646
CB
MET
A
1006
−25.315
53.438
22.773
1.00
73.49
C


ATOM
1647
CG
MET
A
1006
−25.769
53.933
24.131
1.00
78.05
C


ATOM
1648
SD
MET
A
1006
−24.447
54.468
25.254
1.00
74.91
S


ATOM
1649
CE
MET
A
1006
−24.293
53.083
26.396
1.00
86.03
C


ATOM
1650
N
LEU
A
1007
−27.286
55.335
21.179
1.00
72.95
N


ATOM
1651
CA
LEU
A
1007
−28.235
56.464
21.209
1.00
73.25
C


ATOM
1652
C
LEU
A
1007
−29.347
56.268
20.193
1.00
74.44
C


ATOM
1653
O
LEU
A
1007
−30.441
56.795
20.352
1.00
75.83
O


ATOM
1654
CB
LEU
A
1007
−27.539
57.814
21.003
1.00
71.83
C


ATOM
1655
CG
LEU
A
1007
−26.953
58.593
22.197
1.00
76.11
C


ATOM
1656
CD1
LEU
A
1007
−28.031
58.916
23.248
1.00
80.06
C


ATOM
1657
CD2
LEU
A
1007
−25.786
57.886
22.829
1.00
73.36
C


ATOM
1658
N
ARG
A
1008
−29.069
55.514
19.149
1.00
77.47
N


ATOM
1659
CA
ARG
A
1008
−30.095
55.137
18.183
1.00
77.36
C


ATOM
1660
C
ARG
A
1008
−31.138
54.231
18.865
1.00
76.88
C


ATOM
1661
O
ARG
A
1008
−32.311
54.329
18.575
1.00
75.51
O


ATOM
1662
CB
ARG
A
1008
−29.437
54.430
17.040
1.00
72.89
C


ATOM
1663
CG
ARG
A
1008
−30.325
54.077
15.936
1.00
83.81
C


ATOM
1664
CD
ARG
A
1008
−29.530
53.460
14.840
1.00
80.34
C


ATOM
1665
NE
ARG
A
1008
−30.368
52.534
14.093
1.00
81.78
N


ATOM
1666
CZ
ARG
A
1008
−29.944
51.431
13.485
1.00
85.98
C


ATOM
1667
NH1
ARG
A
1008
−28.660
51.081
13.524
1.00
80.09
N


ATOM
1668
NH2
ARG
A
1008
−30.823
50.660
12.823
1.00
93.61
N


ATOM
1669
N
ILE
A
1009
−30.678
53.355
19.773
1.00
78.10
N


ATOM
1670
CA
ILE
A
1009
−31.548
52.465
20.543
1.00
75.58
C


ATOM
1671
C
ILE
A
1009
−32.251
53.251
21.658
1.00
78.69
C


ATOM
1672
O
ILE
A
1009
−33.455
53.134
21.826
1.00
81.42
O


ATOM
1673
CB
ILE
A
1009
−30.736
51.274
21.155
1.00
78.60
C


ATOM
1674
CG1
ILE
A
1009
−30.220
50.329
20.057
1.00
70.72
C


ATOM
1675
CG2
ILE
A
1009
−31.568
50.493
22.202
1.00
73.84
C


ATOM
1676
CD1
ILE
A
1009
−28.924
49.579
20.447
1.00
68.79
C


ATOM
1677
N
ASP
A
1010
−31.492
54.049
22.412
1.00
81.55
N


ATOM
1678
CA
ASP
A
1010
−32.048
54.813
23.555
1.00
80.99
C


ATOM
1679
C
ASP
A
1010
−32.892
56.045
23.199
1.00
82.41
C


ATOM
1680
O
ASP
A
1010
−33.892
56.302
23.876
1.00
83.53
O


ATOM
1681
CB
ASP
A
1010
−30.918
55.254
24.486
1.00
79.05
C


ATOM
1682
CG
ASP
A
1010
−30.273
54.103
25.216
1.00
81.30
C


ATOM
1683
OD1
ASP
A
1010
−30.918
53.076
25.354
1.00
79.79
O


ATOM
1684
OD2
ASP
A
1010
−29.110
54.217
25.660
1.00
68.53
O


ATOM
1685
N
GLU
A
1011
−32.499
56.790
22.163
1.00
81.15
N


ATOM
1686
CA
GLU
A
1011
−33.206
58.046
21.749
1.00
82.09
C


ATOM
1687
C
GLU
A
1011
−34.075
57.898
20.499
1.00
82.84
C


ATOM
1688
O
GLU
A
1011
−35.124
58.541
20.397
1.00
86.08
O


ATOM
1689
CB
GLU
A
1011
−32.189
59.197
21.548
1.00
85.07
C


ATOM
1690
CG
GLU
A
1011
−32.748
60.535
20.987
1.00
86.91
C


ATOM
1691
CD
GLU
A
1011
−33.834
61.166
21.857
1.00
92.06
C


ATOM
1692
OE1
GLU
A
1011
−33.791
60.980
23.098
1.00
92.05
O


ATOM
1693
OE2
GLU
A
1011
−34.736
61.855
21.291
1.00
82.39
O


ATOM
1694
N
GLY
A
1012
−33.648
57.067
19.550
1.00
77.24
N


ATOM
1695
CA
GLY
A
1012
−34.407
56.839
18.336
1.00
75.19
C


ATOM
1696
C
GLY
A
1012
−33.925
57.768
17.246
1.00
74.64
C


ATOM
1697
O
GLY
A
1012
−33.635
58.934
17.494
1.00
71.63
O


ATOM
1698
N
LEU
A
1013
−33.841
57.245
16.032
1.00
74.33
N


ATOM
1699
CA
LEU
A
1013
−33.402
58.024
14.892
1.00
73.75
C


ATOM
1700
C
LEU
A
1013
−34.653
58.394
14.103
1.00
73.66
C


ATOM
1701
O
LEU
A
1013
−35.397
57.511
13.670
1.00
72.82
O


ATOM
1702
CB
LEU
A
1013
−32.419
57.214
14.060
1.00
73.46
C


ATOM
1703
CG
LEU
A
1013
−31.520
58.014
13.118
1.00
77.16
C


ATOM
1704
CD1
LEU
A
1013
−30.400
57.161
12.611
1.00
81.09
C


ATOM
1705
CD2
LEU
A
1013
−32.304
58.573
11.978
1.00
75.18
C


ATOM
1706
N
ARG
A
1014
−34.875
59.696
13.925
1.00
73.47
N


ATOM
1707
CA
ARG
A
1014
−36.005
60.206
13.143
1.00
75.08
C


ATOM
1708
C
ARG
A
1014
−35.492
60.915
11.894
1.00
73.46
C


ATOM
1709
O
ARG
A
1014
−34.665
61.818
11.967
1.00
72.29
O


ATOM
1710
CB
ARG
A
1014
−36.899
61.098
13.994
1.00
73.94
C


ATOM
1711
CG
ARG
A
1014
−37.552
60.286
15.128
1.00
82.18
C


ATOM
1712
CD
ARG
A
1014
−38.783
60.917
15.716
1.00
87.29
C


ATOM
1713
NE
ARG
A
1014
−39.879
61.089
14.746
1.00
93.86
N


ATOM
1714
CZ
ARG
A
1014
−41.129
61.469
15.053
1.00
92.93
C


ATOM
1715
NH1
ARG
A
1014
−41.488
61.729
16.323
1.00
95.92
N


ATOM
1716
NH2
ARG
A
1014
−42.038
61.591
14.080
1.00
92.74
N


ATOM
1717
N
LEU
A
1015
−36.001
60.481
10.746
1.00
73.79
N


ATOM
1718
CA
LEU
A
1015
−35.566
60.969
9.444
1.00
73.57
C


ATOM
1719
C
LEU
A
1015
−36.284
62.239
9.033
1.00
74.92
C


ATOM
1720
O
LEU
A
1015
−35.757
62.988
8.224
1.00
72.14
O


ATOM
1721
CB
LEU
A
1015
−35.781
59.871
8.411
1.00
73.34
C


ATOM
1722
CG
LEU
A
1015
−35.065
58.555
8.749
1.00
71.48
C


ATOM
1723
CD1
LEU
A
1015
−35.240
57.576
7.626
1.00
66.34
C


ATOM
1724
CD2
LEU
A
1015
−33.595
58.783
9.033
1.00
63.66
C


ATOM
1725
N
LYS
A
1016
−37.480
62.472
9.593
1.00
77.95
N


ATOM
1726
CA
LYS
A
1016
−38.274
63.678
9.327
1.00
75.82
C


ATOM
1727
C
LYS
A
1016
−38.338
64.571
10.563
1.00
73.96
C


ATOM
1728
O
LYS
A
1016
−38.379
64.065
11.680
1.00
71.82
O


ATOM
1729
CB
LYS
A
1016
−39.688
63.302
8.908
1.00
75.21
C


ATOM
1730
CG
LYS
A
1016
−39.744
62.451
7.649
1.00
79.49
C


ATOM
1731
CD
LYS
A
1016
−41.159
62.214
7.176
1.00
77.17
C


ATOM
1732
CE
LYS
A
1016
−41.160
61.358
5.900
1.00
81.57
C


ATOM
1733
NZ
LYS
A
1016
−42.521
61.187
5.296
1.00
74.99
N


ATOM
1734
N
ILE
A
1017
−38.347
65.889
10.334
1.00
73.18
N


ATOM
1735
CA
ILE
A
1017
−38.511
66.900
11.372
1.00
72.14
C


ATOM
1736
C
ILE
A
1017
−39.703
66.595
12.265
1.00
73.25
C


ATOM
1737
O
ILE
A
1017
−40.764
66.207
11.774
1.00
74.28
O


ATOM
1738
CB
ILE
A
1017
−38.705
68.320
10.765
1.00
71.53
C


ATOM
1739
CG1
ILE
A
1017
−37.391
68.862
10.210
1.00
74.05
C


ATOM
1740
CG2
ILE
A
1017
−39.204
69.308
11.788
1.00
70.34
C


ATOM
1741
CD1
ILE
A
1017
−37.443
70.318
9.833
1.00
73.51
C


ATOM
1742
N
TYR
A
1018
−39.515
66.773
13.573
1.00
72.91
N


ATOM
1743
CA
TYR
A
1018
−40.571
66.562
14.552
1.00
74.12
C


ATOM
1744
C
TYR
A
1018
−40.391
67.540
15.700
1.00
73.99
C


ATOM
1745
O
TYR
A
1018
−39.304
68.091
15.883
1.00
75.95
O


ATOM
1746
CB
TYR
A
1018
−40.546
65.119
15.070
1.00
75.31
C


ATOM
1747
CG
TYR
A
1018
−39.316
64.769
15.884
1.00
75.01
C


ATOM
1748
CD1
TYR
A
1018
−39.355
64.765
17.283
1.00
77.73
C


ATOM
1749
CD2
TYR
A
1018
−38.113
64.440
15.263
1.00
75.41
C


ATOM
1750
CE1
TYR
A
1018
−38.223
64.442
18.042
1.00
72.04
C


ATOM
1751
CE2
TYR
A
1018
−36.974
64.116
16.016
1.00
77.21
C


ATOM
1752
CZ
TYR
A
1018
−37.042
64.120
17.403
1.00
76.64
C


ATOM
1753
OH
TYR
A
1018
−35.923
63.800
18.142
1.00
79.46
O


ATOM
1754
N
LYS
A
1019
−41.461
67.746
16.459
1.00
73.71
N


ATOM
1755
CA
LYS
A
1019
−41.427
68.597
17.636
1.00
74.41
C


ATOM
1756
C
LYS
A
1019
−41.056
67.729
18.826
1.00
75.37
C


ATOM
1757
O
LYS
A
1019
−41.715
66.718
19.073
1.00
77.13
O


ATOM
1758
CB
LYS
A
1019
−42.773
69.262
17.885
1.00
73.28
C


ATOM
1759
CG
LYS
A
1019
−43.104
70.352
16.899
1.00
72.01
C


ATOM
1760
CD
LYS
A
1019
−44.357
71.099
17.326
1.00
72.42
C


ATOM
1761
CE
LYS
A
1019
−44.649
72.280
16.414
1.00
73.72
C


ATOM
1762
NZ
LYS
A
1019
−45.910
72.993
16.796
1.00
71.74
N


ATOM
1763
N
ASP
A
1020
−40.012
68.112
19.561
1.00
76.71
N


ATOM
1764
CA
ASP
A
1020
−39.610
67.388
20.776
1.00
78.54
C


ATOM
1765
C
ASP
A
1020
−40.629
67.615
21.913
1.00
78.53
C


ATOM
1766
O
ASP
A
1020
−41.693
68.205
21.698
1.00
79.88
O


ATOM
1767
CB
ASP
A
1020
−38.174
67.778
21.198
1.00
79.44
C


ATOM
1768
CG
ASP
A
1020
−38.071
69.183
21.838
1.00
81.53
C


ATOM
1769
OD1
ASP
A
1020
−39.087
69.873
22.069
1.00
77.18
O


ATOM
1770
OD2
ASP
A
1020
−36.929
69.600
22.116
1.00
89.87
O


ATOM
1771
N
THR
A
1021
−40.297
67.147
23.112
1.00
81.31
N


ATOM
1772
CA
THR
A
1021
−41.163
67.303
24.299
1.00
81.40
C


ATOM
1773
C
THR
A
1021
−41.464
68.771
24.674
1.00
81.89
C


ATOM
1774
O
THR
A
1021
−42.562
69.085
25.152
1.00
83.48
O


ATOM
1775
CB
THR
A
1021
−40.532
66.624
25.514
1.00
83.23
C


ATOM
1776
OG1
THR
A
1021
−39.212
67.149
25.716
1.00
86.46
O


ATOM
1777
CG2
THR
A
1021
−40.454
65.100
25.305
1.00
83.63
C


ATOM
1778
N
GLU
A
1022
−40.488
69.653
24.447
1.00
81.25
N


ATOM
1779
CA
GLU
A
1022
−40.617
71.091
24.720
1.00
80.42
C


ATOM
1780
C
GLU
A
1022
−41.256
71.863
23.534
1.00
79.28
C


ATOM
1781
O
GLU
A
1022
−41.339
73.093
23.576
1.00
79.62
O


ATOM
1782
CB
GLU
A
1022
−39.243
71.710
25.032
1.00
81.04
C


ATOM
1783
CG
GLU
A
1022
−38.301
70.940
26.011
1.00
82.43
C


ATOM
1784
CD
GLU
A
1022
−38.788
70.891
27.448
1.00
92.01
C


ATOM
1785
OE1
GLU
A
1022
−39.749
71.611
27.806
1.00
94.63
O


ATOM
1786
OE2
GLU
A
1022
−38.192
70.117
28.231
1.00
97.00
O


ATOM
1787
N
GLY
A
1023
−41.696
71.146
22.491
1.00
79.51
N


ATOM
1788
CA
GLY
A
1023
−42.310
71.743
21.302
1.00
78.42
C


ATOM
1789
C
GLY
A
1023
−41.324
72.232
20.244
1.00
78.89
C


ATOM
1790
O
GLY
A
1023
−41.750
72.842
19.255
1.00
79.69
O


ATOM
1791
N
TYR
A
1024
−40.021
71.971
20.436
1.00
77.49
N


ATOM
1792
CA
TYR
A
1024
−38.981
72.476
19.532
1.00
76.94
C


ATOM
1793
C
TYR
A
1024
−38.694
71.548
18.363
1.00
77.71
C


ATOM
1794
O
TYR
A
1024
−38.686
70.321
18.524
1.00
79.61
O


ATOM
1795
CB
TYR
A
1024
−37.663
72.699
20.269
1.00
77.27
C


ATOM
1796
CG
TYR
A
1024
−37.711
73.692
21.400
1.00
79.68
C


ATOM
1797
CD1
TYR
A
1024
−38.145
74.987
21.192
1.00
81.27
C


ATOM
1798
CD2
TYR
A
1024
−37.313
73.337
22.684
1.00
79.56
C


ATOM
1799
CE1
TYR
A
1024
−38.189
75.913
22.232
1.00
83.68
C


ATOM
1800
CE2
TYR
A
1024
−37.353
74.253
23.736
1.00
78.83
C


ATOM
1801
CZ
TYR
A
1024
−37.792
75.541
23.503
1.00
80.59
C


ATOM
1802
OH
TYR
A
1024
−37.836
76.457
24.534
1.00
83.58
O


ATOM
1803
N
TYR
A
1025
−38.452
72.145
17.191
1.00
74.53
N


ATOM
1804
CA
TYR
A
1025
−38.176
71.387
15.969
1.00
73.53
C


ATOM
1805
C
TYR
A
1025
−36.849
70.657
16.050
1.00
68.58
C


ATOM
1806
O
TYR
A
1025
−35.801
71.285
16.188
1.00
69.06
O


ATOM
1807
CB
TYR
A
1025
−38.195
72.295
14.736
1.00
71.74
C


ATOM
1808
CG
TYR
A
1025
−39.564
72.806
14.416
1.00
70.34
C


ATOM
1809
CD1
TYR
A
1025
−39.924
74.104
14.717
1.00
71.87
C


ATOM
1810
CD2
TYR
A
1025
−40.510
71.983
13.810
1.00
71.28
C


ATOM
1811
CE1
TYR
A
1025
−41.186
74.577
14.423
1.00
77.57
C


ATOM
1812
CE2
TYR
A
1025
−41.783
72.445
13.509
1.00
70.46
C


ATOM
1813
CZ
TYR
A
1025
−42.118
73.747
13.819
1.00
72.50
C


ATOM
1814
OH
TYR
A
1025
−43.366
74.222
13.531
1.00
73.02
O


ATOM
1815
N
THR
A
1026
−36.923
69.330
15.960
1.00
68.83
N


ATOM
1816
CA
THR
A
1026
−35.783
68.431
16.129
1.00
69.83
C


ATOM
1817
C
THR
A
1026
−35.730
67.408
15.000
1.00
71.01
C


ATOM
1818
O
THR
A
1026
−36.752
67.153
14.338
1.00
75.69
O


ATOM
1819
CB
THR
A
1026
−35.922
67.741
17.500
1.00
68.73
C


ATOM
1820
OG1
THR
A
1026
−36.033
68.752
18.502
1.00
71.36
O


ATOM
1821
CG2
THR
A
1026
−34.750
66.824
17.843
1.00
65.86
C


ATOM
1822
N
ILE
A
1027
−34.552
66.825
14.769
1.00
69.80
N


ATOM
1823
CA
ILE
A
1027
−34.397
65.788
13.737
1.00
72.31
C


ATOM
1824
C
ILE
A
1027
−33.259
64.836
14.091
1.00
73.35
C


ATOM
1825
O
ILE
A
1027
−32.339
65.215
14.794
1.00
74.44
O


ATOM
1826
CB
ILE
A
1027
−34.153
66.428
12.350
1.00
71.44
C


ATOM
1827
CG1
ILE
A
1027
−34.482
65.443
11.224
1.00
73.60
C


ATOM
1828
CG2
ILE
A
1027
−32.713
66.934
12.235
1.00
69.68
C


ATOM
1829
CD1
ILE
A
1027
−34.540
66.100
9.855
1.00
69.11
C


ATOM
1830
N
GLY
A
1028
−33.331
63.596
13.601
1.00
72.50
N


ATOM
1831
CA
GLY
A
1028
−32.287
62.599
13.863
1.00
71.73
C


ATOM
1832
C
GLY
A
1028
−32.366
62.107
15.292
1.00
71.31
C


ATOM
1833
O
GLY
A
1028
−33.455
61.891
15.807
1.00
70.02
O


ATOM
1834
N
ILE
A
1029
−31.202
61.935
15.918
1.00
73.01
N


ATOM
1835
CA
ILE
A
1029
−31.090
61.460
17.295
1.00
72.52
C


ATOM
1836
C
ILE
A
1029
−31.167
62.687
18.232
1.00
74.59
C


ATOM
1837
O
ILE
A
1029
−30.158
63.152
18.782
1.00
77.27
O


ATOM
1838
CB
ILE
A
1029
−29.786
60.637
17.526
1.00
72.24
C


ATOM
1839
CG1
ILE
A
1029
−29.696
59.446
16.569
1.00
71.48
C


ATOM
1840
CG2
ILE
A
1029
−29.716
60.140
18.998
1.00
70.08
C


ATOM
1841
CD1
ILE
A
1029
−28.290
58.898
16.365
1.00
72.58
C


ATOM
1842
N
GLY
A
1030
−32.378
63.195
18.397
1.00
72.49
N


ATOM
1843
CA
GLY
A
1030
−32.622
64.375
19.197
1.00
72.04
C


ATOM
1844
C
GLY
A
1030
−31.760
65.582
18.861
1.00
73.16
C


ATOM
1845
O
GLY
A
1030
−31.349
66.291
19.755
1.00
72.16
O


ATOM
1846
N
HIS
A
1031
−31.480
65.821
17.574
1.00
75.22
N


ATOM
1847
CA
HIS
A
1031
−30.705
67.007
17.192
1.00
74.36
C


ATOM
1848
C
HIS
A
1031
−31.668
68.175
17.134
1.00
75.22
C


ATOM
1849
O
HIS
A
1031
−32.614
68.137
16.371
1.00
78.88
O


ATOM
1850
CB
HIS
A
1031
−29.985
66.864
15.840
1.00
76.09
C


ATOM
1851
CG
HIS
A
1031
−29.190
68.068
15.470
1.00
74.73
C


ATOM
1852
ND1
HIS
A
1031
−27.828
68.140
15.644
1.00
73.37
N


ATOM
1853
CD2
HIS
A
1031
−29.568
69.255
14.941
1.00
70.52
C


ATOM
1854
CE1
HIS
A
1031
−27.397
69.320
15.234
1.00
72.05
C


ATOM
1855
NE2
HIS
A
1031
−28.434
70.016
14.805
1.00
73.60
N


ATOM
1856
N
LEU
A
1032
−31.420
69.209
17.937
1.00
74.86
N


ATOM
1857
CA
LEU
A
1032
−32.271
70.400
17.966
1.00
75.57
C


ATOM
1858
C
LEU
A
1032
−31.871
71.334
16.840
1.00
75.37
C


ATOM
1859
O
LEU
A
1032
−30.681
71.654
16.690
1.00
76.52
O


ATOM
1860
CB
LEU
A
1032
−32.148
71.137
19.305
1.00
74.42
C


ATOM
1861
CG
LEU
A
1032
−33.092
72.326
19.545
1.00
76.35
C


ATOM
1862
CD1
LEU
A
1032
−34.490
71.821
19.722
1.00
81.83
C


ATOM
1863
CD2
LEU
A
1032
−32.682
73.124
20.760
1.00
73.47
C


ATOM
1864
N
LEU
A
1033
−32.857
71.770
16.057
1.00
74.07
N


ATOM
1865
CA
LEU
A
1033
−32.610
72.675
14.928
1.00
73.31
C


ATOM
1866
C
LEU
A
1033
−32.684
74.147
15.331
1.00
72.89
C


ATOM
1867
O
LEU
A
1033
−31.789
74.937
15.003
1.00
70.82
O


ATOM
1868
CB
LEU
A
1033
−33.595
72.374
13.787
1.00
73.71
C


ATOM
1869
CG
LEU
A
1033
−33.380
71.032
13.099
1.00
68.93
C


ATOM
1870
CD1
LEU
A
1033
−34.527
70.727
12.146
1.00
71.95
C


ATOM
1871
CD2
LEU
A
1033
−32.023
71.025
12.372
1.00
66.34
C


ATOM
1872
N
THR
A
1034
−33.751
74.502
16.039
1.00
73.89
N


ATOM
1873
CA
THR
A
1034
−33.986
75.873
16.494
1.00
71.83
C


ATOM
1874
C
THR
A
1034
−35.070
75.849
17.538
1.00
73.47
C


ATOM
1875
O
THR
A
1034
−35.849
74.896
17.591
1.00
74.91
O


ATOM
1876
CB
THR
A
1034
−34.439
76.792
15.325
1.00
71.67
C


ATOM
1877
OG1
THR
A
1034
−34.731
78.108
15.807
1.00
67.14
O


ATOM
1878
CG2
THR
A
1034
−35.685
76.231
14.634
1.00
67.88
C


ATOM
1879
N
LYS
A
1035
−35.120
76.892
18.365
1.00
73.16
N


ATOM
1880
CA
LYS
A
1035
−36.190
77.037
19.352
1.00
74.56
C


ATOM
1881
C
LYS
A
1035
−37.402
77.824
18.786
1.00
74.05
C


ATOM
1882
O
LYS
A
1035
−38.435
77.955
19.456
1.00
73.29
O


ATOM
1883
CB
LYS
A
1035
−35.669
77.666
20.656
1.00
75.36
C


ATOM
1884
CG
LYS
A
1035
−34.711
76.751
21.441
1.00
74.77
C


ATOM
1885
CD
LYS
A
1035
−34.455
77.236
22.866
1.00
77.28
C


ATOM
1886
CE
LYS
A
1035
−33.836
78.641
22.924
1.00
82.03
C


ATOM
1887
NZ
LYS
A
1035
−33.484
79.060
24.331
1.00
81.98
N


ATOM
1888
N
SER
A
1036
−37.256
78.330
17.559
1.00
73.73
N


ATOM
1889
CA
SER
A
1036
−38.296
79.089
16.869
1.00
73.96
C


ATOM
1890
C
SER
A
1036
−39.540
78.248
16.553
1.00
73.14
C


ATOM
1891
O
SER
A
1036
−39.420
77.055
16.331
1.00
75.32
O


ATOM
1892
CB
SER
A
1036
−37.713
79.649
15.561
1.00
74.91
C


ATOM
1893
OG
SER
A
1036
−38.688
80.260
14.743
1.00
72.52
O


ATOM
1894
N
PRO
A
1037
−40.740
78.874
16.532
1.00
73.94
N


ATOM
1895
CA
PRO
A
1037
−41.968
78.169
16.153
1.00
73.36
C


ATOM
1896
C
PRO
A
1037
−42.157
78.061
14.632
1.00
74.22
C


ATOM
1897
O
PRO
A
1037
−43.072
77.365
14.184
1.00
76.00
O


ATOM
1898
CB
PRO
A
1037
−43.054
79.058
16.732
1.00
72.94
C


ATOM
1899
CG
PRO
A
1037
−42.497
80.415
16.588
1.00
73.18
C


ATOM
1900
CD
PRO
A
1037
−41.032
80.284
16.861
1.00
73.91
C


ATOM
1901
N
SER
A
1038
−41.308
78.744
13.855
1.00
73.88
N


ATOM
1902
CA
SER
A
1038
−41.346
78.688
12.406
1.00
73.57
C


ATOM
1903
C
SER
A
1038
−40.709
77.399
11.934
1.00
74.14
C


ATOM
1904
O
SER
A
1038
−39.552
77.122
12.271
1.00
73.16
O


ATOM
1905
CB
SER
A
1038
−40.594
79.872
11.797
1.00
73.20
C


ATOM
1906
OG
SER
A
1038
−40.486
79.739
10.391
1.00
72.62
O


ATOM
1907
N
LEU
A
1039
−41.463
76.617
11.163
1.00
72.03
N


ATOM
1908
CA
LEU
A
1039
−40.961
75.378
10.590
1.00
72.88
C


ATOM
1909
C
LEU
A
1039
−39.905
75.704
9.557
1.00
72.75
C


ATOM
1910
O
LEU
A
1039
−38.836
75.100
9.543
1.00
74.58
O


ATOM
1911
CB
LEU
A
1039
−42.092
74.581
9.944
1.00
72.16
C


ATOM
1912
CG
LEU
A
1039
−41.717
73.267
9.243
1.00
71.92
C


ATOM
1913
CD1
LEU
A
1039
−41.053
72.299
10.198
1.00
68.87
C


ATOM
1914
CD2
LEU
A
1039
−42.951
72.627
8.601
1.00
71.51
C


ATOM
1915
N
ASN
A
1040
−40.218
76.665
8.694
1.00
74.45
N


ATOM
1916
CA
ASN
A
1040
−39.282
77.148
7.665
1.00
74.48
C


ATOM
1917
C
ASN
A
1040
−37.918
77.589
8.246
1.00
74.80
C


ATOM
1918
O
ASN
A
1040
−36.899
77.499
7.561
1.00
76.00
O


ATOM
1919
CB
ASN
A
1040
−39.937
78.277
6.838
1.00
74.13
C


ATOM
1920
CG
ASN
A
1040
−41.135
77.779
5.985
1.00
73.14
C


ATOM
1921
OD1
ASN
A
1040
−41.183
76.619
5.555
1.00
70.14
O


ATOM
1922
ND2
ASN
A
1040
−42.095
78.665
5.748
1.00
66.32
N


ATOM
1923
N
ALA
A
1041
−37.918
78.058
9.500
1.00
74.86
N


ATOM
1924
CA
ALA
A
1041
−36.689
78.402
10.228
1.00
75.02
C


ATOM
1925
C
ALA
A
1041
−35.886
77.139
10.604
1.00
75.73
C


ATOM
1926
O
ALA
A
1041
−34.664
77.155
10.555
1.00
75.98
O


ATOM
1927
CB
ALA
A
1041
−37.011
79.220
11.466
1.00
73.60
C


ATOM
1928
N
ALA
A
1042
−36.583
76.061
10.975
1.00
75.31
N


ATOM
1929
CA
ALA
A
1042
−35.953
74.762
11.236
1.00
74.13
C


ATOM
1930
C
ALA
A
1042
−35.329
74.212
9.956
1.00
75.51
C


ATOM
1931
O
ALA
A
1042
−34.152
73.844
9.946
1.00
75.22
O


ATOM
1932
CB
ALA
A
1042
−36.969
73.782
11.779
1.00
73.73
C


ATOM
1933
N
LYS
A
1043
−36.135
74.164
8.887
1.00
75.74
N


ATOM
1934
CA
LYS
A
1043
−35.689
73.728
7.547
1.00
76.54
C


ATOM
1935
C
LYS
A
1043
−34.445
74.481
7.067
1.00
76.74
C


ATOM
1936
O
LYS
A
1043
−33.526
73.869
6.530
1.00
78.09
O


ATOM
1937
CB
LYS
A
1043
−36.823
73.893
6.527
1.00
75.43
C


ATOM
1938
CG
LYS
A
1043
−37.946
72.879
6.697
1.00
76.23
C


ATOM
1939
CD
LYS
A
1043
−39.136
73.199
5.806
1.00
82.58
C


ATOM
1940
CE
LYS
A
1043
−40.094
72.010
5.671
1.00
85.26
C


ATOM
1941
NZ
LYS
A
1043
−40.465
71.420
6.980
1.00
90.37
N


ATOM
1942
N
SER
A
1044
−34.430
75.798
7.267
1.00
75.27
N


ATOM
1943
CA
SER
A
1044
−33.272
76.626
6.931
1.00
75.10
C


ATOM
1944
C
SER
A
1044
−32.023
76.175
7.694
1.00
75.62
C


ATOM
1945
O
SER
A
1044
−30.957
76.023
7.097
1.00
77.80
O


ATOM
1946
CB
SER
A
1044
−33.557
78.096
7.232
1.00
75.53
C


ATOM
1947
OG
SER
A
1044
−32.465
78.910
6.848
1.00
75.67
O


ATOM
1948
N
GLU
A
1045
−32.166
75.966
9.006
1.00
73.82
N


ATOM
1949
CA
GLU
A
1045
−31.062
75.471
9.836
1.00
73.20
C


ATOM
1950
C
GLU
A
1045
−30.641
74.064
9.456
1.00
71.02
C


ATOM
1951
O
GLU
A
1045
−29.448
73.752
9.494
1.00
71.07
O


ATOM
1952
CB
GLU
A
1045
−31.427
75.494
11.331
1.00
71.62
C


ATOM
1953
CG
GLU
A
1045
−31.659
76.884
11.923
1.00
73.11
C


ATOM
1954
CD
GLU
A
1045
−30.431
77.762
11.949
1.00
76.79
C


ATOM
1955
OE1
GLU
A
1045
−29.308
77.260
11.747
1.00
80.04
O


ATOM
1956
OE2
GLU
A
1045
−30.595
78.980
12.177
1.00
85.26
O


ATOM
1957
N
LEU
A
1046
−31.615
73.224
9.100
1.00
70.49
N


ATOM
1958
CA
LEU
A
1046
−31.342
71.839
8.710
1.00
73.29
C


ATOM
1959
C
LEU
A
1046
−30.492
71.807
7.455
1.00
73.52
C


ATOM
1960
O
LEU
A
1046
−29.453
71.144
7.434
1.00
74.73
O


ATOM
1961
CB
LEU
A
1046
−32.644
71.064
8.469
1.00
75.19
C


ATOM
1962
CG
LEU
A
1046
−32.556
69.562
8.130
1.00
73.72
C


ATOM
1963
CD1
LEU
A
1046
−31.881
68.775
9.243
1.00
74.36
C


ATOM
1964
CD2
LEU
A
1046
−33.942
69.000
7.851
1.00
73.43
C


ATOM
1965
N
ASP
A
1047
−30.945
72.529
6.426
1.00
72.94
N


ATOM
1966
CA
ASP
A
1047
−30.225
72.645
5.148
1.00
75.39
C


ATOM
1967
C
ASP
A
1047
−28.812
73.161
5.343
1.00
74.85
C


ATOM
1968
O
ASP
A
1047
−27.875
72.634
4.758
1.00
76.38
O


ATOM
1969
CB
ASP
A
1047
−30.953
73.595
4.160
1.00
74.34
C


ATOM
1970
CG
ASP
A
1047
−32.327
73.076
3.713
1.00
75.72
C


ATOM
1971
OD1
ASP
A
1047
−32.707
71.938
4.072
1.00
72.63
O


ATOM
1972
OD2
ASP
A
1047
−33.029
73.824
2.995
1.00
80.04
O


ATOM
1973
N
LYS
A
1048
−28.677
74.193
6.168
1.00
75.86
N


ATOM
1974
CA
LYS
A
1048
−27.379
74.779
6.494
1.00
75.59
C


ATOM
1975
C
LYS
A
1048
−26.464
73.779
7.248
1.00
75.44
C


ATOM
1976
O
LYS
A
1048
−25.241
73.810
7.088
1.00
75.09
O


ATOM
1977
CB
LYS
A
1048
−27.603
76.061
7.296
1.00
75.61
C


ATOM
1978
CG
LYS
A
1048
−26.368
76.896
7.555
1.00
77.43
C


ATOM
1979
CD
LYS
A
1048
−26.729
78.377
7.838
1.00
78.34
C


ATOM
1980
CE
LYS
A
1048
−27.671
78.567
9.048
1.00
80.49
C


ATOM
1981
NZ
LYS
A
1048
−27.074
78.073
10.322
1.00
79.34
N


ATOM
1982
N
ALA
A
1049
−27.073
72.903
8.055
1.00
73.58
N


ATOM
1983
CA
ALA
A
1049
−26.345
71.870
8.804
1.00
74.82
C


ATOM
1984
C
ALA
A
1049
−25.950
70.699
7.909
1.00
75.13
C


ATOM
1985
O
ALA
A
1049
−24.797
70.271
7.913
1.00
75.27
O


ATOM
1986
CB
ALA
A
1049
−27.188
71.364
9.973
1.00
73.33
C


ATOM
1987
N
ILE
A
1050
−26.924
70.194
7.152
1.00
73.31
N


ATOM
1988
CA
ILE
A
1050
−26.746
69.038
6.255
1.00
73.24
C


ATOM
1989
C
ILE
A
1050
−25.994
69.378
4.951
1.00
72.49
C


ATOM
1990
O
ILE
A
1050
−25.155
68.602
4.500
1.00
72.34
O


ATOM
1991
CB
ILE
A
1050
−28.124
68.397
5.931
1.00
73.95
C


ATOM
1992
CG1
ILE
A
1050
−28.758
67.828
7.207
1.00
72.34
C


ATOM
1993
CG2
ILE
A
1050
−27.996
67.297
4.907
1.00
70.31
C


ATOM
1994
CD1
ILE
A
1050
−27.968
66.650
7.819
1.00
75.79
C


ATOM
1995
N
GLY
A
1051
−26.304
70.530
4.362
1.00
70.45
N


ATOM
1996
CA
GLY
A
1051
−25.628
71.022
3.152
1.00
69.29
C


ATOM
1997
C
GLY
A
1051
−26.371
70.810
1.841
1.00
68.84
C


ATOM
1998
O
GLY
A
1051
−25.741
70.624
0.800
1.00
64.99
O


ATOM
1999
N
ARG
A
1052
−27.703
70.841
1.900
1.00
66.56
N


ATOM
2000
CA
ARG
A
1052
−28.563
70.680
0.730
1.00
67.90
C


ATOM
2001
C
ARG
A
1052
−29.995
71.012
1.090
1.00
68.30
C


ATOM
2002
O
ARG
A
1052
−30.309
71.175
2.261
1.00
71.80
O


ATOM
2003
CB
ARG
A
1052
−28.502
69.251
0.162
1.00
66.98
C


ATOM
2004
CG
ARG
A
1052
−28.971
68.148
1.076
1.00
72.44
C


ATOM
2005
CD
ARG
A
1052
−29.034
66.833
0.330
1.00
70.28
C


ATOM
2006
NE
ARG
A
1052
−29.469
65.745
1.210
1.00
74.46
N


ATOM
2007
CZ
ARG
A
1052
−30.740
65.449
1.540
1.00
75.81
C


ATOM
2008
NH1
ARG
A
1052
−31.772
66.146
1.076
1.00
75.31
N


ATOM
2009
NH2
ARG
A
1052
−30.991
64.430
2.354
1.00
78.34
N


ATOM
2010
N
ASN
A
1053
−30.856
71.108
0.083
1.00
67.63
N


ATOM
2011
CA
ASN
A
1053
−32.289
71.307
0.299
1.00
69.73
C


ATOM
2012
C
ASN
A
1053
−32.876
69.984
0.719
1.00
69.35
C


ATOM
2013
O
ASN
A
1053
−33.005
69.075
−0.095
1.00
69.80
O


ATOM
2014
CB
ASN
A
1053
−32.996
71.829
−0.957
1.00
70.79
C


ATOM
2015
CG
ASN
A
1053
−32.759
73.310
−1.201
1.00
72.54
C


ATOM
2016
OD1
ASN
A
1053
−33.223
73.847
−2.196
1.00
78.00
O


ATOM
2017
ND2
ASN
A
1053
−32.037
73.975
−0.292
1.00
81.34
N


ATOM
2018
N
THR
A
1054
−33.228
69.880
1.996
1.00
72.38
N


ATOM
2019
CA
THR
A
1054
−33.753
68.647
2.552
1.00
72.53
C


ATOM
2020
C
THR
A
1054
−35.266
68.588
2.510
1.00
73.02
C


ATOM
2021
O
THR
A
1054
−35.826
67.516
2.305
1.00
77.21
O


ATOM
2022
CB
THR
A
1054
−33.251
68.458
3.982
1.00
71.32
C


ATOM
2023
OG1
THR
A
1054
−33.557
69.619
4.758
1.00
71.59
O


ATOM
2024
CG2
THR
A
1054
−31.748
68.254
3.981
1.00
73.10
C


ATOM
2025
N
ASN
A
1055
−35.908
69.737
2.702
1.00
75.24
N


ATOM
2026
CA
ASN
A
1055
−37.360
69.842
2.815
1.00
76.78
C


ATOM
2027
C
ASN
A
1055
−37.876
69.053
4.046
1.00
76.94
C


ATOM
2028
O
ASN
A
1055
−38.985
68.522
4.044
1.00
82.59
O


ATOM
2029
CB
ASN
A
1055
−38.062
69.398
1.517
1.00
76.21
C


ATOM
2030
CG
ASN
A
1055
−39.511
69.867
1.431
1.00
78.54
C


ATOM
2031
OD1
ASN
A
1055
−39.930
70.812
2.109
1.00
77.71
O


ATOM
2032
ND2
ASN
A
1055
−40.285
69.199
0.587
1.00
87.37
N


ATOM
2033
N
GLY
A
1056
−37.054
68.984
5.091
1.00
76.49
N


ATOM
2034
CA
GLY
A
1056
−37.403
68.283
6.314
1.00
76.60
C


ATOM
2035
C
GLY
A
1056
−36.995
66.832
6.409
1.00
74.80
C


ATOM
2036
O
GLY
A
1056
−37.028
66.298
7.489
1.00
77.03
O


ATOM
2037
N
VAL
A
1057
−36.610
66.192
5.301
1.00
74.21
N


ATOM
2038
CA
VAL
A
1057
−36.246
64.770
5.320
1.00
73.68
C


ATOM
2039
C
VAL
A
1057
−34.745
64.554
5.095
1.00
73.15
C


ATOM
2040
O
VAL
A
1057
−34.181
65.114
4.151
1.00
77.13
O


ATOM
2041
CB
VAL
A
1057
−37.029
63.981
4.261
1.00
72.56
C


ATOM
2042
CG1
VAL
A
1057
−36.711
62.498
4.368
1.00
74.94
C


ATOM
2043
CG2
VAL
A
1057
−38.523
64.201
4.432
1.00
70.86
C


ATOM
2044
N
ILE
A
1058
−34.120
63.742
5.963
1.00
71.83
N


ATOM
2045
CA
ILE
A
1058
−32.698
63.347
5.843
1.00
71.92
C


ATOM
2046
C
ILE
A
1058
−32.520
61.833
5.872
1.00
73.30
C


ATOM
2047
O
ILE
A
1058
−33.382
61.111
6.370
1.00
76.64
O


ATOM
2048
CB
ILE
A
1058
−31.815
63.953
6.956
1.00
67.96
C


ATOM
2049
CG1
ILE
A
1058
−32.220
63.448
8.364
1.00
71.45
C


ATOM
2050
CG2
ILE
A
1058
−31.883
65.457
6.899
1.00
65.81
C


ATOM
2051
CD1
ILE
A
1058
−31.205
63.816
9.445
1.00
65.73
C


ATOM
2052
N
THR
A
1059
−31.392
61.362
5.343
1.00
72.25
N


ATOM
2053
CA
THR
A
1059
−31.070
59.932
5.345
1.00
71.10
C


ATOM
2054
C
THR
A
1059
−30.533
59.545
6.712
1.00
70.11
C


ATOM
2055
O
THR
A
1059
−30.166
60.405
7.495
1.00
71.12
O


ATOM
2056
CB
THR
A
1059
−29.990
59.595
4.353
1.00
70.42
C


ATOM
2057
OG1
THR
A
1059
−28.775
60.228
4.776
1.00
67.63
O


ATOM
2058
CG2
THR
A
1059
−30.377
60.056
2.874
1.00
63.19
C


ATOM
2059
N
LYS
A
1060
−30.493
58.244
6.972
1.00
70.88
N


ATOM
2060
CA
LYS
A
1060
−29.950
57.684
8.208
1.00
71.95
C


ATOM
2061
C
LYS
A
1060
−28.481
58.087
8.416
1.00
72.94
C


ATOM
2062
O
LYS
A
1060
−28.082
58.452
9.536
1.00
71.54
O


ATOM
2063
CB
LYS
A
1060
−30.074
56.151
8.215
1.00
70.63
C


ATOM
2064
CG
LYS
A
1060
−29.325
55.466
9.387
1.00
75.49
C


ATOM
2065
CD
LYS
A
1060
−29.500
53.953
9.419
1.00
73.88
C


ATOM
2066
CE
LYS
A
1060
−28.422
53.322
10.308
1.00
71.79
C


ATOM
2067
NZ
LYS
A
1060
−28.584
51.848
10.427
1.00
80.16
N


ATOM
2068
N
ASP
A
1061
−27.706
58.010
7.330
1.00
68.84
N


ATOM
2069
CA
ASP
A
1061
−26.282
58.339
7.320
1.00
68.20
C


ATOM
2070
C
ASP
A
1061
−26.043
59.814
7.586
1.00
66.58
C


ATOM
2071
O
ASP
A
1061
−25.076
60.148
8.255
1.00
70.66
O


ATOM
2072
CB
ASP
A
1061
−25.622
57.923
5.983
1.00
64.64
C


ATOM
2073
CG
ASP
A
1061
−25.572
56.386
5.788
1.00
73.92
C


ATOM
2074
OD1
ASP
A
1061
−25.501
55.619
6.782
1.00
83.01
O


ATOM
2075
OD2
ASP
A
1061
−25.603
55.935
4.630
1.00
82.92
O


ATOM
2076
N
GLU
A
1062
−26.909
60.687
7.071
1.00
60.10
N


ATOM
2077
CA
GLU
A
1062
−26.856
62.113
7.437
1.00
62.56
C


ATOM
2078
C
GLU
A
1062
−27.203
62.317
8.955
1.00
64.45
C


ATOM
2079
O
GLU
A
1062
−26.559
63.127
9.619
1.00
62.28
O


ATOM
2080
CB
GLU
A
1062
−27.770
62.910
6.572
1.00
60.74
C


ATOM
2081
CG
GLU
A
1062
−27.243
63.018
5.142
1.00
60.93
C


ATOM
2082
CD
GLU
A
1062
−28.230
63.606
4.198
1.00
68.90
C


ATOM
2083
OE1
GLU
A
1062
−29.446
63.498
4.488
1.00
67.42
O


ATOM
2084
OE2
GLU
A
1062
−27.795
64.182
3.155
1.00
73.08
O


ATOM
2085
N
ALA
A
1063
−28.197
61.590
9.485
1.00
64.40
N


ATOM
2086
CA
ALA
A
1063
−28.494
61.647
10.955
1.00
68.01
C


ATOM
2087
C
ALA
A
1063
−27.251
61.296
11.748
1.00
69.72
C


ATOM
2088
O
ALA
A
1063
−26.948
61.940
12.745
1.00
79.20
O


ATOM
2089
CB
ALA
A
1063
−29.637
60.723
11.331
1.00
65.57
C


ATOM
2090
N
GLU
A
1064
−26.529
60.269
11.298
1.00
73.91
N


ATOM
2091
CA
GLU
A
1064
−25.261
59.873
11.920
1.00
71.50
C


ATOM
2092
C
GLU
A
1064
−24.218
60.973
11.815
1.00
72.04
C


ATOM
2093
O
GLU
A
1064
−23.465
61.158
12.747
1.00
76.73
O


ATOM
2094
CB
GLU
A
1064
−24.746
58.584
11.294
1.00
70.30
C


ATOM
2095
CG
GLU
A
1064
−23.391
58.090
11.772
1.00
71.10
C


ATOM
2096
CD
GLU
A
1064
−23.012
56.715
11.195
1.00
81.17
C


ATOM
2097
OE1
GLU
A
1064
−23.711
56.194
10.251
1.00
87.16
O


ATOM
2098
OE2
GLU
A
1064
−22.006
56.150
11.688
1.00
89.77
O


ATOM
2099
N
LYS
A
1065
−24.168
61.701
10.695
1.00
73.67
N


ATOM
2100
CA
LYS
A
1065
−23.204
62.821
10.568
1.00
73.69
C


ATOM
2101
C
LYS
A
1065
−23.466
63.903
11.620
1.00
70.10
C


ATOM
2102
O
LYS
A
1065
−22.553
64.325
12.310
1.00
68.11
O


ATOM
2103
CB
LYS
A
1065
−23.232
63.461
9.190
1.00
75.31
C


ATOM
2104
CG
LYS
A
1065
−22.891
62.546
8.022
1.00
80.59
C


ATOM
2105
CD
LYS
A
1065
−21.422
62.223
7.938
1.00
85.80
C


ATOM
2106
CE
LYS
A
1065
−21.098
61.379
6.680
1.00
81.37
C


ATOM
2107
NZ
LYS
A
1065
−21.661
61.944
5.425
1.00
72.55
N


ATOM
2108
N
LEU
A
1066
−24.718
64.335
11.723
1.00
69.54
N


ATOM
2109
CA
LEU
A
1066
−25.133
65.305
12.748
1.00
71.70
C


ATOM
2110
C
LEU
A
1066
−24.854
64.804
14.165
1.00
70.08
C


ATOM
2111
O
LEU
A
1066
−24.476
65.572
15.033
1.00
68.08
O


ATOM
2112
CB
LEU
A
1066
−26.621
65.605
12.636
1.00
71.78
C


ATOM
2113
CG
LEU
A
1066
−27.056
66.466
11.468
1.00
76.18
C


ATOM
2114
CD1
LEU
A
1066
−28.565
66.357
11.261
1.00
78.42
C


ATOM
2115
CD2
LEU
A
1066
−26.625
67.899
11.715
1.00
77.95
C


ATOM
2116
N
PHE
A
1067
−25.049
63.513
14.373
1.00
71.17
N


ATOM
2117
CA
PHE
A
1067
−24.844
62.899
15.673
1.00
71.75
C


ATOM
2118
C
PHE
A
1067
−23.383
62.902
16.104
1.00
71.56
C


ATOM
2119
O
PHE
A
1067
−23.098
63.171
17.275
1.00
76.85
O


ATOM
2120
CB
PHE
A
1067
−25.378
61.480
15.669
1.00
74.38
C


ATOM
2121
CG
PHE
A
1067
−25.046
60.740
16.880
1.00
75.45
C


ATOM
2122
CD1
PHE
A
1067
−25.810
60.897
18.012
1.00
78.29
C


ATOM
2123
CD2
PHE
A
1067
−23.957
59.877
16.909
1.00
81.22
C


ATOM
2124
CE1
PHE
A
1067
−25.509
60.221
19.138
1.00
77.84
C


ATOM
2125
CE2
PHE
A
1067
−23.650
59.194
18.043
1.00
81.99
C


ATOM
2126
CZ
PHE
A
1067
−24.425
59.361
19.165
1.00
78.68
C


ATOM
2127
N
ASN
A
1068
−22.464
62.611
15.188
1.00
72.06
N


ATOM
2128
CA
ASN
A
1068
−21.035
62.658
15.520
1.00
72.87
C


ATOM
2129
C
ASN
A
1068
−20.611
64.092
15.888
1.00
74.75
C


ATOM
2130
O
ASN
A
1068
−19.722
64.272
16.716
1.00
76.41
O


ATOM
2131
CB
ASN
A
1068
−20.134
62.151
14.400
1.00
70.30
C


ATOM
2132
CG
ASN
A
1068
−20.477
60.741
13.936
1.00
77.74
C


ATOM
2133
OD1
ASN
A
1068
−20.902
59.903
14.722
1.00
71.67
O


ATOM
2134
ND2
ASN
A
1068
−20.286
60.479
12.617
1.00
72.67
N


ATOM
2135
N
GLN
A
1069
−21.244
65.094
15.275
1.00
73.62
N


ATOM
2136
CA
GLN
A
1069
−20.982
66.487
15.631
1.00
73.62
C


ATOM
2137
C
GLN
A
1069
−21.541
66.803
17.020
1.00
73.06
C


ATOM
2138
O
GLN
A
1069
−20.874
67.455
17.802
1.00
68.92
O


ATOM
2139
CB
GLN
A
1069
−21.586
67.449
14.634
1.00
71.42
C


ATOM
2140
CG
GLN
A
1069
−20.967
67.379
13.249
1.00
81.86
C


ATOM
2141
CD
GLN
A
1069
−21.717
68.235
12.239
1.00
81.55
C


ATOM
2142
OE1
GLN
A
1069
−22.504
69.116
12.614
1.00
87.86
O


ATOM
2143
NE2
GLN
A
1069
−21.480
67.980
10.948
1.00
91.37
N


ATOM
2144
N
ASP
A
1070
−22.759
66.341
17.304
1.00
69.64
N


ATOM
2145
CA
ASP
A
1070
−23.362
66.549
18.615
1.00
70.15
C


ATOM
2146
C
ASP
A
1070
−22.510
65.879
19.689
1.00
70.56
C


ATOM
2147
O
ASP
A
1070
−22.272
66.486
20.737
1.00
71.26
O


ATOM
2148
CB
ASP
A
1070
−24.795
66.038
18.664
1.00
68.69
C


ATOM
2149
CG
ASP
A
1070
−25.742
66.828
17.756
1.00
77.13
C


ATOM
2150
OD1
ASP
A
1070
−25.444
67.997
17.392
1.00
75.90
O


ATOM
2151
OD2
ASP
A
1070
−26.794
66.277
17.400
1.00
74.40
O


ATOM
2152
N
VAL
A
1071
−22.058
64.649
19.426
1.00
67.27
N


ATOM
2153
CA
VAL
A
1071
−21.150
63.948
20.341
1.00
69.23
C


ATOM
2154
C
VAL
A
1071
−19.906
64.780
20.577
1.00
70.51
C


ATOM
2155
O
VAL
A
1071
−19.509
64.968
21.714
1.00
71.56
O


ATOM
2156
CB
VAL
A
1071
−20.737
62.563
19.820
1.00
66.16
C


ATOM
2157
CG1
VAL
A
1071
−19.561
62.086
20.548
1.00
63.68
C


ATOM
2158
CG2
VAL
A
1071
−21.887
61.570
19.952
1.00
68.51
C


ATOM
2159
N
ASP
A
1072
−19.294
65.280
19.500
1.00
74.44
N


ATOM
2160
CA
ASP
A
1072
−18.128
66.154
19.623
1.00
73.07
C


ATOM
2161
C
ASP
A
1072
−18.410
67.321
20.555
1.00
74.85
C


ATOM
2162
O
ASP
A
1072
−17.628
67.585
21.464
1.00
78.47
O


ATOM
2163
CB
ASP
A
1072
−17.688
66.703
18.269
1.00
77.99
C


ATOM
2164
CG
ASP
A
1072
−16.502
67.645
18.387
1.00
82.49
C


ATOM
2165
OD1
ASP
A
1072
−15.355
67.156
18.521
1.00
93.79
O


ATOM
2166
OD2
ASP
A
1072
−16.730
68.874
18.341
1.00
88.07
O


ATOM
2167
N
ALA
A
1073
−19.530
68.003
20.316
1.00
70.34
N


ATOM
2168
CA
ALA
A
1073
−19.981
69.126
21.153
1.00
71.13
C


ATOM
2169
C
ALA
A
1073
−20.178
68.742
22.643
1.00
72.31
C


ATOM
2170
O
ALA
A
1073
−19.771
69.495
23.520
1.00
72.64
O


ATOM
2171
CB
ALA
A
1073
−21.261
69.675
20.620
1.00
68.05
C


ATOM
2172
N
ALA
A
1074
−20.797
67.584
22.890
1.00
69.54
N


ATOM
2173
CA
ALA
A
1074
−21.085
67.096
24.244
1.00
71.93
C


ATOM
2174
C
ALA
A
1074
−19.795
66.903
25.028
1.00
72.36
C


ATOM
2175
O
ALA
A
1074
−19.684
67.378
26.149
1.00
77.33
O


ATOM
2176
CB
ALA
A
1074
−21.851
65.807
24.193
1.00
71.13
C


ATOM
2177
N
VAL
A
1075
−18.836
66.207
24.415
1.00
72.46
N


ATOM
2178
CA
VAL
A
1075
−17.511
65.973
24.995
1.00
74.25
C


ATOM
2179
C
VAL
A
1075
−16.752
67.262
25.348
1.00
74.70
C


ATOM
2180
O
VAL
A
1075
−16.163
67.345
26.429
1.00
74.40
O


ATOM
2181
CB
VAL
A
1075
−16.648
65.102
24.073
1.00
74.74
C


ATOM
2182
CG1
VAL
A
1075
−15.208
65.076
24.539
1.00
75.33
C


ATOM
2183
CG2
VAL
A
075
−17.205
63.696
24.033
1.00
76.41
C


ATOM
2184
N
ARG
A
1076
−16.768
68.246
24.455
1.00
72.53
N


ATOM
2185
CA
ARG
A
1076
−16.124
69.546
24.726
1.00
74.21
C


ATOM
2186
C
ARG
A
1076
−16.766
70.230
25.911
1.00
74.53
C


ATOM
2187
O
ARG
A
1076
−16.070
70.838
26.735
1.00
74.38
O


ATOM
2188
CB
ARG
A
1076
−16.204
70.487
23.533
1.00
70.53
C


ATOM
2189
CG
ARG
A
1076
−15.356
70.070
22.367
1.00
76.54
C


ATOM
2190
CD
ARG
A
1076
−15.465
71.070
21.205
1.00
82.85
C


ATOM
2191
NE
ARG
A
1076
−15.065
70.430
19.951
1.00
91.84
N


ATOM
2192
CZ
ARG
A
1076
−13.802
70.183
19.569
1.00
96.99
C


ATOM
2193
NH1
ARG
A
1076
−12.746
70.517
20.334
1.00
97.76
N


ATOM
2194
NH2
ARG
A
1076
−13.580
69.588
18.393
1.00
103.37
N


ATOM
2195
N
GLY
A
1077
−18.091
70.120
25.977
1.00
72.90
N


ATOM
2196
CA
GLY
A
1077
−18.865
70.656
27.059
1.00
73.02
C


ATOM
2197
C
GLY
A
1077
−18.543
69.995
28.392
1.00
72.32
C


ATOM
2198
O
GLY
A
1077
−18.322
70.690
29.387
1.00
73.12
O


ATOM
2199
N
ILE
A
1078
−18.523
68.661
28.399
1.00
72.41
N


ATOM
2200
CA
ILE
A
1078
−18.104
67.858
29.564
1.00
73.34
C


ATOM
2201
C
ILE
A
1078
−16.739
68.293
30.083
1.00
71.54
C


ATOM
2202
O
ILE
A
1078
−16.577
68.522
31.280
1.00
76.16
O


ATOM
2203
CB
ILE
A
1078
−18.044
66.363
29.210
1.00
74.38
C


ATOM
2204
CG1
ILE
A
1078
−19.453
65.757
29.198
1.00
79.15
C


ATOM
2205
CG2
ILE
A
1078
−17.205
65.616
30.174
1.00
64.66
C


ATOM
2206
CD1
ILE
A
1078
−19.514
64.385
28.554
1.00
75.65
C


ATOM
2207
N
LEU
A
1079
−15.775
68.407
29.177
1.00
72.22
N


ATOM
2208
CA
LEU
A
1079
−14.407
68.819
29.512
1.00
73.11
C


ATOM
2209
C
LEU
A
1079
−14.278
70.275
30.001
1.00
73.92
C


ATOM
2210
O
LEU
A
1079
−13.281
70.613
30.627
1.00
78.75
O


ATOM
2211
CB
LEU
A
1079
−13.462
68.578
28.317
1.00
72.14
C


ATOM
2212
CG
LEU
A
1079
−13.192
67.115
27.909
1.00
72.35
C


ATOM
2213
CD1
LEU
A
1079
−12.301
67.074
26.679
1.00
59.40
C


ATOM
2214
CD2
LEU
A
1079
−12.543
66.309
29.031
1.00
62.86
C


ATOM
2215
N
ARG
A
1080
−15.269
71.118
29.714
1.00
73.61
N


ATOM
2216
CA
ARG
A
1080
−15.330
72.490
30.251
1.00
74.19
C


ATOM
2217
C
ARG
A
1080
−16.039
72.537
31.609
1.00
72.82
C


ATOM
2218
O
ARG
A
1080
−15.894
73.505
32.331
1.00
72.73
O


ATOM
2219
CB
ARG
A
1080
−16.061
73.439
29.301
1.00
74.26
C


ATOM
2220
CG
ARG
A
1080
−15.236
73.955
28.140
1.00
79.49
C


ATOM
2221
CD
ARG
A
1080
−15.964
75.121
27.460
1.00
79.38
C


ATOM
2222
NE
ARG
A
1080
−17.260
74.691
26.922
1.00
88.39
N


ATOM
2223
CZ
ARG
A
1080
−17.535
74.387
25.636
1.00
87.86
C


ATOM
2224
NH1
ARG
A
1080
−16.608
74.451
24.665
1.00
87.22
N


ATOM
2225
NH2
ARG
A
1080
−18.777
74.009
25.314
1.00
81.94
N


ATOM
2226
N
ASN
A
1081
−16.800
71.495
31.946
1.00
72.20
N


ATOM
2227
CA
ASN
A
1081
−17.553
71.464
33.183
1.00
70.14
C


ATOM
2228
C
ASN
A
1081
−16.680
70.903
34.293
1.00
70.99
C


ATOM
2229
O
ASN
A
1081
−16.295
69.733
34.252
1.00
70.87
O


ATOM
2230
CB
ASN
A
1081
−18.817
70.642
33.002
1.00
68.83
C


ATOM
2231
CG
ASN
A
1081
−19.789
70.813
34.137
1.00
74.52
C


ATOM
2232
OD1
ASN
A
1081
−19.521
70.405
35.275
1.00
81.88
O


ATOM
2233
ND2
ASN
A
1081
−20.937
71.417
33.848
1.00
65.69
N


ATOM
2234
N
ALA
A
1082
−16.374
71.750
35.281
1.00
69.81
N


ATOM
2235
CA
ALA
A
1082
−15.539
71.386
36.459
1.00
71.34
C


ATOM
2236
C
ALA
A
1082
−15.942
70.101
37.203
1.00
70.68
C


ATOM
2237
O
ALA
A
1082
−15.081
69.426
37.748
1.00
77.40
O


ATOM
2238
CB
ALA
A
1082
−15.508
72.546
37.451
1.00
64.36
C


ATOM
2239
N
LYS
A
1083
−17.233
69.784
37.213
1.00
68.55
N


ATOM
2240
CA
LYS
A
1083
−17.776
68.623
37.931
1.00
69.05
C


ATOM
2241
C
LYS
A
1083
−17.798
67.337
37.093
1.00
72.30
C


ATOM
2242
O
LYS
A
1083
−17.732
66.243
37.649
1.00
76.42
O


ATOM
2243
CB
LYS
A
1083
−19.193
68.923
38.398
1.00
70.45
C


ATOM
2244
CG
LYS
A
1083
−19.359
70.297
39.063
1.00
75.92
C


ATOM
2245
CD
LYS
A
1083
−20.720
70.447
39.698
1.00
81.44
C


ATOM
2246
CE
LYS
A
1083
−20.926
71.864
40.217
1.00
85.50
C


ATOM
2247
NZ
LYS
A
1083
−22.148
71.994
41.068
1.00
88.29
N


ATOM
2248
N
LEU
A
1084
−17.891
67.460
35.765
1.00
68.40
N


ATOM
2249
CA
LEU
A
1084
−17.898
66.286
34.894
1.00
70.90
C


ATOM
2250
C
LEU
A
1084
−16.518
65.924
34.402
1.00
70.17
C


ATOM
2251
O
LEU
A
1084
−16.247
64.740
34.205
1.00
72.79
O


ATOM
2252
CB
LEU
A
1084
−18.843
66.497
33.694
1.00
68.64
C


ATOM
2253
CG
LEU
A
1084
−20.304
66.801
34.051
1.00
71.12
C


ATOM
2254
CD1
LEU
A
1084
−21.213
66.916
32.787
1.00
59.19
C


ATOM
2255
CD2
LEU
A
1084
−20.847
65.747
35.010
1.00
56.68
C


ATOM
2256
N
LYS
A
1085
−15.650
66.917
34.205
1.00
68.34
N


ATOM
2257
CA
LYS
A
1085
−14.316
66.663
33.652
1.00
68.63
C


ATOM
2258
C
LYS
A
1085
−13.538
65.559
34.383
1.00
68.76
C


ATOM
2259
O
LYS
A
1085
−13.070
64.643
33.723
1.00
75.06
O


ATOM
2260
CB
LYS
A
1085
−13.482
67.951
33.565
1.00
67.89
C


ATOM
2261
CG
LYS
A
1085
−12.110
67.770
32.917
1.00
69.24
C


ATOM
2262
CD
LYS
A
1085
−11.322
69.054
32.925
1.00
72.42
C


ATOM
2263
CE
LYS
A
1085
−10.040
68.905
32.138
1.00
77.84
C


ATOM
2264
NZ
LYS
A
1085
−9.131
67.881
32.745
1.00
85.14
N


ATOM
2265
N
PRO
A
1086
−13.399
65.636
35.740
1.00
69.24
N


ATOM
2266
CA
PRO
A
1086
−12.637
64.586
36.440
1.00
69.18
C


ATOM
2267
C
PRO
A
1086
−13.226
63.190
36.261
1.00
68.97
C


ATOM
2268
O
PRO
A
1086
−12.488
62.197
36.199
1.00
69.99
O


ATOM
2269
CB
PRO
A
1086
−12.771
64.966
37.923
1.00
67.72
C


ATOM
2270
CG
PRO
A
1086
−13.201
66.357
37.950
1.00
72.53
C


ATOM
2271
CD
PRO
A
1086
−13.918
66.646
36.679
1.00
66.17
C


ATOM
2272
N
VAL
A
1087
−14.549
63.136
36.184
1.00
68.84
N


ATOM
2273
CA
VAL
A
1087
−15.265
61.885
36.092
1.00
72.28
C


ATOM
2274
C
VAL
A
1087
−14.985
61.291
34.743
1.00
73.91
C


ATOM
2275
O
VAL
A
1087
−14.480
60.176
34.672
1.00
77.11
O


ATOM
2276
CB
VAL
A
1087
−16.769
62.062
36.305
1.00
72.36
C


ATOM
2277
CG1
VAL
A
1087
−17.431
60.720
36.368
1.00
78.50
C


ATOM
2278
CG2
VAL
A
1087
−17.032
62.832
37.602
1.00
75.48
C


ATOM
2279
N
TYR
A
1088
−15.310
62.047
33.691
1.00
76.42
N


ATOM
2280
CA
TYR
A
1088
−15.087
61.660
32.290
1.00
74.22
C


ATOM
2281
C
TYR
A
1088
−13.655
61.241
32.054
1.00
73.79
C


ATOM
2282
O
TYR
A
1088
−13.414
60.184
31.502
1.00
71.26
O


ATOM
2283
CB
TYR
A
1088
−15.420
62.840
31.382
1.00
76.80
C


ATOM
2284
CG
TYR
A
1088
−15.345
62.536
29.909
1.00
78.30
C


ATOM
2285
CD1
TYR
A
1088
−16.447
61.994
29.225
1.00
82.88
C


ATOM
2286
CD2
TYR
A
1088
−14.181
62.787
29.185
1.00
82.17
C


ATOM
2287
CE1
TYR
A
1088
−16.368
61.712
27.838
1.00
78.90
C


ATOM
2288
CE2
TYR
A
1088
−14.102
62.511
27.823
1.00
73.71
C


ATOM
2289
CZ
TYR
A
1088
−15.199
61.976
27.162
1.00
71.96
C


ATOM
2290
OH
TYR
A
1088
−15.099
61.705
25.804
1.00
90.19
O


ATOM
2291
N
ASP
A
1089
−12.708
62.082
32.483
1.00
73.28
N


ATOM
2292
CA
ASP
A
1089
−11.262
61.780
32.399
1.00
72.57
C


ATOM
2293
C
ASP
A
1089
−10.880
60.403
32.936
1.00
71.20
C


ATOM
2294
O
ASP
A
1089
−9.987
59.755
32.407
1.00
70.55
O


ATOM
2295
CB
ASP
A
1089
−10.438
62.802
33.208
1.00
72.85
C


ATOM
2296
CG
ASP
A
1089
−10.162
64.118
32.468
1.00
79.93
C


ATOM
2297
OD1
ASP
A
1089
−10.551
64.287
31.274
1.00
78.98
O


ATOM
2298
OD2
ASP
A
1089
−9.528
65.004
33.120
1.00
69.59
O


ATOM
2299
N
SER
A
1090
−11.562
59.969
33.993
1.00
72.25
N


ATOM
2300
CA
SER
A
1090
−11.244
58.711
34.670
1.00
71.13
C


ATOM
2301
C
SER
A
1090
−11.893
57.495
34.027
1.00
69.01
C


ATOM
2302
O
SER
A
1090
−11.411
56.383
34.199
1.00
69.79
O


ATOM
2303
CB
SER
A
1090
−11.684
58.791
36.124
1.00
67.58
C


ATOM
2304
OG
SER
A
1090
−13.085
58.665
36.232
1.00
65.84
O


ATOM
2305
N
LEU
A
1091
−12.979
57.703
33.293
1.00
72.26
N


ATOM
2306
CA
LEU
A
1091
−13.727
56.593
32.729
1.00
70.30
C


ATOM
2307
C
LEU
A
1091
−13.092
56.023
31.462
1.00
73.59
C


ATOM
2308
O
LEU
A
1091
−12.420
56.715
30.705
1.00
75.14
O


ATOM
2309
CB
LEU
A
1091
−15.172
57.014
32.457
1.00
73.21
C


ATOM
2310
CG
LEU
A
1091
−16.060
57.298
33.687
1.00
69.70
C


ATOM
2311
CD1
LEU
A
1091
−17.374
57.869
33.211
1.00
74.59
C


ATOM
2312
CD2
LEU
A
1091
−16.287
56.051
34.482
1.00
61.23
C


ATOM
2313
N
ASP
A
1092
−13.324
54.735
31.257
1.00
73.36
N


ATOM
2314
CA
ASP
A
1092
−12.955
54.046
30.017
1.00
74.54
C


ATOM
2315
C
ASP
A
1092
−13.910
54.480
28.927
1.00
72.07
C


ATOM
2316
O
ASP
A
1092
−14.938
55.088
29.230
1.00
69.09
O


ATOM
2317
CB
ASP
A
1092
−13.093
52.547
30.180
1.00
74.48
C


ATOM
2318
CG
ASP
A
1092
−14.502
52.157
30.522
1.00
82.40
C


ATOM
2319
OD1
ASP
A
1092
−15.393
52.059
29.643
1.00
78.10
O


ATOM
2320
OD2
ASP
A
1092
−14.702
51.956
31.702
1.00
93.39
O


ATOM
2321
N
ALA
A
1093
−13.561
54.156
27.677
1.00
71.26
N


ATOM
2322
CA
ALA
A
1093
−14.332
54.530
26.455
1.00
72.91
C


ATOM
2323
C
ALA
A
1093
−15.817
54.185
26.482
1.00
72.49
C


ATOM
2324
O
ALA
A
1093
−16.642
55.016
26.143
1.00
77.77
O


ATOM
2325
CB
ALA
A
1093
−13.682
53.884
25.213
1.00
68.92
C


ATOM
2326
N
VAL
A
1094
−16.152
52.959
26.882
1.00
74.25
N


ATOM
2327
CA
VAL
A
1094
−17.566
52.517
26.970
1.00
76.09
C


ATOM
2328
C
VAL
A
1094
−18.388
53.297
28.021
1.00
74.99
C


ATOM
2329
O
VAL
A
1094
−19.550
53.686
27.760
1.00
74.98
O


ATOM
2330
CB
VAL
A
1094
−17.680
51.014
27.268
1.00
77.56
C


ATOM
2331
CG1
VAL
A
1094
−19.124
50.634
27.518
1.00
80.02
C


ATOM
2332
CG2
VAL
A
1094
−17.091
50.192
26.098
1.00
77.82
C


ATOM
2333
N
ARG
A
1095
−17.804
53.527
29.192
1.00
74.55
N


ATOM
2334
CA
ARG
A
1095
−18.502
54.306
30.245
1.00
72.87
C


ATOM
2335
C
ARG
A
1095
−18.564
55.792
29.877
1.00
70.86
C


ATOM
2336
O
ARG
A
1095
−19.529
56.471
30.224
1.00
70.60
O


ATOM
2337
CB
ARG
A
1095
−17.855
54.090
31.598
1.00
74.90
C


ATOM
2338
CG
ARG
A
1095
−17.944
52.648
32.049
1.00
74.88
C


ATOM
2339
CD
ARG
A
1095
−17.456
52.519
33.465
1.00
76.13
C


ATOM
2340
NE
ARG
A
1095
−17.765
51.222
34.039
1.00
68.67
N


ATOM
2341
CZ
ARG
A
1095
−17.035
50.111
33.935
1.00
75.88
C


ATOM
2342
NH1
ARG
A
1095
−15.900
50.077
33.261
1.00
77.10
N


ATOM
2343
NH2
ARG
A
1095
−17.456
48.999
34.523
1.00
78.82
N


ATOM
2344
N
ARG
A
1096
−17.550
56.304
29.176
1.00
70.40
N


ATOM
2345
CA
ARG
A
1096
−17.646
57.654
28.616
1.00
72.78
C


ATOM
2346
C
ARG
A
1096
−18.895
57.781
27.683
1.00
75.95
C


ATOM
2347
O
ARG
A
1096
−19.603
58.790
27.741
1.00
76.20
O


ATOM
2348
CB
ARG
A
1096
−16.382
58.032
27.849
1.00
74.96
C


ATOM
2349
CG
ARG
A
1096
−15.212
58.381
28.729
1.00
76.80
C


ATOM
2350
CD
ARG
A
1096
−13.987
58.699
27.888
1.00
73.69
C


ATOM
2351
NE
ARG
A
1096
−12.821
58.877
28.724
1.00
75.05
N


ATOM
2352
CZ
ARG
A
1096
−11.617
59.276
28.334
1.00
76.09
C


ATOM
2353
NH1
ARG
A
1096
−11.358
59.568
27.062
1.00
79.66
N


ATOM
2354
NH2
ARG
A
1096
−10.642
59.388
29.244
1.00
73.76
N


ATOM
2355
N
ALA
A
1097
−19.154
56.767
26.842
1.00
74.52
N


ATOM
2356
CA
ALA
A
1097
−20.381
56.762
25.993
1.00
75.40
C


ATOM
2357
C
ALA
A
1097
−21.606
56.787
26.899
1.00
74.68
C


ATOM
2358
O
ALA
A
1097
−22.596
57.444
26.594
1.00
75.19
O


ATOM
2359
CB
ALA
A
1097
−20.428
55.556
25.084
1.00
65.57
C


ATOM
2360
N
ALA
A
1098
−21.538
56.068
28.019
1.00
77.29
N


ATOM
2361
CA
ALA
A
1098
−22.627
56.110
29.004
1.00
77.63
C


ATOM
2362
C
ALA
A
1098
−22.855
57.549
29.425
1.00
75.46
C


ATOM
2363
O
ALA
A
1098
−23.953
58.037
29.306
1.00
77.90
O


ATOM
2364
CB
ALA
A
1098
−22.319
55.252
30.201
1.00
72.80
C


ATOM
2365
N
LEU
A
1099
−21.808
58.215
29.906
1.00
74.27
N


ATOM
2366
CA
LEU
A
1099
−21.935
59.610
30.389
1.00
73.67
C


ATOM
2367
C
LEU
A
1099
−22.493
60.542
29.329
1.00
73.73
C


ATOM
2368
O
LEU
A
1099
−23.412
61.328
29.602
1.00
73.92
O


ATOM
2369
CB
LEU
A
1099
−20.590
60.144
30.886
1.00
76.16
C


ATOM
2370
CG
LEU
A
1099
−20.612
61.381
31.794
1.00
76.75
C


ATOM
2371
CD1
LEU
A
1099
−21.495
61.167
33.000
1.00
72.22
C


ATOM
2372
CD2
LEU
A
1099
−19.187
61.741
32.236
1.00
66.69
C


ATOM
2373
N
ILE
A
1100
−21.941
60.454
28.115
1.00
72.03
N


ATOM
2374
CA
ILE
A
1100
−22.430
61.252
26.986
1.00
70.39
C


ATOM
2375
C
ILE
A
1100
−23.908
60.998
26.758
1.00
69.34
C


ATOM
2376
O
ILE
A
1100
−24.674
61.941
26.552
1.00
70.67
O


ATOM
2377
CB
ILE
A
1100
−21.672
60.964
25.677
1.00
74.51
C


ATOM
2378
CG1
ILE
A
1100
−20.208
61.411
25.789
1.00
74.12
C


ATOM
2379
CG2
ILE
A
1100
−22.368
61.684
24.523
1.00
66.55
C


ATOM
2380
CD1
ILE
A
1100
−19.273
60.643
24.910
1.00
71.96
C


ATOM
2381
N
ASN
A
1101
−24.305
59.727
26.798
1.00
69.64
N


ATOM
2382
CA
ASN
A
1101
−25.716
59.340
26.656
1.00
71.15
C


ATOM
2383
C
ASN
A
1101
−26.587
60.154
27.596
1.00
73.26
C


ATOM
2384
O
ASN
A
1101
−27.606
60.720
27.158
1.00
73.01
O


ATOM
2385
CB
ASN
A
1101
−25.859
57.819
26.880
1.00
74.91
C


ATOM
2386
CG
ASN
A
1101
−27.242
57.286
26.621
1.00
72.41
C


ATOM
2387
OD1
ASN
A
1101
−28.218
57.895
26.974
1.00
68.21
O


ATOM
2388
ND2
ASN
A
1101
−27.321
56.110
25.995
1.00
66.91
N


ATOM
2389
N
MET
A
1102
−26.191
60.217
28.876
1.00
73.18
N


ATOM
2390
CA
MET
A
1102
−26.922
60.986
29.894
1.00
73.53
C


ATOM
2391
C
MET
A
1102
−26.897
62.472
29.579
1.00
72.72
C


ATOM
2392
O
MET
A
1102
−27.909
63.130
29.719
1.00
75.34
O


ATOM
2393
CB
MET
A
1102
−26.341
60.788
31.315
1.00
69.92
C


ATOM
2394
CG
MET
A
1102
−26.311
59.343
31.846
1.00
71.61
C


ATOM
2395
SD
MET
A
1102
−25.639
59.205
33.558
1.00
75.01
S


ATOM
2396
CE
MET
A
1102
−26.903
60.133
34.436
1.00
72.22
C


ATOM
2397
N
VAL
A
1103
−25.743
62.997
29.160
1.00
73.08
N


ATOM
2398
CA
VAL
A
1103
−25.650
64.430
28.774
1.00
75.10
C


ATOM
2399
C
VAL
A
1103
−26.605
64.736
27.585
1.00
74.50
C


ATOM
2400
O
VAL
A
1103
−27.280
65.769
27.587
1.00
74.33
O


ATOM
2401
CB
VAL
A
1103
−24.203
64.859
28.445
1.00
74.63
C


ATOM
2402
CG1
VAL
A
1103
−24.181
66.214
27.773
1.00
74.66
C


ATOM
2403
CG2
VAL
A
1103
−23.345
64.892
29.715
1.00
73.28
C


ATOM
2404
N
PHE
A
1104
−26.648
63.838
26.598
1.00
72.50
N


ATOM
2405
CA
PHE
A
1104
−27.641
63.937
25.507
1.00
77.14
C


ATOM
2406
C
PHE
A
1104
−29.050
64.133
26.087
1.00
77.59
C


ATOM
2407
O
PHE
A
1104
−29.746
65.023
25.684
1.00
78.04
O


ATOM
2408
CB
PHE
A
1104
−27.615
62.712
24.557
1.00
78.31
C


ATOM
2409
CG
PHE
A
1104
−26.870
62.933
23.259
1.00
81.99
C


ATOM
2410
CD1
PHE
A
1104
−25.489
63.029
23.238
1.00
78.79
C


ATOM
2411
CD2
PHE
A
1104
−27.580
63.037
22.027
1.00
90.20
C


ATOM
2412
CE1
PHE
A
1104
−24.800
63.227
22.021
1.00
77.16
C


ATOM
2413
CE2
PHE
A
1104
−26.910
63.237
20.821
1.00
84.00
C


ATOM
2414
CZ
PHE
A
1104
−25.509
63.332
20.823
1.00
86.09
C


ATOM
2415
N
GLN
A
1105
−29.446
63.296
27.039
1.00
76.01
N


ATOM
2416
CA
GLN
A
1105
−30.818
63.335
27.575
1.00
75.44
C


ATOM
2417
C
GLN
A
1105
−31.159
64.480
28.544
1.00
74.63
C


ATOM
2418
O
GLN
A
1105
−32.249
65.043
28.455
1.00
75.26
O


ATOM
2419
CB
GLN
A
1105
−31.100
62.012
28.270
1.00
77.85
C


ATOM
2420
CG
GLN
A
1105
−32.567
61.757
28.615
1.00
75.54
C


ATOM
2421
CD
GLN
A
1105
−32.781
60.378
29.168
1.00
75.71
C


ATOM
2422
OE1
GLN
A
1105
−31.834
59.620
29.340
1.00
71.29
O


ATOM
2423
NE2
GLN
A
1105
−34.025
60.039
29.450
1.00
70.96
N


ATOM
2424
N
MET
A
1106
−30.250
64.819
29.456
1.00
76.62
N


ATOM
2425
CA
MET
A
1106
−30.529
65.838
30.524
1.00
77.20
C


ATOM
2426
C
MET
A
1106
−29.666
67.100
30.498
1.00
75.43
C


ATOM
2427
O
MET
A
1106
−29.901
68.010
31.296
1.00
78.05
O


ATOM
2428
CB
MET
A
1106
−30.319
65.216
31.902
1.00
79.39
C


ATOM
2429
CG
MET
A
1106
−30.886
63.832
32.124
1.00
85.85
C


ATOM
2430
SD
MET
A
1106
−30.091
63.064
33.542
1.00
85.72
S


ATOM
2431
CE
MET
A
1106
−29.933
61.419
32.917
1.00
81.10
C


ATOM
2432
N
GLY
A
1107
−28.675
67.166
29.609
1.00
75.52
N


ATOM
2433
CA
GLY
A
1107
−27.748
68.294
29.560
1.00
76.26
C


ATOM
2434
C
GLY
A
1107
−26.702
68.166
30.651
1.00
78.60
C


ATOM
2435
O
GLY
A
1107
−26.827
67.330
31.555
1.00
78.11
O


ATOM
2436
N
GLU
A
1108
−25.666
68.993
30.573
1.00
79.86
N


ATOM
2437
CA
GLU
A
1108
−24.556
68.931
31.548
1.00
82.66
C


ATOM
2438
C
GLU
A
1108
−24.966
69.224
32.966
1.00
82.97
C


ATOM
2439
O
GLU
A
1108
−24.495
68.560
33.891
1.00
86.76
O


ATOM
2440
CB
GLU
A
1108
−23.454
69.901
31.185
1.00
82.78
C


ATOM
2441
CG
GLU
A
1108
−22.714
69.512
29.969
1.00
86.49
C


ATOM
2442
CD
GLU
A
1108
−21.722
70.556
29.554
1.00
90.68
C


ATOM
2443
OE1
GLU
A
1108
−21.148
71.271
30.409
1.00
90.93
O


ATOM
2444
OE2
GLU
A
1108
−21.523
70.659
28.348
1.00
103.73
O


ATOM
2445
N
THR
A
1109
−25.837
70.214
33.132
1.00
82.33
N


ATOM
2446
CA
THR
A
1109
−26.324
70.597
34.451
1.00
80.83
C


ATOM
2447
C
THR
A
1109
−27.061
69.455
35.112
1.00
79.03
C


ATOM
2448
O
THR
A
1109
−26.755
69.098
36.247
1.00
84.70
O


ATOM
2449
CB
THR
A
1109
−27.270
71.802
34.386
1.00
79.62
C


ATOM
2450
OG1
THR
A
1109
−26.591
72.907
33.776
1.00
80.39
O


ATOM
2451
CG2
THR
A
1109
−27.731
72.196
35.790
1.00
81.01
C


ATOM
2452
N
GLY
A
1110
−28.028
68.889
34.395
1.00
78.14
N


ATOM
2453
CA
GLY
A
1110
−28.794
67.749
34.888
1.00
77.59
C


ATOM
2454
C
GLY
A
1110
−27.893
66.637
35.399
1.00
76.91
C


ATOM
2455
O
GLY
A
1110
−28.147
66.069
36.450
1.00
77.84
O


ATOM
2456
N
VAL
A
1111
−26.838
66.348
34.639
1.00
74.81
N


ATOM
2457
CA
VAL
A
1111
−25.901
65.274
34.952
1.00
73.76
C


ATOM
2458
C
VAL
A
1111
−24.901
65.663
36.063
1.00
74.74
C


ATOM
2459
O
VAL
A
1111
−24.475
64.809
36.863
1.00
70.56
O


ATOM
2460
CB
VAL
A
1111
−25.094
64.844
33.715
1.00
68.49
C


ATOM
2461
CG1
VAL
A
1111
−24.173
63.695
34.081
1.00
64.99
C


ATOM
2462
CG2
VAL
A
1111
−26.023
64.438
32.582
1.00
68.09
C


ATOM
2463
N
ALA
A
1112
−24.531
66.949
36.096
1.00
74.29
N


ATOM
2464
CA
ALA
A
1112
−23.631
67.491
37.119
1.00
77.63
C


ATOM
2465
C
ALA
A
1112
−24.255
67.423
38.496
1.00
77.53
C


ATOM
2466
O
ALA
A
1112
−23.542
67.410
39.483
1.00
82.24
O


ATOM
2467
CB
ALA
A
1112
−23.256
68.931
36.798
1.00
77.58
C


ATOM
2468
N
GLY
A
1113
−25.591
67.381
38.553
1.00
79.76
N


ATOM
2469
CA
GLY
A
1113
−26.323
67.270
39.808
1.00
77.64
C


ATOM
2470
C
GLY
A
1113
−26.267
65.900
40.461
1.00
78.25
C


ATOM
2471
O
GLY
A
1113
−26.687
65.780
41.588
1.00
76.50
O


ATOM
2472
N
PHE
A
1114
−25.757
64.861
39.774
1.00
77.88
N


ATOM
2473
CA
PHE
A
1114
−25.586
63.533
40.383
1.00
76.03
C


ATOM
2474
C
PHE
A
1114
−24.276
63.500
41.179
1.00
75.36
C


ATOM
2475
O
PHE
A
1114
−23.399
62.693
40.887
1.00
73.89
O


ATOM
2476
CB
PHE
A
1114
−25.559
62.437
39.310
1.00
81.97
C


ATOM
2477
CG
PHE
A
1114
−26.845
62.247
38.594
1.00
84.39
C


ATOM
2478
CD1
PHE
A
1114
−27.906
61.609
39.202
1.00
93.33
C


ATOM
2479
CD2
PHE
A
1114
−27.001
62.696
37.310
1.00
90.99
C


ATOM
2480
CE1
PHE
A
1114
−29.108
61.433
38.521
1.00
91.37
C


ATOM
2481
CE2
PHE
A
1114
−28.187
62.526
36.627
1.00
91.29
C


ATOM
2482
CZ
PHE
A
1114
−29.244
61.894
37.229
1.00
89.69
C


ATOM
2483
N
THR
A
1115
−24.139
64.364
42.186
1.00
70.77
N


ATOM
2484
CA
THR
A
1115
−22.860
64.550
42.845
1.00
71.53
C


ATOM
2485
C
THR
A
1115
−22.292
63.256
43.420
1.00
72.52
C


ATOM
2486
O
THR
A
1115
−21.124
62.964
43.206
1.00
74.92
O


ATOM
2487
CB
THR
A
1115
−22.950
65.613
43.957
1.00
74.18
C


ATOM
2488
OG1
THR
A
1115
−23.638
66.761
43.454
1.00
81.29
O


ATOM
2489
CG2
THR
A
1115
−21.562
66.027
44.419
1.00
65.46
C


ATOM
2490
N
ASN
A
1116
−23.112
62.494
44.138
1.00
73.53
N


ATOM
2491
CA
ASN
A
1116
−22.644
61.260
44.779
1.00
74.43
C


ATOM
2492
C
ASN
A
1116
−22.327
60.176
43.753
1.00
74.36
C


ATOM
2493
O
ASN
A
1116
−21.286
59.523
43.854
1.00
73.98
O


ATOM
2494
CB
ASN
A
1116
−23.652
60.723
45.803
1.00
76.08
C


ATOM
2495
CG
ASN
A
1116
−23.916
61.701
46.958
1.00
81.08
C


ATOM
2496
OD1
ASN
A
1116
−22.994
62.125
47.647
1.00
76.24
O


ATOM
2497
ND2
ASN
A
1116
−25.181
62.050
47.163
1.00
82.05
N


ATOM
2498
N
SER
A
1117
−23.218
59.986
42.776
1.00
71.66
N


ATOM
2499
CA
SER
A
1117
−22.990
59.014
41.697
1.00
71.35
C


ATOM
2500
C
SER
A
1117
−21.736
59.307
40.895
1.00
72.60
C


ATOM
2501
O
SER
A
1117
−21.010
58.386
40.567
1.00
73.79
O


ATOM
2502
CB
SER
A
1117
−24.170
58.962
40.748
1.00
74.52
C


ATOM
2503
OG
SER
A
1117
−25.271
58.329
41.351
1.00
74.10
O


ATOM
2504
N
LEU
A
1118
−21.484
60.580
40.586
1.00
74.22
N


ATOM
2505
CA
LEU
A
1118
−20.253
60.997
39.863
1.00
74.12
C


ATOM
2506
C
LEU
A
1118
−18.961
60.660
40.618
1.00
73.96
C


ATOM
2507
O
LEU
A
1118
−17.984
60.195
40.025
1.00
78.17
O


ATOM
2508
CB
LEU
A
1118
−20.299
62.503
39.566
1.00
74.92
C


ATOM
2509
CG
LEU
A
1118
−21.337
62.937
38.500
1.00
77.06
C


ATOM
2510
CD1
LEU
A
1118
−21.516
64.484
38.518
1.00
79.91
C


ATOM
2511
CD2
LEU
A
1118
−20.950
62.456
37.120
1.00
79.15
C


ATOM
2512
N
ARG
A
1119
−18.972
60.901
41.917
1.00
73.81
N


ATOM
2513
CA
ARG
A
1119
−17.857
60.571
42.806
1.00
76.37
C


ATOM
2514
C
ARG
A
1119
−17.586
59.069
42.837
1.00
73.72
C


ATOM
2515
O
ARG
A
1119
−16.439
58.633
42.760
1.00
71.54
O


ATOM
2516
CB
ARG
A
1119
−18.179
61.041
44.229
1.00
72.72
C


ATOM
2517
CG
ARG
A
1119
−17.067
60.873
45.241
1.00
81.26
C


ATOM
2518
CD
ARG
A
1119
−17.608
60.978
46.712
1.00
88.03
C


ATOM
2519
NE
ARG
A
1119
−18.622
62.027
46.876
1.00
97.80
N


ATOM
2520
CZ
ARG
A
1119
−18.387
63.350
46.852
1.00
107.35
C


ATOM
2521
NH1
ARG
A
1119
−17.149
63.849
46.666
1.00
110.91
N


ATOM
2522
NH2
ARG
A
1119
−19.407
64.207
47.016
1.00
108.55
N


ATOM
2523
N
MET
A
1120
−18.653
58.296
42.958
1.00
72.40
N


ATOM
2524
CA
MET
A
1120
−18.554
56.843
42.948
1.00
75.44
C


ATOM
2525
C
MET
A
1120
−18.059
56.323
41.610
1.00
72.59
C


ATOM
2526
O
MET
A
1120
−17.199
55.455
41.580
1.00
74.45
O


ATOM
2527
CB
MET
A
1120
−19.884
56.228
43.338
1.00
74.73
C


ATOM
2528
CG
MET
A
1120
−20.116
56.407
44.861
1.00
79.31
C


ATOM
2529
SD
MET
A
1120
−21.698
55.810
45.341
1.00
86.73
S


ATOM
2530
CE
MET
A
1120
−22.843
56.943
44.571
1.00
82.05
C


ATOM
2531
N
LEU
A
1121
−18.592
56.851
40.516
1.00
70.51
N


ATOM
2532
CA
LEU
A
1121
−18.067
56.495
39.211
1.00
73.43
C


ATOM
2533
C
LEU
A
1121
−16.581
56.816
39.149
1.00
71.65
C


ATOM
2534
O
LEU
A
1121
−15.810
55.970
38.728
1.00
74.87
O


ATOM
2535
CB
LEU
A
1121
−18.802
57.213
38.086
1.00
67.49
C


ATOM
2536
CG
LEU
A
1121
−20.247
56.734
37.877
1.00
72.80
C


ATOM
2537
CD1
LEU
A
1121
−20.990
57.724
36.968
1.00
65.17
C


ATOM
2538
CD2
LEU
A
1121
−20.343
55.241
37.364
1.00
62.04
C


ATOM
2539
N
GLN
A
1122
−16.186
58.020
39.565
1.00
69.51
N


ATOM
2540
CA
GLN
A
1122
−14.760
58.419
39.551
1.00
69.43
C


ATOM
2541
C
GLN
A
1122
−13.852
57.422
40.302
1.00
69.96
C


ATOM
2542
O
GLN
A
1122
−12.726
57.188
39.889
1.00
72.67
O


ATOM
2543
CB
GLN
A
1122
−14.602
59.828
40.123
1.00
69.75
C


ATOM
2544
CG
GLN
A
1122
−13.203
60.432
39.975
1.00
73.87
C


ATOM
2545
CD
GLN
A
1122
−13.125
61.883
40.443
1.00
79.31
C


ATOM
2546
OE1
GLN
A
1122
−14.139
62.505
40.758
1.00
88.60
O


ATOM
2547
NE2
GLN
A
1122
−11.907
62.426
40.490
1.00
84.85
N


ATOM
2548
N
GLN
A
1123
−14.364
56.850
41.395
1.00
66.33
N


ATOM
2549
CA
GLN
A
1123
−13.659
55.833
42.182
1.00
71.40
C


ATOM
2550
C
GLN
A
1123
−13.857
54.373
41.688
1.00
70.37
C


ATOM
2551
O
GLN
A
1123
−13.323
53.450
42.300
1.00
68.04
O


ATOM
2552
CB
GLN
A
1123
−14.083
55.958
43.662
1.00
66.15
C


ATOM
2553
CG
GLN
A
1123
−13.701
57.318
44.267
1.00
65.21
C


ATOM
2554
CD
GLN
A
1123
−14.267
57.566
45.652
1.00
69.71
C


ATOM
2555
OE1
GLN
A
1123
−15.294
57.018
46.035
1.00
77.32
O


ATOM
2556
NE2
GLN
A
1123
−13.585
58.404
46.411
1.00
63.29
N


ATOM
2557
N
LYS
A
1124
−14.609
54.174
40.604
1.00
69.50
N


ATOM
2558
CA
LYS
A
1124
−14.896
52.830
40.066
1.00
74.09
C


ATOM
2559
C
LYS
A
1124
−15.719
51.959
41.048
1.00
75.70
C


ATOM
2560
O
LYS
A
1124
−15.562
50.733
41.084
1.00
75.06
O


ATOM
2561
CB
LYS
A
1124
−13.603
52.089
39.688
1.00
79.02
C


ATOM
2562
CG
LYS
A
1124
−12.571
52.892
38.892
1.00
83.65
C


ATOM
2563
CD
LYS
A
1124
−12.682
52.701
37.399
1.00
89.63
C


ATOM
2564
CE
LYS
A
1124
−11.978
53.758
36.540
1.00
92.96
C


ATOM
2565
NZ
LYS
A
1124
−12.833
55.011
36.416
1.00
104.91
N


ATOM
2566
N
ARG
A
1125
−16.586
52.609
41.828
1.00
74.41
N


ATOM
2567
CA
ARG
A
1125
−17.489
51.958
42.758
1.00
73.39
C


ATOM
2568
C
ARG
A
1125
−18.787
51.862
41.975
1.00
72.92
C


ATOM
2569
O
ARG
A
1125
−19.755
52.569
42.238
1.00
75.78
O


ATOM
2570
CB
ARG
A
1125
−17.625
52.765
44.049
1.00
72.14
C


ATOM
2571
CG
ARG
A
1125
−16.304
52.959
44.776
1.00
74.10
C


ATOM
2572
CD
ARG
A
1125
−16.448
53.860
45.974
1.00
76.04
C


ATOM
2573
NE
ARG
A
1125
−17.144
53.226
47.096
1.00
83.94
N


ATOM
2574
CZ
ARG
A
1125
−17.567
53.859
48.203
1.00
87.62
C


ATOM
2575
NH1
ARG
A
1125
−17.373
55.179
48.365
1.00
91.97
N


ATOM
2576
NH2
ARG
A
1125
−18.195
53.171
49.172
1.00
84.34
N


ATOM
2577
N
TRP
A
1126
−18.778
50.963
41.005
1.00
74.20
N


ATOM
2578
CA
TRP
A
1126
−19.839
50.847
39.981
1.00
72.97
C


ATOM
2579
C
TRP
A
1126
−21.220
50.507
40.541
1.00
71.90
C


ATOM
2580
O
TRP
A
1126
−22.203
51.187
40.232
1.00
67.72
O


ATOM
2581
CB
TRP
A
1126
−19.473
49.781
38.959
1.00
69.76
C


ATOM
2582
CG
TRP
A
1126
−18.124
49.894
38.335
1.00
70.87
C


ATOM
2583
CD1
TRP
A
1126
−17.183
48.917
38.255
1.00
66.79
C


ATOM
2584
CD2
TRP
A
1126
−17.564
51.047
37.699
1.00
70.96
C


ATOM
2585
NE1
TRP
A
1126
−16.068
49.386
37.610
1.00
69.94
N


ATOM
2586
CE2
TRP
A
1126
−16.280
50.692
37.260
1.00
70.31
C


ATOM
2587
CE3
TRP
A
1126
−18.026
52.351
37.457
1.00
71.68
C


ATOM
2588
CZ2
TRP
A
1126
−15.460
51.576
36.600
1.00
74.24
C


ATOM
2589
CZ3
TRP
A
1126
−17.205
53.235
36.801
1.00
71.20
C


ATOM
2590
CH2
TRP
A
1126
−15.939
52.850
36.379
1.00
73.75
C


ATOM
2591
N
ASP
A
1127
−21.273
49.458
41.357
1.00
72.11
N


ATOM
2592
CA
ASP
A
1127
−22.537
49.012
41.994
1.00
74.28
C


ATOM
2593
C
ASP
A
1127
−23.168
50.100
42.830
1.00
72.96
C


ATOM
2594
O
ASP
A
1127
−24.377
50.283
42.781
1.00
77.35
O


ATOM
2595
CB
ASP
A
1127
−22.326
47.768
42.874
1.00
73.85
C


ATOM
2596
CG
ASP
A
1127
−22.177
46.475
42.065
1.00
80.36
C


ATOM
2597
OD1
ASP
A
1127
−22.034
46.530
40.836
1.00
81.11
O


ATOM
2598
OD2
ASP
A
1127
−22.204
45.386
42.681
1.00
97.24
O


ATOM
2599
N
GLU
A
1128
−22.349
50.813
43.586
1.00
70.74
N


ATOM
2600
CA
GLU
A
1128
−22.840
51.886
44.452
1.00
72.33
C


ATOM
2601
C
GLU
A
1128
−23.299
53.089
43.646
1.00
70.63
C


ATOM
2602
O
GLU
A
1128
−24.276
53.740
44.017
1.00
70.95
O


ATOM
2603
CB
GLU
A
1128
−21.764
52.293
45.420
1.00
70.41
C


ATOM
2604
CG
GLU
A
1128
−21.384
51.186
46.364
1.00
79.81
C


ATOM
2605
CD
GLU
A
1128
−20.232
51.570
47.219
1.00
81.59
C


ATOM
2606
OE1
GLU
A
1128
−20.445
52.476
48.059
1.00
90.24
O


ATOM
2607
OE2
GLU
A
1128
−19.131
50.969
47.053
1.00
85.83
O


ATOM
2608
N
ALA
A
1129
−22.587
53.372
42.561
1.00
67.55
N


ATOM
2609
CA
ALA
A
1129
−23.000
54.375
41.574
1.00
71.98
C


ATOM
2610
C
ALA
A
1129
−24.378
54.000
41.058
1.00
70.13
C


ATOM
2611
O
ALA
A
1129
−25.263
54.827
41.057
1.00
76.48
O


ATOM
2612
CB
ALA
A
1129
−21.989
54.462
40.410
1.00
65.05
C


ATOM
2613
N
ALA
A
1130
−24.534
52.742
40.634
1.00
72.74
N


ATOM
2614
CA
ALA
A
1130
−25.821
52.182
40.126
1.00
70.99
C


ATOM
2615
C
ALA
A
1130
−26.956
52.249
41.142
1.00
70.01
C


ATOM
2616
O
ALA
A
1130
−28.122
52.487
40.776
1.00
68.38
O


ATOM
2617
CB
ALA
A
1130
−25.623
50.735
39.676
1.00
66.12
C


ATOM
2618
N
VAL
A
1131
−26.632
52.039
42.416
1.00
68.58
N


ATOM
2619
CA
VAL
A
1131
−27.663
52.124
43.473
1.00
67.85
C


ATOM
2620
C
VAL
A
1131
−28.222
53.535
43.503
1.00
69.11
C


ATOM
2621
O
VAL
A
1131
−29.445
53.719
43.551
1.00
67.62
O


ATOM
2622
CB
VAL
A
1131
−27.149
51.685
44.851
1.00
69.41
C


ATOM
2623
CG1
VAL
A
1131
−28.070
52.236
46.024
1.00
60.58
C


ATOM
2624
CG2
VAL
A
1131
−27.016
50.147
44.886
1.00
64.52
C


ATOM
2625
N
ASN
A
1132
−27.323
54.517
43.466
1.00
71.57
N


ATOM
2626
CA
ASN
A
1132
−27.706
55.935
43.510
1.00
73.21
C


ATOM
2627
C
ASN
A
1132
−28.408
56.412
42.232
1.00
70.74
C


ATOM
2628
O
ASN
A
1132
−29.422
57.104
42.307
1.00
68.21
O


ATOM
2629
CB
ASN
A
1132
−26.486
56.817
43.828
1.00
72.85
C


ATOM
2630
CG
ASN
A
1132
−26.112
56.776
45.296
1.00
78.49
C


ATOM
2631
OD1
ASN
A
1132
−26.088
57.807
45.962
1.00
83.67
O


ATOM
2632
ND2
ASN
A
1132
−25.822
55.588
45.808
1.00
84.32
N


ATOM
2633
N
LEU
A
1133
−27.869
56.040
41.076
1.00
69.80
N


ATOM
2634
CA
LEU
A
1133
−28.465
56.421
39.798
1.00
72.25
C


ATOM
2635
C
LEU
A
1133
−29.883
55.859
39.633
1.00
73.77
C


ATOM
2636
O
LEU
A
1133
−30.748
56.528
39.065
1.00
78.51
O


ATOM
2637
CB
LEU
A
1133
−27.569
55.971
38.637
1.00
74.37
C


ATOM
2638
CG
LEU
A
1133
−26.186
56.632
38.565
1.00
74.55
C


ATOM
2639
CD1
LEU
A
1133
−25.327
56.045
37.436
1.00
81.95
C


ATOM
2640
CD2
LEU
A
1133
−26.336
58.139
38.408
1.00
79.93
C


ATOM
2641
N
ALA
A
1134
−30.121
54.639
40.132
1.00
72.16
N


ATOM
2642
CA
ALA
A
1134
−31.455
54.022
40.090
1.00
71.47
C


ATOM
2643
C
ALA
A
1134
−32.505
54.776
40.950
1.00
72.41
C


ATOM
2644
O
ALA
A
1134
−33.701
54.586
40.741
1.00
70.87
O


ATOM
2645
CB
ALA
A
1134
−31.378
52.533
40.511
1.00
68.95
C


ATOM
2646
N
LYS
A
1135
−32.052
55.620
41.901
1.00
68.74
N


ATOM
2647
CA
LYS
A
1135
−32.946
56.436
42.773
1.00
72.99
C


ATOM
2648
C
LYS
A
1135
−33.266
57.857
42.231
1.00
72.10
C


ATOM
2649
O
LYS
A
1135
−33.585
58.762
43.000
1.00
74.92
O


ATOM
2650
CB
LYS
A
1135
−32.343
56.536
44.199
1.00
69.32
C


ATOM
2651
CG
LYS
A
1135
−32.402
55.253
45.012
1.00
76.21
C


ATOM
2652
CD
LYS
A
1135
−31.674
55.410
46.367
1.00
77.13
C


ATOM
2653
CE
LYS
A
1135
−31.950
54.245
47.310
1.00
77.91
C


ATOM
2654
NZ
LYS
A
1135
−30.938
54.134
48.434
1.00
77.31
N


ATOM
2655
N
SER
A
1136
−33.185
58.051
40.916
1.00
76.65
N


ATOM
2656
CA
SER
A
1136
−33.404
59.364
40.298
1.00
74.00
C


ATOM
2657
C
SER
A
1136
−34.753
59.516
39.594
1.00
76.99
C


ATOM
2658
O
SER
A
1136
−35.464
58.536
39.320
1.00
79.35
O


ATOM
2659
CB
SER
A
1136
−32.289
59.635
39.304
1.00
75.82
C


ATOM
2660
OG
SER
A
1136
−32.139
58.573
38.368
1.00
74.98
O


ATOM
2661
N
ARG
A
1137
−35.081
60.775
39.313
1.00
74.80
N


ATOM
2662
CA
ARG
A
1137
−36.216
61.141
38.485
1.00
72.91
C


ATOM
2663
C
ARG
A
1137
−35.965
60.639
37.043
1.00
71.62
C


ATOM
2664
O
ARG
A
1137
−36.884
60.312
36.325
1.00
70.32
O


ATOM
2665
CB
ARG
A
1137
−36.395
62.661
38.504
1.00
71.58
C


ATOM
2666
CG
ARG
A
1137
−37.567
63.220
37.702
1.00
76.14
C


ATOM
2667
CD
ARG
A
1137
−38.896
62.715
38.231
1.00
84.99
C


ATOM
2668
NE
ARG
A
1137
−40.034
63.267
37.497
1.00
85.80
N


ATOM
2669
CZ
ARG
A
1137
−41.305
62.894
37.664
1.00
83.56
C


ATOM
2670
NH1
ARG
A
1137
−41.640
61.951
38.547
1.00
74.66
N


ATOM
2671
NH2
ARG
A
1137
−42.263
63.469
36.933
1.00
88.55
N


ATOM
2672
N
TRP
A
1138
−34.703
60.592
36.651
1.00
70.67
N


ATOM
2673
CA
TRP
A
1138
−34.289
60.023
35.392
1.00
72.64
C


ATOM
2674
C
TRP
A
1138
−34.759
58.569
35.248
1.00
74.44
C


ATOM
2675
O
TRP
A
1138
−35.428
58.233
34.279
1.00
74.91
O


ATOM
2676
CB
TRP
A
1138
−32.778
60.118
35.298
1.00
72.63
C


ATOM
2677
CG
TRP
A
1138
−32.166
59.434
34.164
1.00
72.19
C


ATOM
2678
CD1
TRP
A
1138
−32.556
59.491
32.862
1.00
72.21
C


ATOM
2679
CD2
TRP
A
1138
−31.024
58.574
34.207
1.00
68.92
C


ATOM
2680
NE1
TRP
A
1138
−31.724
58.713
32.092
1.00
73.58
N


ATOM
2681
CE2
TRP
A
1138
−30.780
58.139
32.887
1.00
67.95
C


ATOM
2682
CE3
TRP
A
1138
−30.190
58.125
35.223
1.00
66.16
C


ATOM
2683
CZ2
TRP
A
1138
−29.749
57.293
32.570
1.00
69.41
C


ATOM
2684
CZ3
TRP
A
1138
−29.142
57.262
34.893
1.00
71.01
C


ATOM
2685
CH2
TRP
A
1138
−28.934
56.863
33.600
1.00
71.24
C


ATOM
2686
N
TYR
A
1139
−34.407
57.727
36.212
1.00
73.44
N


ATOM
2687
CA
TYR
A
1139
−34.828
56.318
36.196
1.00
70.88
C


ATOM
2688
C
TYR
A
1139
−36.337
56.170
36.397
1.00
71.38
C


ATOM
2689
O
TYR
A
1139
−36.940
55.218
35.922
1.00
72.03
O


ATOM
2690
CB
TYR
A
1139
−34.064
55.516
37.258
1.00
74.06
C


ATOM
2691
CG
TYR
A
1139
−34.530
54.095
37.404
1.00
70.82
C


ATOM
2692
CD1
TYR
A
1139
−34.012
53.101
36.601
1.00
76.39
C


ATOM
2693
CD2
TYR
A
1139
−35.496
53.746
38.351
1.00
76.13
C


ATOM
2694
CE1
TYR
A
1139
−34.429
51.793
36.720
1.00
73.02
C


ATOM
2695
CE2
TYR
A
1139
−35.923
52.436
38.481
1.00
79.81
C


ATOM
2696
CZ
TYR
A
1139
−35.376
51.461
37.651
1.00
77.76
C


ATOM
2697
OH
TYR
A
1139
−35.772
50.154
37.747
1.00
78.31
O


ATOM
2698
N
ASN
A
1140
−36.943
57.111
37.106
1.00
72.72
N


ATOM
2699
CA
ASN
A
1140
−38.381
57.111
37.312
1.00
71.50
C


ATOM
2700
C
ASN
A
1140
−39.175
57.391
36.028
1.00
70.46
C


ATOM
2701
O
ASN
A
1140
−40.307
56.929
35.882
1.00
72.15
O


ATOM
2702
CB
ASN
A
1140
−38.755
58.172
38.338
1.00
68.60
C


ATOM
2703
CG
ASN
A
1140
−40.230
58.240
38.572
1.00
72.49
C


ATOM
2704
OD1
ASN
A
1140
−40.864
59.264
38.335
1.00
72.97
O


ATOM
2705
ND2
ASN
A
1140
−40.801
57.138
39.040
1.00
83.45
N


ATOM
2706
N
GLN
A
1141
−38.573
58.145
35.111
1.00
71.15
N


ATOM
2707
CA
GLN
A
1141
−39.248
58.616
33.896
1.00
71.46
C


ATOM
2708
C
GLN
A
1141
−38.919
57.755
32.677
1.00
72.46
C


ATOM
2709
O
GLN
A
1141
−39.828
57.378
31.930
1.00
69.45
O


ATOM
2710
CB
GLN
A
1141
−38.887
60.091
33.663
1.00
71.54
C


ATOM
2711
CG
GLN
A
1141
−39.493
61.040
34.694
1.00
74.06
C


ATOM
2712
CD
GLN
A
1141
−40.987
61.142
34.576
1.00
84.66
C


ATOM
2713
OE1
GLN
A
1141
−41.726
60.867
35.539
1.00
72.30
O


ATOM
2714
NE2
GLN
A
1141
−41.461
61.535
33.387
1.00
78.96
N


ATOM
2715
N
THR
A
1142
−37.633
57.454
32.490
1.00
69.68
N


ATOM
2716
CA
THR
A
1142
−37.167
56.582
31.409
1.00
71.24
C


ATOM
2717
C
THR
A
1142
−36.399
55.382
31.997
1.00
71.57
C


ATOM
2718
O
THR
A
1142
−35.177
55.274
31.783
1.00
73.67
O


ATOM
2719
CB
THR
A
1142
−36.318
57.373
30.396
1.00
73.10
C


ATOM
2720
OG1
THR
A
1142
−35.335
58.134
31.100
1.00
65.73
O


ATOM
2721
CG2
THR
A
1142
−37.223
58.326
29.569
1.00
63.92
C


ATOM
2722
N
PRO
A
1143
−37.120
54.475
32.744
1.00
68.91
N


ATOM
2723
CA
PRO
A
1143
−36.537
53.299
33.404
1.00
67.95
C


ATOM
2724
C
PRO
A
1143
−35.762
52.372
32.497
1.00
71.67
C


ATOM
2725
O
PRO
A
1143
−34.638
52.018
32.834
1.00
70.47
O


ATOM
2726
CB
PRO
A
1143
−37.760
52.553
33.937
1.00
70.48
C


ATOM
2727
CG
PRO
A
1143
−38.920
53.137
33.252
1.00
70.91
C


ATOM
2728
CD
PRO
A
1143
−38.570
54.528
33.001
1.00
68.98
C


ATOM
2729
N
ASN
A
1144
−36.357
51.993
31.361
1.00
70.87
N


ATOM
2730
CA
ASN
A
1144
−35.706
51.083
30.410
1.00
71.14
C


ATOM
2731
C
ASN
A
1144
−34.382
51.613
29.946
1.00
72.08
C


ATOM
2732
O
ASN
A
1144
−33.369
50.921
30.071
1.00
71.77
O


ATOM
2733
CB
ASN
A
1144
−36.582
50.818
29.180
1.00
71.11
C


ATOM
2734
CG
ASN
A
1144
−37.859
50.077
29.515
1.00
74.27
C


ATOM
2735
OD1
ASN
A
1144
−37.919
49.323
30.480
1.00
76.06
O


ATOM
2736
ND2
ASN
A
1144
−38.895
50.288
28.709
1.00
86.76
N


ATOM
2737
N
ARG
A
1145
−34.388
52.837
29.409
1.00
70.29
N


ATOM
2738
CA
ARG
A
1145
−33.147
53.500
28.979
1.00
69.78
C


ATOM
2739
C
ARG
A
1145
−32.142
53.627
30.132
1.00
70.79
C


ATOM
2740
O
ARG
A
1145
−30.950
53.299
29.984
1.00
71.02
O


ATOM
2741
CB
ARG
A
1145
−33.450
54.880
28.462
1.00
69.40
C


ATOM
2742
CG
ARG
A
1145
−32.215
55.697
28.103
1.00
72.46
C


ATOM
2743
CD
ARG
A
1145
−32.619
56.971
27.368
1.00
71.18
C


ATOM
2744
NE
ARG
A
1145
−31.446
57.732
26.931
1.00
74.68
N


ATOM
2745
CZ
ARG
A
1145
−31.488
58.840
26.186
1.00
81.63
C


ATOM
2746
NH1
ARG
A
1145
−32.661
59.353
25.769
1.00
76.56
N


ATOM
2747
NH2
ARG
A
1145
−30.354
59.453
25.844
1.00
80.74
N


ATOM
2748
N
ALA
A
1146
−32.632
54.105
31.272
1.00
68.56
N


ATOM
2749
CA
ALA
A
1146
−31.782
54.265
32.437
1.00
70.12
C


ATOM
2750
C
ALA
A
1146
−31.211
52.906
32.844
1.00
68.68
C


ATOM
2751
O
ALA
A
1146
−30.039
52.815
33.194
1.00
70.80
O


ATOM
2752
CB
ALA
A
1146
−32.534
54.894
33.547
1.00
66.71
C


ATOM
2753
N
LYS
A
1147
−32.029
51.862
32.786
1.00
69.92
N


ATOM
2754
CA
LYS
A
1147
−31.583
50.524
33.161
1.00
72.10
C


ATOM
2755
C
LYS
A
1147
−30.365
50.177
32.322
1.00
72.04
C


ATOM
2756
O
LYS
A
1147
−29.317
49.794
32.881
1.00
66.93
O


ATOM
2757
CB
LYS
A
1147
−32.712
49.514
32.992
1.00
73.45
C


ATOM
2758
CG
LYS
A
1147
−32.536
48.177
33.665
1.00
78.51
C


ATOM
2759
CD
LYS
A
1147
−33.835
47.348
33.477
1.00
82.18
C


ATOM
2760
CE
LYS
A
1147
−33.762
45.956
34.103
1.00
93.88
C


ATOM
2761
NZ
LYS
A
1147
−33.816
45.967
35.617
1.00
96.78
N


ATOM
2762
N
ARG
A
1148
−30.494
50.323
30.986
1.00
73.00
N


ATOM
2763
CA
ARG
A
1148
−29.393
50.016
30.051
1.00
70.89
C


ATOM
2764
C
ARG
A
1148
−28.135
50.860
30.239
1.00
74.78
C


ATOM
2765
O
ARG
A
1148
−27.034
50.334
30.098
1.00
80.02
O


ATOM
2766
CB
ARG
A
1148
−29.832
50.150
28.590
1.00
72.03
C


ATOM
2767
CG
ARG
A
1148
−30.780
49.068
28.099
1.00
70.33
C


ATOM
2768
CD
ARG
A
1148
−30.936
49.102
26.596
1.00
72.06
C


ATOM
2769
NE
ARG
A
1148
−31.674
50.290
26.158
1.00
71.65
N


ATOM
2770
CZ
ARG
A
1148
−33.006
50.391
26.084
1.00
72.14
C


ATOM
2771
NH1
ARG
A
1148
−33.801
49.382
26.416
1.00
78.11
N


ATOM
2772
NH2
ARG
A
1148
−33.568
51.523
25.671
1.00
71.64
N


ATOM
2773
N
VAL
A
1149
−28.270
52.150
30.547
1.00
74.24
N


ATOM
2774
CA
VAL
A
1149
−27.072
52.990
30.752
1.00
71.90
C


ATOM
2775
C
VAL
A
1149
−26.399
52.632
32.098
1.00
74.21
C


ATOM
2776
O
VAL
A
1149
−25.188
52.478
32.164
1.00
77.27
O


ATOM
2777
CB
VAL
A
1149
−27.374
54.510
30.676
1.00
78.88
C


ATOM
2778
CG1
VAL
A
1149
−26.122
55.316
31.006
1.00
78.08
C


ATOM
2779
CG2
VAL
A
1149
−27.939
54.922
29.282
1.00
63.48
C


ATOM
2780
N
ILE
A
1150
−27.182
52.498
33.158
1.00
70.73
N


ATOM
2781
CA
ILE
A
1150
−26.623
52.164
34.467
1.00
70.91
C


ATOM
2782
C
ILE
A
1150
−25.901
50.823
34.437
1.00
69.25
C


ATOM
2783
O
ILE
A
1150
−24.744
50.727
34.864
1.00
74.32
O


ATOM
2784
CB
ILE
A
1150
−27.689
52.145
35.570
1.00
71.99
C


ATOM
2785
CG1
ILE
A
1150
−28.264
53.541
35.793
1.00
69.99
C


ATOM
2786
CG2
ILE
A
1150
−27.077
51.645
36.848
1.00
70.14
C


ATOM
2787
CD1
ILE
A
1150
−29.550
53.539
36.637
1.00
76.32
C


ATOM
2788
N
THR
A
1151
−26.578
49.793
33.938
1.00
67.61
N


ATOM
2789
CA
THR
A
1151
−25.954
48.476
33.707
1.00
67.20
C


ATOM
2790
C
THR
A
1151
−24.648
48.608
32.920
1.00
70.88
C


ATOM
2791
O
THR
A
1151
−23.683
47.887
33.190
1.00
74.26
O


ATOM
2792
CB
THR
A
1151
−26.876
47.558
32.905
1.00
68.20
C


ATOM
2793
OG1
THR
A
1151
−28.158
47.491
33.541
1.00
72.09
O


ATOM
2794
CG2
THR
A
1151
−26.270
46.158
32.776
1.00
64.27
C


ATOM
2795
N
THR
A
1152
−24.619
49.527
31.941
1.00
67.75
N


ATOM
2796
CA
THR
A
1152
−23.399
49.788
31.157
1.00
70.23
C


ATOM
2797
C
THR
A
1152
−22.322
50.417
32.042
1.00
69.27
C


ATOM
2798
O
THR
A
1152
−21.145
50.068
31.924
1.00
71.67
O


ATOM
2799
CB
THR
A
1152
−23.676
50.696
29.941
1.00
66.28
C


ATOM
2800
OG1
THR
A
1152
−24.680
50.076
29.121
1.00
70.58
O


ATOM
2801
CG2
THR
A
1152
−22.396
50.939
29.157
1.00
69.86
C


ATOM
2802
N
PHE
A
1153
−22.719
51.337
32.913
1.00
68.71
N


ATOM
2803
CA
PHE
A
1153
−21.791
51.796
33.990
1.00
74.06
C


ATOM
2804
C
PHE
A
1153
−21.345
50.649
34.890
1.00
70.59
C


ATOM
2805
O
PHE
A
1153
−20.232
50.662
35.389
1.00
76.73
O


ATOM
2806
CB
PHE
A
1153
−22.391
52.892
34.872
1.00
71.58
C


ATOM
2807
CG
PHE
A
1153
−22.359
54.274
34.258
1.00
75.33
C


ATOM
2808
CD1
PHE
A
1153
−21.180
54.807
33.778
1.00
76.55
C


ATOM
2809
CD2
PHE
A
1153
−23.508
55.034
34.170
1.00
79.26
C


ATOM
2810
CE1
PHE
A
1153
−21.147
56.059
33.224
1.00
73.15
C


ATOM
2811
CE2
PHE
A
1153
−23.479
56.282
33.622
1.00
72.03
C


ATOM
2812
CZ
PHE
A
1153
−22.296
56.799
33.147
1.00
71.84
C


ATOM
2813
N
ARG
A
1154
−22.198
49.655
35.108
1.00
70.31
N


ATOM
2814
CA
ARG
A
1154
−21.801
48.490
35.940
1.00
72.56
C


ATOM
2815
C
ARG
A
1154
−20.774
47.559
35.312
1.00
74.07
C


ATOM
2816
O
ARG
A
1154
−19.826
47.158
35.994
1.00
75.46
O


ATOM
2817
CB
ARG
A
1154
−23.013
47.661
36.354
1.00
71.60
C


ATOM
2818
CG
ARG
A
1154
−23.926
48.392
37.315
1.00
78.93
C


ATOM
2819
CD
ARG
A
1154
−24.313
47.484
38.485
1.00
80.49
C


ATOM
2820
NE
ARG
A
1154
−25.312
46.492
38.126
1.00
81.98
N


ATOM
2821
CZ
ARG
A
1154
−25.657
45.436
38.870
1.00
84.48
C


ATOM
2822
NH1
ARG
A
1154
−25.082
45.186
40.063
1.00
90.99
N


ATOM
2823
NH2
ARG
A
1154
−26.595
44.601
38.421
1.00
88.13
N


ATOM
2824
N
THR
A
1155
−20.956
47.219
34.029
1.00
73.12
N


ATOM
2825
CA
THR
A
1155
−20.095
46.232
33.346
1.00
74.07
C


ATOM
2826
C
THR
A
1155
−18.969
46.828
32.501
1.00
76.10
C


ATOM
2827
O
THR
A
1155
−17.912
46.218
32.361
1.00
76.98
O


ATOM
2828
CB
THR
A
1155
−20.940
45.324
32.422
1.00
75.54
C


ATOM
2829
OG1
THR
A
1155
−21.368
46.063
31.251
1.00
76.27
O


ATOM
2830
CG2
THR
A
1155
−22.152
44.777
33.176
1.00
73.31
C


ATOM
2831
N
GLY
A
1156
−19.185
48.008
31.936
1.00
75.88
N


ATOM
2832
CA
GLY
A
1156
−18.198
48.591
31.038
1.00
76.99
C


ATOM
2833
C
GLY
A
1156
−18.113
47.829
29.703
1.00
78.89
C


ATOM
2834
O
GLY
A
1156
−17.054
47.785
29.073
1.00
77.75
O


ATOM
2835
N
THR
A
1157
−19.240
47.235
29.297
1.00
79.45
N


ATOM
2836
CA
THR
A
1157
−19.388
46.573
28.025
1.00
75.30
C


ATOM
2837
C
THR
A
1157
−20.713
47.007
27.435
1.00
76.85
C


ATOM
2838
O
THR
A
1157
−21.568
47.591
28.121
1.00
72.71
O


ATOM
2839
CB
THR
A
1157
−19.491
45.069
28.150
1.00
77.35
C


ATOM
2840
OG1
THR
A
1157
−20.771
44.747
28.718
1.00
78.06
O


ATOM
2841
CG2
THR
A
1157
−18.332
44.460
29.016
1.00
73.49
C


ATOM
2842
N
TRP
A
1158
−20.880
46.706
26.149
1.00
75.57
N


ATOM
2843
CA
TRP
A
1158
−22.102
47.000
25.412
1.00
72.41
C


ATOM
2844
C
TRP
A
1158
−23.213
45.956
25.579
1.00
72.74
C


ATOM
2845
O
TRP
A
1158
−24.243
46.070
24.904
1.00
74.46
O


ATOM
2846
CB
TRP
A
1158
−21.769
47.101
23.927
1.00
76.07
C


ATOM
2847
CG
TRP
A
1158
−20.844
48.147
23.646
1.00
74.03
C


ATOM
2848
CD1
TRP
A
1158
−19.563
48.007
23.274
1.00
71.12
C


ATOM
2849
CD2
TRP
A
1158
−21.107
49.539
23.717
1.00
72.83
C


ATOM
2850
NE1
TRP
A
1158
−18.986
49.238
23.096
1.00
76.80
N


ATOM
2851
CE2
TRP
A
1158
−19.916
50.200
23.362
1.00
66.00
C


ATOM
2852
CE3
TRP
A
1158
−22.238
50.297
24.049
1.00
74.60
C


ATOM
2853
CZ2
TRP
A
1158
−19.811
51.582
23.322
1.00
71.92
C


ATOM
2854
CZ3
TRP
A
1158
−22.138
51.673
24.012
1.00
76.31
C


ATOM
2855
CH2
TRP
A
1158
−20.927
52.307
23.649
1.00
74.45
C


ATOM
2856
N
ASP
A
1159
−23.019
44.955
26.460
1.00
72.02
N


ATOM
2857
CA
ASP
A
1159
−23.967
43.835
26.664
1.00
71.28
C


ATOM
2858
C
ASP
A
1159
−25.450
44.207
26.837
1.00
72.00
C


ATOM
2859
O
ASP
A
1159
−26.316
43.422
26.450
1.00
73.98
O


ATOM
2860
CB
ASP
A
1159
−23.527
42.966
27.852
1.00
73.56
C


ATOM
2861
CG
ASP
A
1159
−22.254
42.143
27.570
1.00
78.31
C


ATOM
2862
OD1
ASP
A
1159
−21.715
42.178
26.450
1.00
78.77
O


ATOM
2863
OD2
ASP
A
1159
−21.791
41.449
28.499
1.00
92.78
O


ATOM
2864
N
ALA
A
1160
−25.746
45.381
27.405
1.00
72.33
N


ATOM
2865
CA
ALA
A
1160
−27.154
45.828
27.567
1.00
72.44
C


ATOM
2866
C
ALA
A
1160
−27.815
46.215
26.238
1.00
72.65
C


ATOM
2867
O
ALA
A
1160
−29.037
46.285
26.169
1.00
75.90
O


ATOM
2868
CB
ALA
A
1160
−27.254
46.998
28.560
1.00
69.43
C


ATOM
2869
N
TYR
A
1161
−27.010
46.465
25.200
1.00
73.56
N


ATOM
2870
CA
TYR
A
1161
−27.500
46.850
23.870
1.00
70.85
C


ATOM
2871
C
TYR
A
1161
−27.500
45.693
22.845
1.00
71.12
C


ATOM
2872
O
TYR
A
1161
−27.779
45.928
21.684
1.00
74.87
O


ATOM
2873
CB
TYR
A
1161
−26.712
48.067
23.381
1.00
70.71
C


ATOM
2874
CG
TYR
A
1161
−26.977
49.224
24.282
1.00
69.57
C


ATOM
2875
CD1
TYR
A
1161
−28.020
50.090
24.037
1.00
67.53
C


ATOM
2876
CD2
TYR
A
1161
−26.187
49.451
25.400
1.00
71.83
C


ATOM
2877
CE1
TYR
A
1161
−28.269
51.157
24.878
1.00
69.80
C


ATOM
2878
CE2
TYR
A
1161
−26.432
50.509
26.236
1.00
64.36
C


ATOM
2879
CZ
TYR
A
1161
−27.470
51.366
25.980
1.00
73.12
C


ATOM
2880
OH
TYR
A
1161
−27.695
52.436
26.850
1.00
77.23
O


ATOM
2881
N
LYS
A
263
−27.193
44.468
23.289
1.00
68.62
N


ATOM
2882
CA
LYS
A
263
−27.211
43.302
22.437
1.00
68.39
C


ATOM
2883
C
LYS
A
263
−28.617
42.992
22.039
1.00
68.68
C


ATOM
2884
O
LYS
A
263
−29.497
43.032
22.873
1.00
71.28
O


ATOM
2885
CB
LYS
A
263
−26.716
42.052
23.152
1.00
70.24
C


ATOM
2886
CG
LYS
A
263
−25.247
41.957
23.391
1.00
76.63
C


ATOM
2887
CD
LYS
A
263
−24.916
40.567
23.966
1.00
77.14
C


ATOM
2888
CE
LYS
A
263
−23.433
40.257
23.926
1.00
86.04
C


ATOM
2889
NZ
LYS
A
263
−23.139
38.781
24.083
1.00
88.05
N


ATOM
2890
N
PHE
A
264
−28.823
42.683
20.766
1.00
66.31
N


ATOM
2891
CA
PHE
A
264
−30.129
42.267
20.240
1.00
67.14
C


ATOM
2892
C
PHE
A
264
−31.247
43.272
20.499
1.00
66.08
C


ATOM
2893
O
PHE
A
264
−32.385
42.892
20.754
1.00
66.16
O


ATOM
2894
CB
PHE
A
264
−30.490
40.877
20.774
1.00
65.00
C


ATOM
2895
CG
PHE
A
264
−29.455
39.843
20.461
1.00
68.77
C


ATOM
2896
CD1
PHE
A
264
−28.761
39.182
21.465
1.00
69.39
C


ATOM
2897
CD2
PHE
A
264
−29.166
39.530
19.135
1.00
69.77
C


ATOM
2898
CE1
PHE
A
264
−27.800
38.223
21.148
1.00
67.15
C


ATOM
2899
CE2
PHE
A
264
−28.210
38.578
18.825
1.00
68.88
C


ATOM
2900
CZ
PHE
A
264
−27.531
37.925
19.832
1.00
67.43
C


ATOM
2901
N
CYS
A
265
−30.891
44.551
20.430
1.00
66.76
N


ATOM
2902
CA
CYS
A
265
−31.837
45.636
20.494
1.00
66.33
C


ATOM
2903
C
CYS
A
265
−32.192
46.034
19.051
1.00
66.21
C


ATOM
2904
O
CYS
A
265
−33.355
46.217
18.742
1.00
67.25
O


ATOM
2905
CB
CYS
A
265
−31.269
46.818
21.274
1.00
65.09
C


ATOM
2906
SG
CYS
A
265
−31.066
46.521
23.110
1.00
69.25
S


ATOM
2907
N
LEU
A
266
−31.183
46.163
18.186
1.00
63.62
N


ATOM
2908
CA
LEU
A
266
−31.385
46.560
16.806
1.00
65.11
C


ATOM
2909
C
LEU
A
266
−32.130
45.487
16.037
1.00
65.91
C


ATOM
2910
O
LEU
A
266
−31.815
44.312
16.172
1.00
65.41
O


ATOM
2911
CB
LEU
A
266
−30.044
46.787
16.116
1.00
63.47
C


ATOM
2912
CG
LEU
A
266
−29.137
47.928
16.567
1.00
66.69
C


ATOM
2913
CD1
LEU
A
266
−27.791
47.827
15.848
1.00
64.50
C


ATOM
2914
CD2
LEU
A
266
−29.796
49.271
16.325
1.00
66.85
C


ATOM
2915
N
LYS
A
267
−33.112
45.898
15.238
1.00
68.75
N


ATOM
2916
CA
LYS
A
267
−33.870
44.973
14.350
1.00
71.90
C


ATOM
2917
C
LYS
A
267
−32.973
44.007
13.566
1.00
69.09
C


ATOM
2918
O
LYS
A
267
−33.274
42.829
13.463
1.00
72.39
O


ATOM
2919
CB
LYS
A
267
−34.768
45.762
13.373
1.00
74.97
C


ATOM
2920
CG
LYS
A
267
−34.008
46.721
12.419
1.00
82.81
C


ATOM
2921
CD
LYS
A
267
−34.913
47.656
11.634
1.00
84.17
C


ATOM
2922
CE
LYS
A
267
−34.071
48.658
10.816
1.00
88.72
C


ATOM
2923
NZ
LYS
A
267
−34.904
49.777
10.290
1.00
88.47
N


ATOM
2924
N
GLU
A
268
−31.877
44.519
13.024
1.00
68.14
N


ATOM
2925
CA
GLU
A
268
−30.929
43.706
12.257
1.00
68.69
C


ATOM
2926
C
GLU
A
268
−30.232
42.665
13.125
1.00
69.97
C


ATOM
2927
O
GLU
A
268
−30.010
41.554
12.679
1.00
71.10
O


ATOM
2928
CB
GLU
A
268
−29.897
44.568
11.492
1.00
71.25
C


ATOM
2929
CG
GLU
A
268
−29.063
45.591
12.318
1.00
82.60
C


ATOM
2930
CD
GLU
A
268
−29.720
46.975
12.491
1.00
84.41
C


ATOM
2931
OE1
GLU
A
268
−30.934
47.164
12.219
1.00
81.45
O


ATOM
2932
OE2
GLU
A
268
−28.992
47.877
12.911
1.00
83.78
O


ATOM
2933
N
HIS
A
269
−29.891
43.023
14.366
1.00
70.32
N


ATOM
2934
CA
HIS
A
269
−29.274
42.065
15.287
1.00
68.22
C


ATOM
2935
C
HIS
A
269
−30.290
41.039
15.757
1.00
66.82
C


ATOM
2936
O
HIS
A
269
−29.946
39.856
15.882
1.00
65.02
O


ATOM
2937
CB
HIS
A
269
−28.612
42.794
16.451
1.00
71.92
C


ATOM
2938
CG
HIS
A
269
−27.390
43.554
16.047
1.00
71.60
C


ATOM
2939
ND1
HIS
A
269
−26.547
44.134
16.958
1.00
68.24
N


ATOM
2940
CD2
HIS
A
269
−26.867
43.824
14.824
1.00
75.13
C


ATOM
2941
CE1
HIS
A
269
−25.557
44.734
16.325
1.00
73.61
C


ATOM
2942
NE2
HIS
A
269
−25.725
44.562
15.027
1.00
78.78
N


ATOM
2943
N
LYS
A
270
−31.526
41.477
16.014
1.00
64.48
N


ATOM
2944
CA
LYS
A
270
−32.619
40.536
16.296
1.00
67.98
C


ATOM
2945
C
LYS
A
270
−32.761
39.521
15.146
1.00
68.82
C


ATOM
2946
O
LYS
A
270
−32.861
38.328
15.394
1.00
66.36
O


ATOM
2947
CB
LYS
A
270
−33.948
41.247
16.519
1.00
66.79
C


ATOM
2948
CG
LYS
A
270
−33.987
42.127
17.773
1.00
73.23
C


ATOM
2949
CD
LYS
A
270
−35.412
42.556
18.088
1.00
71.51
C


ATOM
2950
CE
LYS
A
270
−35.487
43.405
19.345
1.00
75.87
C


ATOM
2951
NZ
LYS
A
270
−35.017
42.669
20.545
1.00
77.55
N


ATOM
2952
N
ALA
A
271
−32.764
40.009
13.897
1.00
69.99
N


ATOM
2953
CA
ALA
A
271
−32.855
39.121
12.713
1.00
71.38
C


ATOM
2954
C
ALA
A
271
−31.731
38.111
12.727
1.00
71.12
C


ATOM
2955
O
ALA
A
271
−31.975
36.946
12.466
1.00
73.66
O


ATOM
2956
CB
ALA
A
271
−32.855
39.918
11.399
1.00
64.88
C


ATOM
2957
N
LEU
A
272
−30.502
38.548
13.034
1.00
72.07
N


ATOM
2958
CA
LEU
A
272
−29.359
37.599
13.151
1.00
74.58
C


ATOM
2959
C
LEU
A
272
−29.561
36.513
14.240
1.00
75.85
C


ATOM
2960
O
LEU
A
272
−29.137
35.367
14.051
1.00
81.93
O


ATOM
2961
CB
LEU
A
272
−28.025
38.324
13.408
1.00
73.91
C


ATOM
2962
CG
LEU
A
272
−27.406
39.228
12.329
1.00
78.65
C


ATOM
2963
CD1
LEU
A
272
−26.129
39.864
12.873
1.00
79.09
C


ATOM
2964
CD2
LEU
A
272
−27.118
38.465
11.080
1.00
65.55
C


ATOM
2965
N
LYS
A
273
−30.195
36.864
15.358
1.00
76.42
N


ATOM
2966
CA
LYS
A
273
−30.509
35.866
16.403
1.00
77.14
C


ATOM
2967
C
LYS
A
273
−31.512
34.845
15.857
1.00
77.91
C


ATOM
2968
O
LYS
A
273
−31.346
33.663
16.083
1.00
76.67
O


ATOM
2969
CB
LYS
A
273
−31.049
36.529
17.656
1.00
78.11
C


ATOM
2970
CG
LYS
A
273
−31.226
35.608
18.867
1.00
80.19
C


ATOM
2971
CD
LYS
A
273
−31.473
36.443
20.100
1.00
79.05
C


ATOM
2972
CE
LYS
A
273
−31.752
35.624
21.329
1.00
82.30
C


ATOM
2973
NZ
LYS
A
273
−31.953
36.544
22.492
1.00
84.12
N


ATOM
2974
N
THR
A
274
−32.541
35.313
15.139
1.00
80.30
N


ATOM
2975
CA
THR
A
274
−33.525
34.414
14.487
1.00
78.23
C


ATOM
2976
C
THR
A
274
−32.827
33.369
13.651
1.00
76.51
C


ATOM
2977
O
THR
A
274
−33.082
32.191
13.826
1.00
75.03
O


ATOM
2978
CB
THR
A
274
−34.520
35.166
13.564
1.00
81.00
C


ATOM
2979
OG1
THR
A
274
−35.198
36.197
14.293
1.00
83.35
O


ATOM
2980
CG2
THR
A
274
−35.553
34.206
12.963
1.00
79.62
C


ATOM
2981
N
LEU
A
275
−31.950
33.800
12.754
1.00
73.92
N


ATOM
2982
CA
LEU
A
275
−31.183
32.862
11.925
1.00
74.39
C


ATOM
2983
C
LEU
A
275
−30.330
31.927
12.779
1.00
75.45
C


ATOM
2984
O
LEU
A
275
−30.279
30.721
12.524
1.00
76.13
O


ATOM
2985
CB
LEU
A
275
−30.301
33.605
10.922
1.00
75.70
C


ATOM
2986
CG
LEU
A
275
−30.975
34.417
9.808
1.00
75.16
C


ATOM
2987
CD1
LEU
A
275
−29.909
35.169
8.981
1.00
67.26
C


ATOM
2988
CD2
LEU
A
275
−31.846
33.533
8.910
1.00
70.95
C


ATOM
2989
N
GLY
A
276
−29.663
32.476
13.797
1.00
78.78
N


ATOM
2990
CA
GLY
A
276
−28.890
31.661
14.740
1.00
73.44
C


ATOM
2991
C
GLY
A
276
−29.740
30.605
15.422
1.00
73.61
C


ATOM
2992
O
GLY
A
276
−29.247
29.515
15.743
1.00
74.08
O


ATOM
2993
N
ILE
A
277
−31.017
30.921
15.645
1.00
73.26
N


ATOM
2994
CA
ILE
A
277
−31.955
29.987
16.286
1.00
76.95
C


ATOM
2995
C
ILE
A
277
−32.354
28.862
15.334
1.00
75.85
C


ATOM
2996
O
ILE
A
277
−32.540
27.720
15.768
1.00
77.29
O


ATOM
2997
CB
ILE
A
277
−33.202
30.721
16.866
1.00
74.67
C


ATOM
2998
CG1
ILE
A
277
−32.797
31.558
18.081
1.00
76.85
C


ATOM
2999
CG2
ILE
A
277
−34.275
29.747
17.287
1.00
72.00
C


ATOM
3000
CD1
ILE
A
277
−33.970
32.239
18.788
1.00
76.44
C


ATOM
3001
N
ILE
A
278
−32.481
29.188
14.048
1.00
79.83
N


ATOM
3002
CA
ILE
A
278
−32.787
28.208
13.010
1.00
77.27
C


ATOM
3003
C
ILE
A
278
−31.630
27.225
12.931
1.00
79.77
C


ATOM
3004
O
ILE
A
278
−31.833
26.004
12.877
1.00
80.80
O


ATOM
3005
CB
ILE
A
278
−33.004
28.892
11.637
1.00
77.96
C


ATOM
3006
CG1
ILE
A
278
−34.244
29.798
11.651
1.00
79.43
C


ATOM
3007
CG2
ILE
A
278
−33.174
27.888
10.557
1.00
78.57
C


ATOM
3008
CD1
ILE
A
278
−35.544
29.087
11.775
1.00
83.47
C


ATOM
3009
N
MET
A
279
−30.412
27.761
12.933
1.00
79.21
N


ATOM
3010
CA
MET
A
279
−29.206
26.942
12.826
1.00
77.35
C


ATOM
3011
C
MET
A
279
−29.019
26.044
14.018
1.00
75.85
C


ATOM
3012
O
MET
A
279
−28.798
24.844
13.852
1.00
73.53
O


ATOM
3013
CB
MET
A
279
−27.982
27.830
12.632
1.00
76.42
C


ATOM
3014
CG
MET
A
279
−27.992
28.535
11.284
1.00
79.03
C


ATOM
3015
SD
MET
A
279
−26.852
29.897
11.156
1.00
80.66
S


ATOM
3016
CE
MET
A
279
−27.061
30.304
9.407
1.00
79.86
C


ATOM
3017
N
GLY
A
280
−29.112
26.625
15.214
1.00
78.82
N


ATOM
3018
CA
GLY
A
280
−28.937
25.880
16.463
1.00
76.24
C


ATOM
3019
C
GLY
A
280
−29.970
24.785
16.660
1.00
79.31
C


ATOM
3020
O
GLY
A
280
−29.640
23.694
17.120
1.00
80.01
O


ATOM
3021
N
THR
A
281
−31.219
25.084
16.309
1.00
80.59
N


ATOM
3022
CA
THR
A
281
−32.309
24.123
16.408
1.00
78.83
C


ATOM
3023
C
THR
A
281
−32.057
22.935
15.492
1.00
77.28
C


ATOM
3024
O
THR
A
281
−32.190
21.801
15.923
1.00
76.07
O


ATOM
3025
CB
THR
A
281
−33.657
24.780
16.070
1.00
81.71
C


ATOM
3026
OG1
THR
A
281
−33.964
25.771
17.065
1.00
79.40
O


ATOM
3027
CG2
THR
A
281
−34.769
23.760
16.038
1.00
82.36
C


ATOM
3028
N
PHE
A
282
−31.694
23.205
14.237
1.00
79.67
N


ATOM
3029
CA
PHE
A
282
−31.347
22.147
13.279
1.00
77.00
C


ATOM
3030
C
PHE
A
282
−30.221
21.282
13.817
1.00
76.25
C


ATOM
3031
O
PHE
A
282
−30.266
20.069
13.697
1.00
73.80
O


ATOM
3032
CB
PHE
A
282
−30.917
22.731
11.930
1.00
77.95
C


ATOM
3033
CG
PHE
A
282
−30.691
21.679
10.853
1.00
76.73
C


ATOM
3034
CD1
PHE
A
282
−31.723
21.314
9.995
1.00
75.49
C


ATOM
3035
CD2
PHE
A
282
−29.460
21.064
10.706
1.00
76.37
C


ATOM
3036
CE1
PHE
A
282
−31.526
20.343
8.997
1.00
76.36
C


ATOM
3037
CE2
PHE
A
282
−29.254
20.097
9.720
1.00
84.43
C


ATOM
3038
CZ
PHE
A
282
−30.297
19.737
8.857
1.00
76.69
C


ATOM
3039
N
THR
A
283
−29.214
21.923
14.406
1.00
77.11
N


ATOM
3040
CA
THR
A
283
−28.058
21.218
14.945
1.00
76.40
C


ATOM
3041
C
THR
A
283
−28.460
20.311
16.078
1.00
76.62
C


ATOM
3042
O
THR
A
283
−28.105
19.152
16.073
1.00
76.69
O


ATOM
3043
CB
THR
A
283
−26.981
22.183
15.448
1.00
76.06
C


ATOM
3044
OG1
THR
A
283
−26.544
23.023
14.367
1.00
80.64
O


ATOM
3045
CG2
THR
A
283
−25.784
21.412
16.018
1.00
75.59
C


ATOM
3046
N
LEU
A
284
−29.203
20.844
17.041
1.00
78.59
N


ATOM
3047
CA
LEU
A
284
−29.636
20.065
18.204
1.00
77.27
C


ATOM
3048
C
LEU
A
284
−30.578
18.920
17.845
1.00
77.57
C


ATOM
3049
O
LEU
A
284
−30.560
17.886
18.500
1.00
76.25
O


ATOM
3050
CB
LEU
A
284
−30.300
20.986
19.236
1.00
80.83
C


ATOM
3051
CG
LEU
A
284
−29.359
22.013
19.916
1.00
89.13
C


ATOM
3052
CD1
LEU
A
284
−30.162
23.029
20.724
1.00
93.20
C


ATOM
3053
CD2
LEU
A
284
−28.290
21.337
20.801
1.00
91.45
C


ATOM
3054
N
CYS
A
285
−31.395
19.108
16.806
1.00
77.86
N


ATOM
3055
CA
CYS
A
285
−32.341
18.073
16.367
1.00
76.99
C


ATOM
3056
C
CYS
A
285
−31.677
16.920
15.620
1.00
75.92
C


ATOM
3057
O
CYS
A
285
−32.095
15.782
15.783
1.00
75.32
O


ATOM
3058
CB
CYS
A
285
−33.462
18.678
15.513
1.00
76.74
C


ATOM
3059
SG
CYS
A
285
−34.727
19.541
16.511
1.00
86.35
S


ATOM
3060
N
TRP
A
286
−30.657
17.219
14.810
1.00
76.29
N


ATOM
3061
CA
TRP
A
286
−29.938
16.192
14.020
1.00
75.01
C


ATOM
3062
C
TRP
A
286
−28.635
15.671
14.635
1.00
75.15
C


ATOM
3063
O
TRP
A
286
−28.250
14.544
14.330
1.00
75.31
O


ATOM
3064
CB
TRP
A
286
−29.641
16.703
12.599
1.00
72.75
C


ATOM
3065
CG
TRP
A
286
−30.853
16.738
11.766
1.00
70.37
C


ATOM
3066
CD1
TRP
A
286
−31.543
17.832
11.381
1.00
69.67
C


ATOM
3067
CD2
TRP
A
286
−31.533
15.613
11.212
1.00
67.81
C


ATOM
3068
NE1
TRP
A
286
−32.621
17.469
10.614
1.00
76.08
N


ATOM
3069
CE2
TRP
A
286
−32.636
16.107
10.496
1.00
67.36
C


ATOM
3070
CE3
TRP
A
286
−31.314
14.228
11.254
1.00
69.38
C


ATOM
3071
CZ2
TRP
A
286
−33.523
15.274
9.822
1.00
73.52
C


ATOM
3072
CZ3
TRP
A
286
−32.202
13.391
10.581
1.00
71.27
C


ATOM
3073
CH2
TRP
A
286
−33.291
13.920
9.875
1.00
72.45
C


ATOM
3074
N
LEU
A
287
−27.958
16.454
15.474
1.00
71.19
N


ATOM
3075
CA
LEU
A
287
−26.645
16.020
16.001
1.00
72.17
C


ATOM
3076
C
LEU
A
287
−26.669
14.651
16.667
1.00
70.61
C


ATOM
3077
O
LEU
A
287
−25.822
13.834
16.347
1.00
73.46
O


ATOM
3078
CB
LEU
A
287
−26.019
17.049
16.953
1.00
74.35
C


ATOM
3079
CG
LEU
A
287
−24.542
16.884
17.277
1.00
76.88
C


ATOM
3080
CD1
LEU
A
287
−23.682
16.964
16.013
1.00
78.66
C


ATOM
3081
CD2
LEU
A
287
−24.130
17.956
18.279
1.00
79.06
C


ATOM
3082
N
PRO
A
288
−27.627
14.395
17.583
1.00
69.58
N


ATOM
3083
CA
PRO
A
288
−27.682
13.074
18.227
1.00
68.99
C


ATOM
3084
C
PRO
A
288
−27.767
11.903
17.248
1.00
68.90
C


ATOM
3085
O
PRO
A
288
−27.052
10.906
17.406
1.00
67.39
O


ATOM
3086
CB
PRO
A
288
−28.979
13.140
19.057
1.00
68.33
C


ATOM
3087
CG
PRO
A
288
−29.207
14.546
19.305
1.00
66.28
C


ATOM
3088
CD
PRO
A
288
−28.701
15.269
18.091
1.00
70.69
C


ATOM
3089
N
PHE
A
289
−28.641
12.042
16.255
1.00
67.58
N


ATOM
3090
CA
PHE
A
289
−28.807
11.038
15.217
1.00
67.91
C


ATOM
3091
C
PHE
A
289
−27.479
10.746
14.519
1.00
69.22
C


ATOM
3092
O
PHE
A
289
−27.072
9.576
14.412
1.00
68.30
O


ATOM
3093
CB
PHE
A
289
−29.853
11.505
14.189
1.00
69.82
C


ATOM
3094
CG
PHE
A
289
−30.014
10.578
13.047
1.00
67.38
C


ATOM
3095
CD1
PHE
A
289
−30.850
9.475
13.157
1.00
64.32
C


ATOM
3096
CD2
PHE
A
289
−29.335
10.795
11.859
1.00
71.12
C


ATOM
3097
CE1
PHE
A
289
−31.008
8.610
12.114
1.00
70.14
C


ATOM
3098
CE2
PHE
A
289
−29.489
9.926
10.798
1.00
76.34
C


ATOM
3099
CZ
PHE
A
289
−30.327
8.829
10.923
1.00
75.79
C


ATOM
3100
N
PHE
A
290
−26.818
11.811
14.052
1.00
69.03
N


ATOM
3101
CA
PHE
A
290
−25.522
11.686
13.347
1.00
70.29
C


ATOM
3102
C
PHE
A
290
−24.346
11.254
14.222
1.00
70.41
C


ATOM
3103
O
PHE
A
290
−23.386
10.677
13.697
1.00
72.74
O


ATOM
3104
CB
PHE
A
290
−25.180
12.971
12.585
1.00
73.06
C


ATOM
3105
CG
PHE
A
290
−25.942
13.108
11.295
1.00
73.80
C


ATOM
3106
CD1
PHE
A
290
−27.103
13.864
11.226
1.00
70.64
C


ATOM
3107
CD2
PHE
A
290
−25.494
12.467
10.149
1.00
76.58
C


ATOM
3108
CE1
PHE
A
290
−27.800
13.990
10.058
1.00
79.74
C


ATOM
3109
CE2
PHE
A
290
−26.191
12.586
8.960
1.00
75.30
C


ATOM
3110
CZ
PHE
A
290
−27.349
13.352
8.914
1.00
77.36
C


ATOM
3111
N
ILE
A
291
−24.404
11.522
15.528
1.00
69.93
N


ATOM
3112
CA
ILE
A
291
−23.391
11.003
16.454
1.00
71.11
C


ATOM
3113
C
ILE
A
291
−23.517
9.476
16.516
1.00
72.55
C


ATOM
3114
O
ILE
A
291
−22.507
8.765
16.534
1.00
76.20
O


ATOM
3115
CB
ILE
A
291
−23.511
11.621
17.873
1.00
71.52
C


ATOM
3116
CG1
ILE
A
291
−23.073
13.081
17.853
1.00
73.15
C


ATOM
3117
CG2
ILE
A
291
−22.642
10.888
18.894
1.00
65.02
C


ATOM
3118
CD1
ILE
A
291
−23.309
13.807
19.140
1.00
72.16
C


ATOM
3119
N
VAL
A
292
−24.757
8.984
16.545
1.00
74.02
N


ATOM
3120
CA
VAL
A
292
−25.023
7.539
16.619
1.00
74.95
C


ATOM
3121
C
VAL
A
292
−24.586
6.782
15.340
1.00
73.29
C


ATOM
3122
O
VAL
A
292
−24.192
5.619
15.426
1.00
74.16
O


ATOM
3123
CB
VAL
A
292
−26.505
7.253
16.998
1.00
74.89
C


ATOM
3124
CG1
VAL
A
292
−26.814
5.775
16.915
1.00
73.02
C


ATOM
3125
CG2
VAL
A
292
−26.781
7.774
18.427
1.00
73.75
C


ATOM
3126
N
ASN
A
293
−24.651
7.431
14.176
1.00
73.79
N


ATOM
3127
CA
ASN
A
293
−24.112
6.835
12.936
1.00
72.79
C


ATOM
3128
C
ASN
A
293
−22.641
6.495
13.085
1.00
71.66
C


ATOM
3129
O
ASN
A
293
−22.206
5.409
12.709
1.00
72.11
O


ATOM
3130
CB
ASN
A
293
−24.250
7.782
11.745
1.00
72.08
C


ATOM
3131
CG
ASN
A
293
−25.673
7.961
11.287
1.00
76.70
C


ATOM
3132
OD1
ASN
A
293
−26.584
7.306
11.777
1.00
77.56
O


ATOM
3133
ND2
ASN
A
293
−25.871
8.863
10.327
1.00
72.62
N


ATOM
3134
N
ILE
A
294
−21.887
7.435
13.639
1.00
70.24
N


ATOM
3135
CA
ILE
A
294
−20.451
7.272
13.833
1.00
71.74
C


ATOM
3136
C
ILE
A
294
−20.156
6.296
14.963
1.00
71.03
C


ATOM
3137
O
ILE
A
294
−19.288
5.437
14.807
1.00
69.26
O


ATOM
3138
CB
ILE
A
294
−19.784
8.608
14.115
1.00
71.93
C


ATOM
3139
CG1
ILE
A
294
−19.878
9.502
12.885
1.00
71.27
C


ATOM
3140
CG2
ILE
A
294
−18.313
8.421
14.498
1.00
72.08
C


ATOM
3141
CD1
ILE
A
294
−19.391
10.862
13.168
1.00
79.13
C


ATOM
3142
N
VAL
A
295
−20.872
6.426
16.088
1.00
71.55
N


ATOM
3143
CA
VAL
A
295
−20.735
5.484
17.234
1.00
70.36
C


ATOM
3144
C
VAL
A
295
−20.932
4.029
16.782
1.00
69.42
C


ATOM
3145
O
VAL
A
295
−20.224
3.142
17.243
1.00
66.44
O


ATOM
3146
CB
VAL
A
295
−21.720
5.835
18.396
1.00
70.22
C


ATOM
3147
CG1
VAL
A
295
−21.803
4.721
19.419
1.00
68.33
C


ATOM
3148
CG2
VAL
A
295
−21.304
7.125
19.065
1.00
71.01
C


ATOM
3149
N
HIS
A
296
−21.890
3.805
15.878
1.00
69.19
N


ATOM
3150
CA
HIS
A
296
−22.128
2.462
15.311
1.00
71.67
C


ATOM
3151
C
HIS
A
296
−21.079
1.977
14.297
1.00
72.30
C


ATOM
3152
O
HIS
A
296
−21.022
0.785
13.996
1.00
72.47
O


ATOM
3153
CB
HIS
A
296
−23.545
2.349
14.737
1.00
72.70
C


ATOM
3154
CG
HIS
A
296
−24.591
2.269
15.795
1.00
77.53
C


ATOM
3155
ND1
HIS
A
296
−24.369
1.632
16.991
1.00
78.84
N


ATOM
3156
CD2
HIS
A
296
−25.859
2.736
15.848
1.00
85.05
C


ATOM
3157
CE1
HIS
A
296
−25.448
1.710
17.733
1.00
78.27
C


ATOM
3158
NE2
HIS
A
296
−26.370
2.373
17.069
1.00
81.35
N


ATOM
3159
N
VAL
A
297
−20.267
2.893
13.781
1.00
73.43
N


ATOM
3160
CA
VAL
A
297
−19.118
2.535
12.948
1.00
73.61
C


ATOM
3161
C
VAL
A
297
−17.993
2.051
13.878
1.00
72.62
C


ATOM
3162
O
VAL
A
297
−17.220
1.165
13.505
1.00
73.05
O


ATOM
3163
CB
VAL
A
297
−18.653
3.713
12.050
1.00
73.85
C


ATOM
3164
CG1
VAL
A
297
−17.424
3.338
11.238
1.00
72.56
C


ATOM
3165
CG2
VAL
A
297
−19.786
4.139
11.127
1.00
72.80
C


ATOM
3166
N
ILE
A
298
−17.912
2.636
15.082
1.00
72.35
N


ATOM
3167
CA
ILE
A
298
−16.930
2.236
16.097
1.00
73.03
C


ATOM
3168
C
ILE
A
298
−17.351
0.886
16.688
1.00
73.53
C


ATOM
3169
O
ILE
A
298
−16.612
−0.099
16.597
1.00
74.08
O


ATOM
3170
CB
ILE
A
298
−16.780
3.288
17.246
1.00
73.65
C


ATOM
3171
CG1
ILE
A
298
−16.402
4.687
16.716
1.00
76.60
C


ATOM
3172
CG2
ILE
A
298
−15.757
2.827
18.255
1.00
73.25
C


ATOM
3173
CD1
ILE
A
298
−15.059
4.764
16.007
1.00
80.21
C


ATOM
3174
N
GLN
A
299
−18.542
0.864
17.290
1.00
73.48
N


ATOM
3175
CA
GLN
A
299
−19.132
−0.345
17.862
1.00
74.55
C


ATOM
3176
C
GLN
A
299
−20.619
−0.354
17.524
1.00
75.17
C


ATOM
3177
O
GLN
A
299
−21.368
0.466
18.040
1.00
77.55
O


ATOM
3178
CB
GLN
A
299
−18.917
−0.401
19.378
1.00
75.38
C


ATOM
3179
CG
GLN
A
299
−19.532
−1.630
20.086
1.00
76.95
C


ATOM
3180
CD
GLN
A
299
−18.993
−2.963
19.563
1.00
80.36
C


ATOM
3181
OE1
GLN
A
299
−17.801
−3.255
19.675
1.00
81.59
O


ATOM
3182
NE2
GLN
A
299
−19.883
−3.778
18.992
1.00
79.18
N


ATOM
3183
N
ASP
A
300
−21.035
−1.282
16.662
1.00
75.16
N


ATOM
3184
CA
ASP
A
300
−22.420
−1.357
16.206
1.00
76.26
C


ATOM
3185
C
ASP
A
300
−23.306
−1.994
17.284
1.00
76.70
C


ATOM
3186
O
ASP
A
300
−22.818
−2.756
18.121
1.00
75.98
O


ATOM
3187
CB
ASP
A
300
−22.492
−2.145
14.883
1.00
76.84
C


ATOM
3188
CG
ASP
A
300
−23.781
−1.873
14.069
1.00
78.37
C


ATOM
3189
OD1
ASP
A
300
−24.504
−0.874
14.303
1.00
80.03
O


ATOM
3190
OD2
ASP
A
300
−24.068
−2.688
13.168
1.00
83.02
O


ATOM
3191
N
ASN
A
301
−24.601
−1.661
17.248
1.00
76.86
N


ATOM
3192
CA
ASN
A
301
−25.633
−2.196
18.177
1.00
76.17
C


ATOM
3193
C
ASN
A
301
−25.536
−1.740
19.656
1.00
75.65
C


ATOM
3194
O
ASN
A
301
−26.220
−2.290
20.520
1.00
76.59
O


ATOM
3195
CB
ASN
A
301
−25.700
−3.741
18.089
1.00
75.63
C


ATOM
3196
CG
ASN
A
301
−26.022
−4.235
16.690
1.00
74.71
C


ATOM
3197
OD1
ASN
A
301
−26.917
−3.714
16.030
1.00
64.90
O


ATOM
3198
ND2
ASN
A
301
−25.291
−5.246
16.233
1.00
75.78
N


ATOM
3199
N
LEU
A
302
−24.700
−0.743
19.938
1.00
76.35
N


ATOM
3200
CA
LEU
A
302
−24.547
−0.197
21.298
1.00
78.80
C


ATOM
3201
C
LEU
A
302
−25.815
0.534
21.817
1.00
79.26
C


ATOM
3202
O
LEU
A
302
−26.074
0.566
23.018
1.00
80.59
O


ATOM
3203
CB
LEU
A
302
−23.350
0.764
21.335
1.00
79.16
C


ATOM
3204
CG
LEU
A
302
−22.832
1.213
22.703
1.00
79.37
C


ATOM
3205
CD1
LEU
A
302
−22.199
0.039
23.438
1.00
77.36
C


ATOM
3206
CD2
LEU
A
302
−21.839
2.351
22.545
1.00
79.97
C


ATOM
3207
N
ILE
A
303
−26.572
1.105
20.888
1.00
79.64
N


ATOM
3208
CA
ILE
A
303
−27.765
1.886
21.120
1.00
80.47
C


ATOM
3209
C
ILE
A
303
−28.881
1.166
20.393
1.00
80.12
C


ATOM
3210
O
ILE
A
303
−28.747
0.854
19.208
1.00
80.56
O


ATOM
3211
CB
ILE
A
303
−27.623
3.313
20.573
1.00
79.71
C


ATOM
3212
CG1
ILE
A
303
−26.513
4.056
21.326
1.00
80.55
C


ATOM
3213
CG2
ILE
A
303
−28.930
4.068
20.708
1.00
78.77
C


ATOM
3214
CD1
ILE
A
303
−26.243
5.427
20.826
1.00
83.25
C


ATOM
3215
N
ARG
A
304
−29.973
0.910
21.111
1.00
81.10
N


ATOM
3216
CA
ARG
A
304
−31.132
0.177
20.596
1.00
83.66
C


ATOM
3217
C
ARG
A
304
−31.686
0.776
19.310
1.00
82.48
C


ATOM
3218
O
ARG
A
304
−31.542
1.977
19.070
1.00
83.57
O


ATOM
3219
CB
ARG
A
304
−32.265
0.192
21.608
1.00
84.33
C


ATOM
3220
CG
ARG
A
304
−31.969
−0.426
22.966
1.00
91.17
C


ATOM
3221
CD
ARG
A
304
−33.142
−0.227
23.870
1.00
96.90
C


ATOM
3222
NE
ARG
A
304
−34.321
−0.914
23.347
1.00
107.82
N


ATOM
3223
CZ
ARG
A
304
−35.556
−0.820
23.845
1.00
114.88
C


ATOM
3224
NH1
ARG
A
304
−35.826
−0.056
24.909
1.00
119.98
N


ATOM
3225
NH2
ARG
A
304
−36.539
−1.505
23.268
1.00
117.19
N


ATOM
3226
N
LYS
A
305
−32.317
−0.069
18.496
1.00
80.96
N


ATOM
3227
CA
LYS
A
305
−32.950
0.369
17.249
1.00
82.83
C


ATOM
3228
C
LYS
A
305
−34.024
1.432
17.501
1.00
82.19
C


ATOM
3229
O
LYS
A
305
−34.122
2.399
16.751
1.00
83.88
O


ATOM
3230
CB
LYS
A
305
−33.573
−0.819
16.514
1.00
82.92
C


ATOM
3231
CG
LYS
A
305
−34.091
−0.499
15.109
1.00
86.35
C


ATOM
3232
CD
LYS
A
305
−34.754
−1.709
14.472
1.00
87.13
C


ATOM
3233
CE
LYS
A
305
−35.417
−1.329
13.154
1.00
88.05
C


ATOM
3234
NZ
LYS
A
305
−36.147
−2.481
12.552
1.00
91.16
N


ATOM
3235
N
GLU
A
306
−34.813
1.237
18.553
1.00
79.77
N


ATOM
3236
CA
GLU
A
306
−35.888
2.166
18.928
1.00
78.91
C


ATOM
3237
C
GLU
A
306
−35.388
3.534
19.354
1.00
75.42
C


ATOM
3238
O
GLU
A
306
−36.083
4.526
19.156
1.00
73.51
O


ATOM
3239
CB
GLU
A
306
−36.707
1.586
20.062
1.00
80.27
C


ATOM
3240
CG
GLU
A
306
−37.464
0.323
19.682
1.00
88.15
C


ATOM
3241
CD
GLU
A
306
−38.035
−0.379
20.876
1.00
92.42
C


ATOM
3242
OE1
GLU
A
306
−38.087
0.245
21.964
1.00
103.55
O


ATOM
3243
OE2
GLU
A
306
−38.436
−1.560
20.740
1.00
104.10
O


ATOM
3244
N
VAL
A
307
−34.191
3.586
19.938
1.00
74.31
N


ATOM
3245
CA
VAL
A
307
−33.579
4.864
20.314
1.00
73.09
C


ATOM
3246
C
VAL
A
307
−33.126
5.563
19.040
1.00
72.26
C


ATOM
3247
O
VAL
A
307
−33.415
6.740
18.854
1.00
74.87
O


ATOM
3248
CB
VAL
A
307
−32.398
4.703
21.330
1.00
71.64
C


ATOM
3249
CG1
VAL
A
307
−31.597
5.986
21.441
1.00
63.03
C


ATOM
3250
CG2
VAL
A
307
−32.925
4.287
22.703
1.00
68.24
C


ATOM
3251
N
TYR
A
308
−32.423
4.833
18.177
1.00
71.72
N


ATOM
3252
CA
TYR
A
308
−31.955
5.366
16.892
1.00
72.58
C


ATOM
3253
C
TYR
A
308
−33.106
5.892
16.025
1.00
72.20
C


ATOM
3254
O
TYR
A
308
−32.981
6.951
15.414
1.00
72.72
O


ATOM
3255
CB
TYR
A
308
−31.184
4.294
16.135
1.00
75.16
C


ATOM
3256
CG
TYR
A
308
−30.534
4.770
14.855
1.00
75.61
C


ATOM
3257
CD1
TYR
A
308
−30.984
4.338
13.610
1.00
83.25
C


ATOM
3258
CD2
TYR
A
308
−29.470
5.650
14.894
1.00
77.87
C


ATOM
3259
CE1
TYR
A
308
−30.378
4.780
12.435
1.00
82.87
C


ATOM
3260
CE2
TYR
A
308
−28.859
6.098
13.747
1.00
74.78
C


ATOM
3261
CZ
TYR
A
308
−29.313
5.663
12.513
1.00
80.83
C


ATOM
3262
OH
TYR
A
308
−28.716
6.100
11.357
1.00
79.13
O


ATOM
3263
N
ILE
A
309
−34.214
5.152
15.979
1.00
70.18
N


ATOM
3264
CA
ILE
A
309
−35.412
5.593
15.251
1.00
71.74
C


ATOM
3265
C
ILE
A
309
−35.959
6.873
15.863
1.00
72.46
C


ATOM
3266
O
ILE
A
309
−36.272
7.798
15.139
1.00
73.87
O


ATOM
3267
CB
ILE
A
309
−36.519
4.520
15.243
1.00
70.27
C


ATOM
3268
CG1
ILE
A
309
−36.141
3.375
14.316
1.00
69.96
C


ATOM
3269
CG2
ILE
A
309
−37.839
5.100
14.781
1.00
68.79
C


ATOM
3270
CD1
ILE
A
309
−36.987
2.145
14.516
1.00
72.32
C


ATOM
3271
N
LEU
A
310
−36.067
6.917
17.195
1.00
74.30
N


ATOM
3272
CA
LEU
A
310
−36.561
8.109
17.895
1.00
73.71
C


ATOM
3273
C
LEU
A
310
−35.682
9.324
17.619
1.00
75.46
C


ATOM
3274
O
LEU
A
310
−36.208
10.416
17.414
1.00
76.09
O


ATOM
3275
CB
LEU
A
310
−36.677
7.873
19.405
1.00
76.16
C


ATOM
3276
CG
LEU
A
310
−37.103
9.087
20.269
1.00
79.17
C


ATOM
3277
CD1
LEU
A
310
−38.353
9.787
19.699
1.00
80.03
C


ATOM
3278
CD2
LEU
A
310
−37.344
8.666
21.710
1.00
74.92
C


ATOM
3279
N
LEU
A
311
−34.358
9.142
17.612
1.00
74.63
N


ATOM
3280
CA
LEU
A
311
−33.436
10.249
17.271
1.00
73.90
C


ATOM
3281
C
LEU
A
311
−33.611
10.744
15.829
1.00
73.58
C


ATOM
3282
O
LEU
A
311
−33.346
11.904
15.546
1.00
75.24
O


ATOM
3283
CB
LEU
A
311
−31.976
9.857
17.497
1.00
71.95
C


ATOM
3284
CG
LEU
A
311
−31.583
9.468
18.926
1.00
74.42
C


ATOM
3285
CD1
LEU
A
311
−30.094
9.202
18.969
1.00
78.05
C


ATOM
3286
CD2
LEU
A
311
−31.960
10.525
19.951
1.00
79.68
C


ATOM
3287
N
ASN
A
312
−34.053
9.862
14.932
1.00
73.28
N


ATOM
3288
CA
ASN
A
312
−34.337
10.228
13.551
1.00
74.59
C


ATOM
3289
C
ASN
A
312
−35.602
11.076
13.491
1.00
74.96
C


ATOM
3290
O
ASN
A
312
−35.654
12.057
12.741
1.00
76.71
O


ATOM
3291
CB
ASN
A
312
−34.490
8.970
12.679
1.00
73.89
C


ATOM
3292
CG
ASN
A
312
−34.276
9.231
11.170
1.00
75.40
C


ATOM
3293
OD1
ASN
A
312
−33.933
8.302
10.439
1.00
75.69
O


ATOM
3294
ND2
ASN
A
312
−34.471
10.471
10.709
1.00
73.01
N


ATOM
3295
N
TRP
A
313
−36.614
10.702
14.275
1.00
75.65
N


ATOM
3296
CA
TRP
A
313
−37.880
11.461
14.327
1.00
73.90
C


ATOM
3297
C
TRP
A
313
−37.748
12.844
14.967
1.00
74.02
C


ATOM
3298
O
TRP
A
313
−38.556
13.726
14.676
1.00
74.92
O


ATOM
3299
CB
TRP
A
313
−38.988
10.634
14.986
1.00
75.64
C


ATOM
3300
CG
TRP
A
313
−39.562
9.768
13.984
1.00
77.34
C


ATOM
3301
CD1
TRP
A
313
−39.070
8.583
13.548
1.00
77.95
C


ATOM
3302
CD2
TRP
A
313
−40.757
10.004
13.254
1.00
77.66
C


ATOM
3303
NE1
TRP
A
313
−39.885
8.056
12.589
1.00
77.47
N


ATOM
3304
CE2
TRP
A
313
−40.935
8.910
12.384
1.00
79.44
C


ATOM
3305
CE3
TRP
A
313
−41.701
11.037
13.247
1.00
76.72
C


ATOM
3306
CZ2
TRP
A
313
−42.025
8.813
11.509
1.00
79.40
C


ATOM
3307
CZ3
TRP
A
313
−42.790
10.944
12.374
1.00
78.33
C


ATOM
3308
CH2
TRP
A
313
−42.940
9.835
11.518
1.00
77.48
C


ATOM
3309
N
ILE
A
314
−36.742
13.024
15.822
1.00
71.91
N


ATOM
3310
CA
ILE
A
314
−36.411
14.336
16.368
1.00
71.31
C


ATOM
3311
C
ILE
A
314
−35.847
15.185
15.213
1.00
71.78
C


ATOM
3312
O
ILE
A
314
−36.108
16.396
15.114
1.00
72.42
O


ATOM
3313
CB
ILE
A
314
−35.406
14.239
17.546
1.00
69.93
C


ATOM
3314
CG1
ILE
A
314
−36.040
13.519
18.735
1.00
69.22
C


ATOM
3315
CG2
ILE
A
314
−34.975
15.611
18.002
1.00
66.77
C


ATOM
3316
CD1
ILE
A
314
−35.054
13.195
19.841
1.00
70.69
C


ATOM
3317
N
GLY
A
315
−35.072
14.536
14.350
1.00
71.45
N


ATOM
3318
CA
GLY
A
315
−34.603
15.150
13.118
1.00
73.83
C


ATOM
3319
C
GLY
A
315
−35.780
15.520
12.237
1.00
73.35
C


ATOM
3320
O
GLY
A
315
−35.913
16.673
11.866
1.00
71.64
O


ATOM
3321
N
TYR
A
316
−36.634
14.545
11.909
1.00
74.36
N


ATOM
3322
CA
TYR
A
316
−37.825
14.800
11.050
1.00
74.23
C


ATOM
3323
C
TYR
A
316
−38.668
15.970
11.526
1.00
75.48
C


ATOM
3324
O
TYR
A
316
−38.949
16.889
10.754
1.00
76.19
O


ATOM
3325
CB
TYR
A
316
−38.746
13.570
10.960
1.00
74.65
C


ATOM
3326
CG
TYR
A
316
−38.256
12.359
10.139
1.00
77.26
C


ATOM
3327
CD1
TYR
A
316
−38.869
11.121
10.288
1.00
77.77
C


ATOM
3328
CD2
TYR
A
316
−37.200
12.448
9.231
1.00
75.43
C


ATOM
3329
CE1
TYR
A
316
−38.459
10.025
9.571
1.00
71.53
C


ATOM
3330
CE2
TYR
A
316
−36.787
11.358
8.514
1.00
78.76
C


ATOM
3331
CZ
TYR
A
316
−37.420
10.148
8.688
1.00
77.38
C


ATOM
3332
OH
TYR
A
316
−37.015
9.061
7.979
1.00
77.21
O


ATOM
3333
N
VAL
A
317
−39.057
15.910
12.801
1.00
74.20
N


ATOM
3334
CA
VAL
A
317
−39.872
16.934
13.466
1.00
72.51
C


ATOM
3335
C
VAL
A
317
−39.316
18.355
13.330
1.00
72.64
C


ATOM
3336
O
VAL
A
317
−40.086
19.311
13.253
1.00
72.17
O


ATOM
3337
CB
VAL
A
317
−40.088
16.560
14.984
1.00
72.14
C


ATOM
3338
CG1
VAL
A
317
−40.336
17.760
15.804
1.00
74.78
C


ATOM
3339
CG2
VAL
A
317
−41.253
15.603
15.111
1.00
69.59
C


ATOM
3340
N
ASN
A
318
−37.988
18.491
13.296
1.00
74.61
N


ATOM
3341
CA
ASN
A
318
−37.342
19.793
13.078
1.00
75.44
C


ATOM
3342
C
ASN
A
318
−37.882
20.548
11.857
1.00
75.25
C


ATOM
3343
O
ASN
A
318
−37.912
21.779
11.844
1.00
72.63
O


ATOM
3344
CB
ASN
A
318
−35.843
19.645
12.910
1.00
74.36
C


ATOM
3345
CG
ASN
A
318
−35.171
20.958
12.635
1.00
79.05
C


ATOM
3346
OD1
ASN
A
318
−35.023
21.806
13.522
1.00
80.80
O


ATOM
3347
ND2
ASN
A
318
−34.761
21.142
11.396
1.00
83.57
N


ATOM
3348
N
SER
A
319
−38.308
19.798
10.836
1.00
75.32
N


ATOM
3349
CA
SER
A
319
−38.951
20.365
9.640
1.00
76.04
C


ATOM
3350
C
SER
A
319
−40.218
21.194
9.925
1.00
76.24
C


ATOM
3351
O
SER
A
319
−40.670
21.930
9.046
1.00
77.32
O


ATOM
3352
CB
SER
A
319
−39.285
19.245
8.635
1.00
74.82
C


ATOM
3353
OG
SER
A
319
−38.120
18.509
8.281
1.00
77.99
O


ATOM
3354
N
GLY
A
320
−40.782
21.078
11.133
1.00
75.25
N


ATOM
3355
CA
GLY
A
320
−41.934
21.857
11.544
1.00
74.43
C


ATOM
3356
C
GLY
A
320
−41.642
23.110
12.350
1.00
76.72
C


ATOM
3357
O
GLY
A
320
−42.562
23.884
12.581
1.00
78.81
O


ATOM
3358
N
PHE
A
321
−40.391
23.328
12.777
1.00
75.79
N


ATOM
3359
CA
PHE
A
321
−40.076
24.440
13.706
1.00
77.01
C


ATOM
3360
C
PHE
A
321
−39.761
25.780
13.112
1.00
77.35
C


ATOM
3361
O
PHE
A
321
−40.178
26.799
13.680
1.00
75.78
O


ATOM
3362
CB
PHE
A
321
−38.912
24.051
14.634
1.00
78.53
C


ATOM
3363
CG
PHE
A
321
−39.192
22.851
15.497
1.00
78.83
C


ATOM
3364
CD1
PHE
A
321
−38.154
22.005
15.857
1.00
82.01
C


ATOM
3365
CD2
PHE
A
321
−40.490
22.551
15.953
1.00
81.40
C


ATOM
3366
CE1
PHE
A
321
−38.383
20.891
16.650
1.00
81.76
C


ATOM
3367
CE2
PHE
A
321
−40.722
21.446
16.740
1.00
83.47
C


ATOM
3368
CZ
PHE
A
321
−39.667
20.614
17.091
1.00
85.30
C


ATOM
3369
N
ASN
A
322
−39.040
25.807
11.996
1.00
77.86
N


ATOM
3370
CA
ASN
A
322
−38.637
27.082
11.381
1.00
78.02
C


ATOM
3371
C
ASN
A
322
−39.785
28.061
11.142
1.00
78.43
C


ATOM
3372
O
ASN
A
322
−39.620
29.237
11.440
1.00
77.20
O


ATOM
3373
CB
ASN
A
322
−37.866
26.856
10.087
1.00
79.19
C


ATOM
3374
CG
ASN
A
322
−36.531
26.208
10.315
1.00
81.79
C


ATOM
3375
OD1
ASN
A
322
−36.158
25.901
11.455
1.00
74.61
O


ATOM
3376
ND2
ASN
A
322
−35.791
25.989
9.229
1.00
85.04
N


ATOM
3377
N
PRO
A
323
−40.941
27.582
10.609
1.00
78.55
N


ATOM
3378
CA
PRO
A
323
−42.088
28.479
10.469
1.00
79.23
C


ATOM
3379
C
PRO
A
323
−42.469
29.179
11.779
1.00
80.55
C


ATOM
3380
O
PRO
A
323
−42.795
30.367
11.751
1.00
83.61
O


ATOM
3381
CB
PRO
A
323
−43.200
27.542
9.997
1.00
80.04
C


ATOM
3382
CG
PRO
A
323
−42.474
26.473
9.266
1.00
79.60
C


ATOM
3383
CD
PRO
A
323
−41.271
26.232
10.104
1.00
78.83
C


ATOM
3384
N
LEU
A
324
−42.426
28.454
12.899
1.00
80.72
N


ATOM
3385
CA
LEU
A
324
−42.689
29.050
14.233
1.00
81.22
C


ATOM
3386
C
LEU
A
324
−41.580
30.013
14.651
1.00
78.60
C


ATOM
3387
O
LEU
A
324
−41.851
31.053
15.230
1.00
77.91
O


ATOM
3388
CB
LEU
A
324
−42.847
27.970
15.310
1.00
83.48
C


ATOM
3389
CG
LEU
A
324
−44.017
27.003
15.125
1.00
90.04
C


ATOM
3390
CD1
LEU
A
324
−43.821
25.727
15.948
1.00
91.90
C


ATOM
3391
CD2
LEU
A
324
−45.329
27.707
15.474
1.00
93.47
C


ATOM
3392
N
ILE
A
325
−40.334
29.656
14.355
1.00
77.22
N


ATOM
3393
CA
ILE
A
325
−39.186
30.504
14.689
1.00
76.61
C


ATOM
3394
C
ILE
A
325
−39.244
31.830
13.907
1.00
75.96
C


ATOM
3395
O
ILE
A
325
−38.791
32.850
14.407
1.00
76.16
O


ATOM
3396
CB
ILE
A
325
−37.837
29.776
14.433
1.00
76.87
C


ATOM
3397
CG1
ILE
A
325
−37.701
28.520
15.328
1.00
77.05
C


ATOM
3398
CG2
ILE
A
325
−36.654
30.701
14.689
1.00
73.66
C


ATOM
3399
CD1
ILE
A
325
−36.611
27.536
14.870
1.00
70.46
C


ATOM
3400
N
TYR
A
326
−39.803
31.815
12.690
1.00
76.28
N


ATOM
3401
CA
TYR
A
326
−39.934
33.054
11.884
1.00
76.44
C


ATOM
3402
C
TYR
A
326
−40.978
34.017
12.416
1.00
73.86
C


ATOM
3403
O
TYR
A
326
−40.991
35.153
11.992
1.00
77.86
O


ATOM
3404
CB
TYR
A
326
−40.242
32.772
10.409
1.00
77.72
C


ATOM
3405
CG
TYR
A
326
−39.240
31.893
9.701
1.00
77.26
C


ATOM
3406
CD1
TYR
A
326
−39.666
30.889
8.847
1.00
77.44
C


ATOM
3407
CD2
TYR
A
326
−37.865
32.057
9.885
1.00
81.33
C


ATOM
3408
CE1
TYR
A
326
−38.764
30.077
8.194
1.00
78.66
C


ATOM
3409
CE2
TYR
A
326
−36.961
31.248
9.236
1.00
83.58
C


ATOM
3410
CZ
TYR
A
326
−37.423
30.258
8.389
1.00
80.70
C


ATOM
3411
OH
TYR
A
326
−36.541
29.451
7.742
1.00
81.84
O


ATOM
3412
N
CYS
A
327
−41.848
33.582
13.332
1.00
72.88
N


ATOM
3413
CA
CYS
A
327
−42.782
34.496
13.999
1.00
76.25
C


ATOM
3414
C
CYS
A
327
−42.064
35.537
14.905
1.00
78.27
C


ATOM
3415
O
CYS
A
327
−42.684
36.508
15.330
1.00
79.91
O


ATOM
3416
CB
CYS
A
327
−43.827
33.729
14.805
1.00
77.89
C


ATOM
3417
SG
CYS
A
327
−44.970
32.742
13.778
1.00
82.26
S


ATOM
3418
N
ARG
A
328
−40.770
35.320
15.191
1.00
79.14
N


ATOM
3419
CA
ARG
A
328
−39.919
36.324
15.832
1.00
79.59
C


ATOM
3420
C
ARG
A
328
−39.781
37.596
14.984
1.00
79.99
C


ATOM
3421
O
ARG
A
328
−39.581
38.680
15.524
1.00
79.93
O


ATOM
3422
CB
ARG
A
328
−38.515
35.771
16.078
1.00
77.25
C


ATOM
3423
CG
ARG
A
328
−38.423
34.646
17.130
1.00
76.09
C


ATOM
3424
CD
ARG
A
328
−36.998
34.067
17.220
1.00
77.31
C


ATOM
3425
NE
ARG
A
328
−36.006
35.129
17.048
1.00
75.58
N


ATOM
3426
CZ
ARG
A
328
−35.537
35.945
17.985
1.00
81.23
C


ATOM
3427
NH1
ARG
A
328
−35.953
35.859
19.252
1.00
74.46
N


ATOM
3428
NH2
ARG
A
328
−34.631
36.866
17.640
1.00
81.80
N


ATOM
3429
N
SER
A
329
−39.885
37.449
13.659
1.00
82.57
N


ATOM
3430
CA
SER
A
329
−39.788
38.574
12.743
1.00
84.20
C


ATOM
3431
C
SER
A
329
−41.107
39.324
12.711
1.00
84.79
C


ATOM
3432
O
SER
A
329
−42.151
38.687
12.597
1.00
88.37
O


ATOM
3433
CB
SER
A
329
−39.460
38.097
11.335
1.00
84.66
C


ATOM
3434
OG
SER
A
329
−39.465
39.190
10.429
1.00
90.44
O


ATOM
3435
N
PRO
A
330
−41.076
40.675
12.814
1.00
86.62
N


ATOM
3436
CA
PRO
A
330
−42.339
41.415
12.685
1.00
85.84
C


ATOM
3437
C
PRO
A
330
−42.905
41.369
11.253
1.00
86.45
C


ATOM
3438
O
PRO
A
330
−44.103
41.466
11.079
1.00
85.67
O


ATOM
3439
CB
PRO
A
330
−41.960
42.851
13.080
1.00
85.22
C


ATOM
3440
CG
PRO
A
330
−40.597
42.764
13.669
1.00
85.83
C


ATOM
3441
CD
PRO
A
330
−39.946
41.595
13.045
1.00
85.56
C


ATOM
3442
N
ASP
A
331
−42.031
41.216
10.256
1.00
87.75
N


ATOM
3443
CA
ASP
A
331
−42.440
41.127
8.854
1.00
88.35
C


ATOM
3444
C
ASP
A
331
−43.160
39.831
8.516
1.00
88.54
C


ATOM
3445
O
ASP
A
331
−44.180
39.865
7.824
1.00
87.13
O


ATOM
3446
CB
ASP
A
331
−41.225
41.268
7.929
1.00
91.75
C


ATOM
3447
CG
ASP
A
331
−40.585
42.640
8.000
1.00
99.65
C


ATOM
3448
OD1
ASP
A
331
−41.152
43.526
8.660
1.00
101.57
O


ATOM
3449
OD2
ASP
A
331
−39.507
42.825
7.387
1.00
108.72
O


ATOM
3450
N
PHE
A
332
−42.638
38.697
8.992
1.00
87.91
N


ATOM
3451
CA
PHE
A
332
−43.306
37.387
8.759
1.00
85.83
C


ATOM
3452
C
PHE
A
332
−44.596
37.298
9.525
1.00
84.97
C


ATOM
3453
O
PHE
A
332
−45.583
36.811
9.006
1.00
85.85
O


ATOM
3454
CB
PHE
A
332
−42.419
36.206
9.142
1.00
83.02
C


ATOM
3455
CG
PHE
A
332
−41.421
35.848
8.099
1.00
81.58
C


ATOM
3456
CD1
PHE
A
332
−40.184
36.470
8.064
1.00
79.60
C


ATOM
3457
CD2
PHE
A
332
−41.719
34.884
7.141
1.00
82.96
C


ATOM
3458
CE1
PHE
A
332
−39.251
36.138
7.092
1.00
83.90
C


ATOM
3459
CE2
PHE
A
332
−40.794
34.541
6.159
1.00
81.77
C


ATOM
3460
CZ
PHE
A
332
−39.558
35.166
6.132
1.00
84.78
C


ATOM
3461
N
ARG
A
333
−44.567
37.772
10.764
1.00
85.64
N


ATOM
3462
CA
ARG
A
333
−45.742
37.795
11.618
1.00
86.23
C


ATOM
3463
C
ARG
A
333
−46.875
38.603
10.964
1.00
85.68
C


ATOM
3464
O
ARG
A
333
−48.015
38.171
11.005
1.00
85.47
O


ATOM
3465
CB
ARG
A
333
−45.357
38.362
12.963
1.00
86.76
C


ATOM
3466
CG
ARG
A
333
−46.383
38.222
14.059
1.00
90.70
C


ATOM
3467
CD
ARG
A
333
−45.671
38.263
15.407
1.00
93.90
C


ATOM
3468
NE
ARG
A
333
−44.710
39.377
15.472
1.00
100.54
N


ATOM
3469
CZ
ARG
A
333
−43.693
39.490
16.335
1.00
103.24
C


ATOM
3470
NH1
ARG
A
333
−43.449
38.553
17.262
1.00
104.89
N


ATOM
3471
NH2
ARG
A
333
−42.899
40.566
16.271
1.00
102.64
N


ATOM
3472
N
ILE
A
334
−46.534
39.755
10.373
1.00
84.51
N


ATOM
3473
CA
ILE
A
334
−47.472
40.580
9.584
1.00
83.97
C


ATOM
3474
C
ILE
A
334
−47.898
39.862
8.316
1.00
82.02
C


ATOM
3475
O
ILE
A
334
−49.077
39.871
7.963
1.00
86.53
O


ATOM
3476
CB
ILE
A
334
−46.862
41.959
9.211
1.00
85.26
C


ATOM
3477
CG1
ILE
A
334
−46.777
42.860
10.444
1.00
85.90
C


ATOM
3478
CG2
ILE
A
334
−47.692
42.668
8.142
1.00
82.88
C


ATOM
3479
CD1
ILE
A
334
−45.791
44.015
10.287
1.00
88.19
C


ATOM
3480
N
ALA
A
335
−46.934
39.248
7.635
1.00
80.09
N


ATOM
3481
CA
ALA
A
335
−47.202
38.470
6.433
1.00
79.44
C


ATOM
3482
C
ALA
A
335
−48.199
37.345
6.719
1.00
78.78
C


ATOM
3483
O
ALA
A
335
−49.207
37.251
6.057
1.00
78.15
O


ATOM
3484
CB
ALA
A
335
−45.911
37.896
5.876
1.00
78.92
C


ATOM
3485
N
PHE
A
336
−47.896
36.511
7.714
1.00
78.39
N


ATOM
3486
CA
PHE
A
336
−48.760
35.372
8.092
1.00
77.87
C


ATOM
3487
C
PHE
A
336
−50.184
35.751
8.427
1.00
77.70
C


ATOM
3488
O
PHE
A
336
−51.116
35.205
7.847
1.00
75.76
O


ATOM
3489
CB
PHE
A
336
−48.198
34.602
9.301
1.00
77.26
C


ATOM
3490
CG
PHE
A
336
−46.852
33.943
9.072
1.00
77.36
C


ATOM
3491
CD1
PHE
A
336
−46.408
33.562
7.796
1.00
78.61
C


ATOM
3492
CD2
PHE
A
336
−46.024
33.695
10.152
1.00
76.74
C


ATOM
3493
CE1
PHE
A
336
−45.180
32.962
7.621
1.00
76.21
C


ATOM
3494
CE2
PHE
A
336
−44.787
33.089
9.974
1.00
78.36
C


ATOM
3495
CZ
PHE
A
336
−44.372
32.724
8.700
1.00
76.78
C


ATOM
3496
N
GLN
A
337
−50.346
36.681
9.363
1.00
78.23
N


ATOM
3497
CA
GLN
A
337
−51.691
37.124
9.792
1.00
79.51
C


ATOM
3498
C
GLN
A
337
−52.549
37.711
8.642
1.00
78.03
C


ATOM
3499
O
GLN
A
337
−53.778
37.646
8.694
1.00
78.24
O


ATOM
3500
CB
GLN
A
337
−51.609
38.081
10.997
1.00
80.74
C


ATOM
3501
CG
GLN
A
337
−50.870
39.391
10.802
1.00
86.86
C


ATOM
3502
CD
GLN
A
337
−50.613
40.113
12.143
1.00
89.20
C


ATOM
3503
OE1
GLN
A
337
−51.317
39.877
13.127
1.00
97.34
O


ATOM
3504
NE2
GLN
A
337
−49.610
40.989
12.177
1.00
94.24
N


ATOM
3505
N
GLU
A
338
−51.900
38.271
7.622
1.00
76.38
N


ATOM
3506
CA
GLU
A
338
−52.591
38.694
6.411
1.00
74.47
C


ATOM
3507
C
GLU
A
338
−53.085
37.448
5.670
1.00
73.86
C


ATOM
3508
O
GLU
A
338
−54.258
37.370
5.282
1.00
73.06
O


ATOM
3509
CB
GLU
A
338
−51.659
39.512
5.516
1.00
75.29
C


ATOM
3510
CG
GLU
A
338
−52.343
40.279
4.365
1.00
79.56
C


ATOM
3511
CD
GLU
A
338
−52.695
39.442
3.113
1.00
85.28
C


ATOM
3512
OE1
GLU
A
338
−52.500
38.207
3.087
1.00
91.20
O


ATOM
3513
OE2
GLU
A
338
−53.176
40.048
2.131
1.00
89.12
O


ATOM
3514
N
LEU
A
339
−52.177
36.488
5.477
1.00
70.47
N


ATOM
3515
CA
LEU
A
339
−52.475
35.242
4.763
1.00
71.14
C


ATOM
3516
C
LEU
A
339
−53.527
34.375
5.463
1.00
72.95
C


ATOM
3517
O
LEU
A
339
−54.255
33.650
4.793
1.00
74.46
O


ATOM
3518
CB
LEU
A
339
−51.209
34.406
4.567
1.00
68.65
C


ATOM
3519
CG
LEU
A
339
−50.055
35.021
3.778
1.00
69.52
C


ATOM
3520
CD1
LEU
A
339
−48.805
34.139
3.935
1.00
68.79
C


ATOM
3521
CD2
LEU
A
339
−50.411
35.225
2.315
1.00
62.62
C


ATOM
3522
N
LEU
A
340
−53.599
34.455
6.796
1.00
74.28
N


ATOM
3523
CA
LEU
A
340
−54.598
33.718
7.588
1.00
75.08
C


ATOM
3524
C
LEU
A
340
−55.848
34.573
7.901
1.00
76.80
C


ATOM
3525
O
LEU
A
340
−56.573
34.280
8.860
1.00
76.39
O


ATOM
3526
CB
LEU
A
340
−53.960
33.174
8.878
1.00
74.68
C


ATOM
3527
CG
LEU
A
340
−52.763
32.233
8.701
1.00
73.84
C


ATOM
3528
CD1
LEU
A
340
−52.150
31.887
10.048
1.00
73.83
C


ATOM
3529
CD2
LEU
A
340
−53.167
30.976
7.982
1.00
73.24
C


ATOM
3530
N
CYS
A
341
−56.083
35.619
7.088
1.00
79.59
N


ATOM
3531
CA
CYS
A
341
−57.280
36.477
7.152
1.00
80.75
C


ATOM
3532
C
CYS
A
341
−57.536
37.089
8.530
1.00
81.21
C


ATOM
3533
O
CYS
A
341
−58.613
36.911
9.114
1.00
81.97
O


ATOM
3534
CB
CYS
A
341
−58.500
35.683
6.674
1.00
81.26
C


ATOM
3535
SG
CYS
A
341
−58.243
34.889
5.079
1.00
88.24
S


ATOM
3536
N
LEU
A
342
−56.533
37.809
9.031
1.00
81.84
N


ATOM
3537
CA
LEU
A
342
−56.598
38.483
10.333
1.00
82.60
C


ATOM
3538
C
LEU
A
342
−56.370
39.993
10.158
1.00
83.35
C


ATOM
3539
O
LEU
A
342
−55.715
40.650
10.971
1.00
85.10
O


ATOM
3540
CB
LEU
A
342
−55.575
37.878
11.303
1.00
82.19
C


ATOM
3541
CG
LEU
A
342
−55.513
36.347
11.407
1.00
82.55
C


ATOM
3542
CD1
LEU
A
342
−54.415
35.913
12.368
1.00
82.95
C


ATOM
3543
CD2
LEU
A
342
−56.851
35.782
11.838
1.00
83.45
C


TER
3544

LEU
A
342


HETATM
3545
C1
MAL
A
401
−30.433
67.551
22.604
1.00
116.54
C


HETATM
3546
C2
MAL
A
401
−29.508
68.271
21.613
1.00
116.67
C


HETATM
3547
C3
MAL
A
401
−28.286
67.466
21.250
1.00
115.67
C


HETATM
3548
C4
MAL
A
401
−27.437
67.774
22.475
1.00
113.35
C


HETATM
3549
C5
MAL
A
401
−28.222
67.326
23.742
1.00
114.16
C


HETATM
3550
C6
MAL
A
401
−27.467
67.821
24.997
1.00
112.25
C


HETATM
3551
O1
MAL
A
401
−30.860
66.152
22.481
1.00
118.82
O


HETATM
3552
O2
MAL
A
401
−30.196
68.896
20.430
1.00
113.17
O


HETATM
3553
O3
MAL
A
401
−27.692
67.923
20.042
1.00
118.88
O


HETATM
3554
O4
MAL
A
401
−26.151
67.133
22.417
1.00
110.96
O


HETATM
3555
O5
MAL
A
401
−29.640
67.723
23.796
1.00
116.03
O


HETATM
3556
O6
MAL
A
401
−28.329
68.191
26.091
1.00
111.18
O


HETATM
3557
C1′
MAL
A
401
−34.225
64.365
24.555
1.00
118.64
C


HETATM
3558
C2′
MAL
A
401
−34.062
65.875
24.695
1.00
118.98
C


HETATM
3559
C3′
MAL
A
401
−33.053
66.497
23.732
1.00
118.89
C


HETATM
3560
C4′
MAL
A
401
−31.773
65.653
23.533
1.00
118.41
C


HETATM
3561
C5′
MAL
A
401
−32.192
64.169
23.329
1.00
117.27
C


HETATM
3562
C6′
MAL
A
401
−31.044
63.164
23.154
1.00
114.49
C


HETATM
3563
O1′
MAL
A
401
−34.905
63.850
25.705
1.00
115.98
O


HETATM
3564
O2′
MAL
A
401
−35.328
66.510
24.463
1.00
120.41
O


HETATM
3565
O3′
MAL
A
401
−32.803
67.771
24.346
1.00
118.98
O


HETATM
3566
O5′
MAL
A
401
−32.951
63.737
24.467
1.00
118.26
O


HETATM
3567
O6′
MAL
A
401
−31.319
61.889
23.781
1.00
101.09
O


HETATM
3568
S
SO4
A
402
−30.549
40.587
1.840
1.00
88.35
S


HETATM
3569
O1
SO4
A
402
−31.012
40.657
0.449
1.00
91.49
O


HETATM
3570
O2
SO4
A
402
−31.341
41.518
2.648
1.00
79.98
O


HETATM
3571
O3
SO4
A
402
−30.747
39.233
2.389
1.00
86.89
O


HETATM
3572
O4
SO4
A
402
−29.136
40.942
1.808
1.00
79.16
O


HETATM
3573
S
SO4
A
403
−34.529
39.154
20.841
1.00
91.42
S


HETATM
3574
O1
SO4
A
403
−35.518
40.193
21.131
1.00
84.45
O


HETATM
3575
O2
SO4
A
403
−33.296
39.334
21.612
1.00
93.22
O


HETATM
3576
O3
SO4
A
403
−35.078
37.875
21.232
1.00
84.98
O


HETATM
3577
O4
SO4
A
403
−34.193
39.246
19.438
1.00
95.59
O


HETATM
3578
S
SO4
A
404
−26.677
61.410
43.667
1.00
79.61
S


HETATM
3579
O1
SO4
A
404
−26.224
60.482
42.637
1.00
81.00
O


HETATM
3580
O2
SO4
A
404
−27.968
62.001
43.315
1.00
78.07
O


HETATM
3581
O3
SO4
A
404
−26.852
60.655
44.907
1.00
85.67
O


HETATM
3582
O4
SO4
A
404
−25.700
62.487
43.819
1.00
76.33
O


HETATM
3583
S
SO4
A
405
−38.222
54.167
28.613
1.00
76.15
S


HETATM
3584
O1
SO4
A
405
−36.815
54.454
28.371
1.00
62.35
O


HETATM
3585
O2
SO4
A
405
−38.708
53.202
27.628
1.00
74.38
O


HETATM
3586
O3
SO4
A
405
−38.392
53.561
29.925
1.00
77.47
O


HETATM
3587
O4
SO4
A
405
−38.983
55.412
28.536
1.00
74.20
O


HETATM
3588
S
SO4
A
406
−14.626
46.742
34.955
1.00
133.97
S


HETATM
3589
O1
SO4
A
406
−14.905
46.888
33.527
1.00
132.94
O


HETATM
3590
O2
SO4
A
406
−15.870
46.485
35.690
1.00
131.52
O


HETATM
3591
O3
SO4
A
406
−13.744
45.588
35.141
1.00
137.48
O


HETATM
3592
O4
SO4
A
406
−13.968
47.953
35.460
1.00
129.69
O


HETATM
3593
S
SO4
A
407
−39.375
59.242
10.957
1.00
103.03
S


HETATM
3594
O1
SO4
A
407
−38.112
58.591
10.589
1.00
100.34
O


HETATM
3595
O2
SO4
A
407
−40.365
59.029
9.899
1.00
104.55
O


HETATM
3596
O3
SO4
A
407
−39.865
58.663
12.211
1.00
107.47
O


HETATM
3597
O4
SO4
A
407
−39.174
60.683
11.138
1.00
106.88
O


HETATM
3598
O17
CAU
A
408
−33.477
10.957
8.170
1.00
50.96
O


HETATM
3599
C16
CAU
A
408
−32.267
10.230
8.041
1.00
45.65
C


HETATM
3600
C18
CAU
A
408
−32.478
8.951
7.225
1.00
51.24
C


HETATM
3601
N19
CAU
A
408
−33.702
8.250
7.600
1.00
54.99
N


HETATM
3602
C20
CAU
A
408
−33.806
6.805
7.498
1.00
60.13
C


HETATM
3603
C21
CAU
A
408
−33.533
6.385
6.055
1.00
66.62
C


HETATM
3604
C22
CAU
A
408
−35.184
6.350
7.988
1.00
59.87
C


HETATM
3605
C15
CAU
A
408
−31.242
11.105
7.364
1.00
46.24
C


HETATM
3606
O14
CAU
A
408
−30.049
10.367
7.182
1.00
51.01
O


HETATM
3607
C13
CAU
A
408
−28.931
10.857
6.581
1.00
52.29
C


HETATM
3608
C12
CAU
A
408
−28.911
12.133
6.005
1.00
56.44
C


HETATM
3609
C11
CAU
A
408
−27.768
12.628
5.393
1.00
56.29
C


HETATM
3610
O10
CAU
A
408
−26.608
11.875
5.332
1.00
54.90
C


HETATM
3611
C8
CAU
A
408
−26.565
10.625
5.875
1.00
54.68
C


HETATM
3612
C9
CAU
A
408
−27.768
10.108
6.517
1.00
53.44
C


HETATM
3613
N7
CAU
A
408
−25.593
9.657
5.974
1.00
57.07
N


HETATM
3614
C3
CAU
A
408
−26.096
8.561
6.637
1.00
55.09
C


HETATM
3615
C4
CAU
A
408
−27.482
8.863
6.976
1.00
53.66
C


HETATM
3616
C2
CAU
A
408
−25.589
7.338
7.009
1.00
54.54
C


HETATM
3617
C1
CAU
A
408
−26.395
6.432
7.689
1.00
56.87
C


HETATM
3618
C6
CAU
A
408
−27.717
6.731
8.006
1.00
56.28
C


HETATM
3619
C5
CAU
A
408
−28.269
7.948
7.652
1.00
56.42
C


HETATM
3620
C1
BU1
A
409
−26.142
41.424
19.127
1.00
52.74
C


HETATM
3621
C2
BU1
A
409
−24.673
41.194
19.349
1.00
50.50
C


HETATM
3622
C3
BU1
A
409
−24.361
39.703
19.256
1.00
58.68
C


HETATM
3623
C4
BU1
A
409
−23.639
39.193
20.492
1.00
60.90
C


HETATM
3624
O5
BU1
A
409
−26.449
42.812
19.063
1.00
46.89
O


HETATM
3625
O6
BU1
A
409
−23.397
37.796
20.358
1.00
65.39
O


HETATM
3626
C1
BU1
A
410
−25.492
26.523
17.727
1.00
80.96
C


HETATM
3627
C2
BU1
A
410
−25.259
26.818
16.240
1.00
75.39
C


HETATM
3628
C3
BU1
A
410
−25.569
28.286
15.904
1.00
72.90
C


HETATM
3629
C4
BU1
A
410
−24.368
29.076
15.393
1.00
70.20
C


HETATM
3630
O5
BU1
A
410
−25.036
25.202
18.096
1.00
82.37
O


HETATM
3631
O6
BU1
A
410
−23.728
29.769
16.468
1.00
72.23
O


HETATM
3632
C1
ACM
A
411
−33.365
45.116
23.775
1.00
72.09
C


HETATM
3633
O
ACM
A
411
−34.580
45.100
23.784
1.00
74.74
O


HETATM
3634
N
ACM
A
411
−32.677
43.981
23.897
1.00
71.73
N


HETATM
3635
C2
ACM
A
411
−32.587
46.397
23.626
1.00
69.99
C


HETATM
3636
C1
CLR
A
412
−36.292
27.653
−4.352
1.00
98.97
C


HETATM
3637
C2
CLR
A
412
−36.571
29.133
−4.591
1.00
98.10
C


HETATM
3638
C3
CLR
A
412
−35.425
29.793
−5.354
1.00
98.91
C


HETATM
3639
C4
CLR
A
412
−34.965
29.038
−6.627
1.00
98.80
C


HETATM
3640
C5
CLR
A
412
−35.001
27.515
−6.513
1.00
99.38
C


HETATM
3641
C6
CLR
A
412
−34.088
26.785
−7.209
1.00
99.72
C


HETATM
3642
C7
CLR
A
412
−34.017
25.259
−7.204
1.00
98.91
C


HETATM
3643
C8
CLR
A
412
−35.275
24.631
−6.633
1.00
99.40
C


HETATM
3644
C9
CLR
A
412
−35.651
25.377
−5.337
1.00
99.32
C


HETATM
3645
C10
CLR
A
412
−36.050
26.846
−5.645
1.00
100.31
C


HETATM
3646
C11
CLR
A
412
−36.707
24.655
−4.466
1.00
96.50
C


HETATM
3647
C12
CLR
A
412
−36.536
23.143
−4.365
1.00
96.65
C


HETATM
3648
C13
CLR
A
412
−36.329
22.495
−5.719
1.00
100.10
C


HETATM
3649
C14
CLR
A
412
−35.100
23.138
−6.353
1.00
101.09
C


HETATM
3650
C15
CLR
A
412
−34.782
22.237
−7.542
1.00
101.42
C


HETATM
3651
C16
CLR
A
412
−35.217
20.837
−7.081
1.00
101.37
C


HETATM
3652
C17
CLR
A
412
−35.995
21.000
−5.758
1.00
100.60
C


HETATM
3653
C18
CLR
A
412
−37.562
22.726
−6.571
1.00
103.09
C


HETATM
3654
C19
CLR
A
412
−37.268
26.936
−6.512
1.00
102.37
C


HETATM
3655
C20
CLR
A
412
−37.144
19.961
−5.632
1.00
100.25
C


HETATM
3656
C21
CLR
A
412
−38.032
20.187
−4.406
1.00
98.50
C


HETATM
3657
C22
CLR
A
412
−36.561
18.538
−5.657
1.00
101.01
C


HETATM
3658
C23
CLR
A
412
−37.437
17.457
−5.012
1.00
103.22
C


HETATM
3659
C24
CLR
A
412
−36.748
16.094
−5.099
1.00
105.36
C


HETATM
3660
C25
CLR
A
412
−37.535
15.014
−5.853
1.00
107.48
C


HETATM
3661
C26
CLR
A
412
−36.571
14.080
−6.591
1.00
107.73
C


HETATM
3662
C27
CLR
A
412
−38.402
14.192
−4.899
1.00
106.61
C


HETATM
3663
O1
CLR
A
412
−35.828
31.158
−5.664
1.00
99.70
O


HETATM
3664
C1
CLR
A
413
−52.083
29.442
3.198
1.00
84.76
C


HETATM
3665
C2
CLR
A
413
−52.244
30.951
3.100
1.00
85.75
C


HETATM
3666
C3
CLR
A
413
−53.214
31.296
1.956
1.00
84.48
C


HETATM
3667
C4
CLR
A
413
−54.606
30.718
2.233
1.00
82.59
C


HETATM
3668
C5
CLR
A
413
−54.424
29.218
2.245
1.00
86.97
C


HETATM
3669
C6
CLR
A
413
−55.171
28.468
1.400
1.00
86.01
C


HETATM
3670
C7
CLR
A
413
−55.077
26.968
1.309
1.00
88.00
C


HETATM
3671
C8
CLR
A
413
−54.340
26.362
2.487
1.00
89.33
C


HETATM
3672
C9
CLR
A
413
−53.087
27.161
2.862
1.00
88.72
C


HETATM
3673
C10
CLR
A
413
−53.399
28.636
3.214
1.00
85.54
C


HETATM
3674
C11
CLR
A
413
−52.265
26.440
3.959
1.00
87.79
C


HETATM
3675
C12
CLR
A
413
−51.961
24.981
3.616
1.00
90.38
C


HETATM
3676
C13
CLR
A
413
−53.219
24.202
3.283
1.00
90.23
C


HETATM
3677
C14
CLR
A
413
−53.904
24.933
2.142
1.00
92.32
C


HETATM
3678
C15
CLR
A
413
−54.976
23.971
1.668
1.00
92.65
C


HETATM
3679
C16
CLR
A
413
−54.282
22.616
1.827
1.00
93.79
C


HETATM
3680
C17
CLR
A
413
−53.018
22.820
2.693
1.00
91.53
C


HETATM
3681
C18
CLR
A
413
−54.156
24.093
4.499
1.00
87.86
C


HETATM
3682
C19
CLR
A
413
−54.039
28.774
4.595
1.00
82.64
C


HETATM
3683
C20
CLR
A
413
−52.772
21.624
3.653
1.00
91.51
C


HETATM
3684
C21
CLR
A
413
−51.815
21.878
4.816
1.00
89.13
C


HETATM
3685
C22
CLR
A
413
−52.233
20.430
2.857
1.00
91.91
C


HETATM
3686
C23
CLR
A
413
−52.841
19.099
3.296
1.00
93.30
C


HETATM
3687
C24
CLR
A
413
−52.459
17.993
2.311
1.00
92.99
C


HETATM
3688
C25
CLR
A
413
−51.938
16.746
3.002
1.00
92.39
C


HETATM
3689
C26
CLR
A
413
−52.059
15.541
2.076
1.00
94.36
C


HETATM
3690
C27
CLR
A
413
−50.499
16.952
3.460
1.00
89.59
C


HETATM
3691
O1
CLR
A
413
−53.334
32.684
1.607
1.00
81.53
O


HETATM
3692
C1
CLR
A
414
−41.706
27.953
−4.419
1.00
114.97
C


HETATM
3693
C2
CLR
A
414
−41.768
29.448
−4.709
1.00
114.73
C


HETATM
3694
C3
CLR
A
414
−40.664
29.838
−5.694
1.00
114.23
C


HETATM
3695
C4
CLR
A
414
−40.711
29.022
−7.000
1.00
115.07
C


HETATM
3696
C5
CLR
A
414
−40.836
27.528
−6.714
1.00
115.29
C


HETATM
3697
C6
CLR
A
414
−40.069
26.669
−7.418
1.00
115.44
C


HETATM
3698
C7
CLR
A
414
−40.081
25.169
−7.226
1.00
116.17
C


HETATM
3699
C8
CLR
A
414
−41.303
24.678
−6.473
1.00
115.82
C


HETATM
3700
C9
CLR
A
414
−41.567
25.570
−5.252
1.00
114.14
C


HETATM
3701
C10
CLR
A
414
−41.815
27.052
−5.652
1.00
114.08
C


HETATM
3702
C11
CLR
A
414
−42.685
24.996
−4.348
1.00
113.90
C


HETATM
3703
C12
CLR
A
414
−42.459
23.526
−3.999
1.00
115.68
C


HETATM
3704
C13
CLR
A
414
−42.294
22.667
−5.238
1.00
115.89
C


HETATM
3705
C14
CLR
A
414
−41.123
23.217
−6.042
1.00
116.05
C


HETATM
3706
C15
CLR
A
414
−40.935
22.171
−7.143
1.00
117.57
C


HETATM
3707
C16
CLR
A
414
−41.285
20.849
−6.450
1.00
117.86
C


HETATM
3708
C17
CLR
A
414
−41.874
21.195
−5.060
1.00
116.61
C


HETATM
3709
C18
CLR
A
414
−43.602
22.724
−6.051
1.00
114.74
C


HETATM
3710
C19
CLR
A
414
−43.197
27.247
−6.285
1.00
114.74
C


HETATM
3711
C20
CLR
A
414
−42.937
20.193
−4.516
1.00
116.12
C


HETATM
3712
C21
CLR
A
414
−43.284
20.508
−3.065
1.00
114.86
C


HETATM
3713
C22
CLR
A
414
−42.503
18.733
−4.603
1.00
115.06
C


HETATM
3714
C23
CLR
A
414
−43.586
17.708
−4.216
1.00
115.00
C


HETATM
3715
C24
CLR
A
414
−42.985
16.411
−3.662
1.00
113.44
C


HETATM
3716
C25
CLR
A
414
−42.272
15.571
−4.726
1.00
113.32
C


HETATM
3717
C26
CLR
A
414
−40.869
16.077
−5.018
1.00
111.52
C


HETATM
3718
C27
CLR
A
414
−42.204
14.107
−4.309
1.00
112.05
C


HETATM
3719
O1
CLR
A
414
−40.674
31.263
−5.966
1.00
114.05
O


HETATM
3720
C1
PLM
A
415
−57.959
33.303
4.949
1.00
94.70
C


HETATM
3721
O2
PLM
A
415
−57.047
33.732
4.211
1.00
99.49
O


HETATM
3722
C2
PLM
A
415
−58.012
31.828
5.279
1.00
93.74
C


HETATM
3723
C3
PLM
A
415
−59.131
31.125
4.492
1.00
92.47
C


HETATM
3724
C4
PLM
A
415
−58.642
29.982
3.590
1.00
91.37
C


HETATM
3725
C5
PLM
A
415
−59.532
28.742
3.701
1.00
89.98
C


HETATM
3726
C6
PLM
A
415
−59.310
27.816
2.505
1.00
88.93
C


HETATM
3727
C7
PLM
A
415
−60.003
26.467
2.677
1.00
86.74
C


HETATM
3728
C8
PLM
A
415
−59.012
25.330
2.467
1.00
86.47
C


HETATM
3729
C9
PLM
A
415
−59.709
23.971
2.504
1.00
88.96
C


HETATM
3730
CA
PLM
A
415
−58.837
22.893
3.156
1.00
90.43
C


HETATM
3731
CB
PLM
A
415
−59.094
21.474
2.622
1.00
94.25
C


HETATM
3732
CC
PLM
A
415
−57.863
20.775
2.016
1.00
96.09
C


HETATM
3733
CD
PLM
A
415
−57.471
19.483
2.752
1.00
100.00
C


HETATM
3734
CE
PLM
A
415
−56.635
18.544
1.872
1.00
101.35
C


HETATM
3735
CF
PLM
A
415
−56.391
17.207
2.576
1.00
101.14
C


HETATM
3736
CG
PLM
A
415
−55.695
16.199
1.664
1.00
99.15
C


HETATM
3737
C35
12P
A
416
−12.915
54.897
17.807
1.00
109.60
C


HETATM
3738
O34
12P
A
416
−13.949
54.683
18.777
1.00
110.80
O


HETATM
3739
C33
12P
A
416
−13.451
54.394
20.091
1.00
108.13
C


HETATM
3740
C32
12P
A
416
−13.568
55.622
20.982
1.00
105.37
C


HETATM
3741
O31
12P
A
416
−12.299
56.022
21.539
1.00
101.39
O


HETATM
3742
C30
12P
A
416
−12.283
57.340
22.133
1.00
102.00
C


HETATM
3743
C29
12P
A
416
−13.231
57.476
23.346
1.00
99.93
C


HETATM
3744
O28
12P
A
416
−14.580
57.575
22.874
1.00
94.22
O


HETATM
3745
C27
12P
A
416
−15.580
58.132
23.720
1.00
91.37
C


HETATM
3746
C26
12P
A
416
−16.528
58.912
22.820
1.00
89.49
C


HETATM
3747
O25
12P
A
416
−15.752
59.825
22.016
1.00
91.31
O


HETATM
3748
C24
12P
A
416
−16.081
59.833
20.621
1.00
89.90
C


HETATM
3749
C23
12P
A
416
−14.998
60.574
19.838
1.00
88.02
C


HETATM
3750
O22
12P
A
416
−14.838
61.904
20.352
1.00
86.45
O


HETATM
3751
C21
12P
A
416
−15.839
62.838
19.944
1.00
77.75
C


HETATM
3752
C20
12P
A
416
−15.146
63.916
19.149
1.00
82.56
C


HETATM
3753
O19
12P
A
416
−14.005
64.462
19.834
1.00
79.81
O


HETATM
3754
C18
12P
A
416
−14.323
65.263
20.988
1.00
86.00
C


HETATM
3755
C17
12P
A
416
−13.223
66.275
21.332
1.00
87.84
C


HETATM
3756
O16
12P
A
416
−11.998
65.967
20.653
1.00
93.31
O


HETATM
3757
C15
12P
A
416
−11.011
65.354
21.493
1.00
96.79
C


HETATM
3758
O
HOH
A
501
−23.201
38.168
1.127
1.00
60.95
O


HETATM
3759
O
HOH
A
502
−28.322
45.445
19.106
1.00
62.27
O


HETATM
3760
O
HOH
A
503
−25.663
51.540
11.557
1.00
73.63
O


HETATM
3761
O
HOH
A
504
−18.403
54.490
20.568
1.00
49.22
O


HETATM
3762
O
HOH
A
505
−28.865
62.967
14.476
1.00
49.07
O


HETATM
3763
O
HOH
A
506
−38.344
25.395
6.999
1.00
53.60
O


HETATM
3764
O
HOH
A
507
−24.235
47.669
28.279
1.00
58.12
O


HETATM
3765
O
HOH
A
508
−29.566
58.644
29.505
1.00
53.32
O


HETATM
3766
O
HOH
A
509
−9.129
65.077
35.788
1.00
53.82
O


HETATM
3767
O
HOH
A
510
−31.588
56.235
5.270
1.00
54.83
O


HETATM
3768
O
HOH
A
511
−33.767
−0.936
2.970
1.00
76.89
O


HETATM
3769
O
HOH
A
512
−36.831
52.163
25.686
1.00
59.37
O


HETATM
3770
O
HOH
A
513
−20.293
51.784
5.369
1.00
57.17
O


HETATM
3771
O
HOH
A
514
−33.710
48.038
30.053
1.00
55.19
O


HETATM
3772
O
HOH
A
515
−27.498
71.737
31.097
1.00
72.77
O


HETATM
3773
O
HOH
A
516
−38.419
75.003
17.409
1.00
63.18
O


HETATM
3774
O
HOH
A
517
−13.008
52.291
33.720
1.00
54.29
O


HETATM
3775
O
HOH
A
518
−25.417
49.449
2.526
1.00
84.88
O


HETATM
3776
O
HOH
A
519
−44.492
24.103
10.594
1.00
70.04
O


HETATM
3777
O
HOH
A
520
−53.192
34.656
−0.012
0.50
68.32
O


HETATM
3778
O
HOH
A
521
−33.730
52.334
13.531
1.00
71.71
O


HETATM
3779
O
HOH
A
522
−19.401
44.894
11.284
1.00
82.98
O


HETATM
3780
O
HOH
A
523
−20.706
−4.455
5.029
1.00
74.00
O


HETATM
3781
O
HOH
A
524
−18.878
44.843
24.435
1.00
71.79
O


HETATM
3782
O
HOH
A
525
−26.266
47.546
12.074
1.00
96.88
O


HETATM
3783
O
HOH
A
526
−35.818
54.612
25.448
1.00
65.23
O


HETATM
3784
O
HOH
A
527
−27.911
63.928
17.098
1.00
56.73
O


HETATM
3785
O
HOH
A
528
−34.136
24.300
12.374
1.00
64.85
O


HETATM
3786
O
HOH
A
529
−31.485
13.403
16.427
1.00
55.45
O


HETATM
3787
O
HOH
A
530
−33.229
46.525
27.509
1.00
62.72
O


HETATM
3788
O
HOH
A
531
−37.000
56.927
26.470
1.00
69.85
O


HETATM
3789
O
HOH
A
532
−38.272
23.691
9.991
1.00
74.37
O


HETATM
3790
O
HOH
A
533
−23.738
45.700
30.214
1.00
70.87
O


HETATM
3791
O
HOH
A
534
−35.547
18.917
9.368
1.00
68.76
O


HETATM
3792
O
HOH
A
535
−27.520
46.018
35.709
1.00
73.73
O


HETATM
3793
O
HOH
A
536
−11.169
52.639
27.107
1.00
53.63
O


HETATM
3794
O
HOH
A
537
−35.161
36.603
6.920
1.00
84.70
O


HETATM
3795
O
HOH
A
538
−13.331
64.631
16.826
1.00
78.41
O


HETATM
3796
O
HOH
A
539
−15.737
37.989
13.501
1.00
74.20
O


HETATM
3797
O
HOH
A
540
−17.612
49.885
19.741
1.00
67.69
O


HETATM
3798
O
HOH
A
541
−28.871
56.041
4.930
1.00
62.51
O


HETATM
3799
O
HOH
A
542
−18.100
62.470
17.192
1.00
64.11
O


HETATM
3800
O
HOH
A
543
−39.876
5.502
10.338
1.00
78.73
O


HETATM
3801
O
HOH
A
544
−39.732
−5.317
−4.531
1.00
75.67
O


HETATM
3802
O
HOH
A
545
−19.865
59.621
17.222
1.00
67.78
O


HETATM
3803
O
HOH
A
546
−37.397
27.672
6.120
1.00
59.62
O


HETATM
3804
O
HOH
A
547
−19.199
49.833
44.410
1.00
55.49
O


HETATM
3805
O
HOH
A
548
−35.618
21.645
8.827
1.00
61.27
O













CONECT
597
1283






CONECT
1229
1277


CONECT
1277
1229


CONECT
1283
597


CONECT
2906
3635


CONECT
3535
3720


CONECT
3545
3546
3551
3555


CONECT
3546
3545
3547
3552


CONECT
3547
3546
3548
3553


CONECT
3548
3547
3549
3554


CONECT
3549
3548
3550
3555


CONECT
3550
3549
3556


CONECT
3551
3545
3560


CONECT
3552
3546


CONECT
3553
3547


CONECT
3554
3548


CONECT
3555
3545
3549


CONECT
3556
3550


CONECT
3557
3558
3563
3566


CONECT
3558
3557
3559
3564


CONECT
3559
3558
3560
3565


CONECT
3560
3551
3559
3561


CONECT
3561
3560
3562
3566


CONECT
3562
3561
3567


CONECT
3563
3557


CONECT
3564
3558


CONECT
3565
3559


CONECT
3566
3557
3561


CONECT
3567
3562


CONECT
3568
3569
3570
3571
3572


CONECT
3569
3568


CONECT
3570
3568


CONECT
3571
3568


CONECT
3572
3568


CONECT
3573
3574
3575
3576
3577


CONECT
3574
3573


CONECT
3575
3573


CONECT
3576
3573


CONECT
3577
3573


CONECT
3578
3579
3580
3581
3582


CONECT
3579
3578


CONECT
3580
3578


CONECT
3581
3578


CONECT
3582
3578


CONECT
3583
3584
3585
3586
3587


CONECT
3584
3583


CONECT
3585
3583


CONECT
3586
3583


CONECT
3587
3583


CONECT
3588
3589
3590
3591
3592


CONECT
3589
3588


CONECT
3590
3588


CONECT
3591
3588


CONECT
3592
3588


CONECT
3593
3594
3595
3596
3597


CONECT
3594
3593


CONECT
3595
3593


CONECT
3596
3593


CONECT
3597
3593


CONECT
3598
3599


CONECT
3599
3598
3600
3605


CONECT
3600
3599
3601


CONECT
3601
3600
3602


CONECT
3602
3601
3603
3604


CONECT
3603
3602


CONECT
3604
3602


CONECT
3605
3599
3606


CONECT
3606
3605
3607


CONECT
3607
3606
3608
3612


CONECT
3608
3607
3609


CONECT
3609
3608
3610


CONECT
3610
3609
3611


CONECT
3611
3610
3612
3613


CONECT
3612
3607
3611
3615


CONECT
3613
3611
3614


CONECT
3614
3613
3615
3616


CONECT
3615
3612
3614
3619


CONECT
3616
3614
3617


CONECT
3617
3616
3618


CONECT
3618
3617
3619


CONECT
3619
3615
3618


CONECT
3620
3621
3624


CONECT
3621
3620
3622


CONECT
3622
3621
3623


CONECT
3623
3622
3625


CONECT
3624
3620


CONECT
3625
3623


CONECT
3626
3627
3630


CONECT
3627
3626
3628


CONECT
3628
3627
3629


CONECT
3629
3628
3631


CONECT
3630
3626


CONECT
3631
3629


CONECT
3632
3633
3634
3635


CONECT
3633
3632


CONECT
3634
3632


CONECT
3635
2906
3632


CONECT
3636
3637
3645


CONECT
3637
3636
3638


CONECT
3638
3637
3639
3663


CONECT
3639
3638
3640


CONECT
3640
3639
3641
3645


CONECT
3641
3640
3642


CONECT
3642
3641
3643


CONECT
3643
3642
3644
3649


CONECT
3644
3643
3645
3646


CONECT
3645
3636
3640
3644
3654


CONECT
3646
3644
3647


CONECT
3647
3646
3648


CONECT
3648
3647
3649
3652
3653


CONECT
3649
3643
3648
3650


CONECT
3650
3649
3651


CONECT
3651
3650
3652


CONECT
3652
3648
3651
3655


CONECT
3653
3648


CONECT
3654
3645


CONECT
3655
3652
3656
3657


CONECT
3656
3655


CONECT
3657
3655
3658


CONECT
3658
3657
3659


CONECT
3659
3658
3660


CONECT
3660
3659
3661
3662


CONECT
3661
3660


CONECT
3662
3660


CONECT
3663
3638


CONECT
3664
3665
3673


CONECT
3665
3664
3666


CONECT
3666
3665
3667
3691


CONECT
3667
3666
3668


CONECT
3668
3667
3669
3673


CONECT
3669
3668
3670


CONECT
3670
3669
3671


CONECT
3671
3670
3672
3677


CONECT
3672
3671
3673
3674


CONECT
3673
3664
3668
3672
3682


CONECT
3674
3672
3675


CONECT
3675
3674
3676


CONECT
3676
3675
3677
3680
3681


CONECT
3677
3671
3676
3678


CONECT
3678
3677
3679


CONECT
3679
3678
3680


CONECT
3680
3676
3679
3683


CONECT
3681
3676


CONECT
3682
3673


CONECT
3683
3680
3684
3685


CONECT
3684
3683


CONECT
3685
3683
3686


CONECT
3686
3685
3687


CONECT
3687
3686
3688


CONECT
3688
3687
3689
3690


CONECT
3689
3688


CONECT
3690
3688


CONECT
3691
3666


CONECT
3692
3693
3701


CONECT
3693
3692
3694


CONECT
3694
3693
3695
3719


CONECT
3695
3694
3696


CONECT
3696
3695
3697
3701


CONECT
3697
3696
3698


CONECT
3698
3697
3699


CONECT
3699
3698
3700
3705


CONECT
3700
3699
3701
3702


CONECT
3701
3692
3696
3700
3710


CONECT
3702
3700
3703


CONECT
3703
3702
3704


CONECT
3704
3703
3705
3708
3709


CONECT
3705
3699
3704
3706


CONECT
3706
3705
3707


CONECT
3707
3706
3708


CONECT
3708
3704
3707
3711


CONECT
3709
3704


CONECT
3710
3701


CONECT
3711
3708
3712
3713


CONECT
3712
3711


CONECT
3713
3711
3714


CONECT
3714
3713
3715


CONECT
3715
3714
3716


CONECT
3716
3715
3717
3718


CONECT
3717
3716


CONECT
3718
3716


CONECT
3719
3694


CONECT
3720
3535
3721
3722


CONECT
3721
3720


CONECT
3722
3720
3723


CONECT
3723
3722
3724


CONECT
3724
3723
3725


CONECT
3725
3724
3726


CONECT
3726
3725
3727


CONECT
3727
3726
3728


CONECT
3728
3727
3729


CONECT
3729
3728
3730


CONECT
3730
3729
3731


CONECT
3731
3730
3732


CONECT
3732
3731
3733


CONECT
3733
3732
3734


CONECT
3734
3733
3735


CONECT
3735
3734
3736


CONECT
3736
3735


CONECT
3737
3738


CONECT
3738
3737
3739


CONECT
3739
3738
3740


CONECT
3740
3739
3741


CONECT
3741
3740
3742


CONECT
3742
3741
3743


CONECT
3743
3742
3744


CONECT
3744
3743
3745


CONECT
3745
3744
3746


CONECT
3746
3745
3747


CONECT
3747
3746
3748


CONECT
3748
3747
3749


CONECT
3749
3748
3750


CONECT
3750
3749
3751


CONECT
3751
3750
3752


CONECT
3752
3751
3753


CONECT
3753
3752
3754


CONECT
3754
3753
3755


CONECT
3755
3754
3756


CONECT
3756
3755
3757


CONECT
3757
3756




















MASTER
455
0
16
19
4
0
18
6
3804
1
219
39



END

## Claims

1. A composition for lipidic cubic phase crystallization of a membrane protein, comprising
a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2,000;
0.1 to 0.5 M of a salt;
a host lipid, wherein said host lipid is a monoacylglycerol;
a lipid additive, wherein said lipid additive is a sterol, and wherein said sterol is present at a concentration of 5-20% w/w in said host lipid;
a buffer, wherein said buffer has a pH between 4.5-8.0; and
1 to 100 mg/ml of a membrane protein.

2. The composition of claim 1, wherein said salt is selected from the group consisting of a sulfate salt, a citrate salt, a malonate salt, a tartrate salt, an acetate salt, and a formate salt.

3. The composition of claim 2, wherein said salt is present at a concentration of 0.1-0.2 M.

4. The composition of claim 1, wherein said buffer is present at a concentration of 0.05-0.5 M.

5. The composition of claim 1, further comprising an alcohol present at a concentration of 1-10% v/v.

6. The composition of claim 5, wherein said alcohol is present at a concentration of 5-7% v/v.

7. The composition of claim 5, wherein said alcohol is a diol or triol.

8. The composition of claim 1 wherein said host lipid is selected from the group consisting of monopalmitolein, monovaccenin and monoolein.

9. The composition of claim 1 wherein said protein to be crystallized in said composition is present at a concentration of 40-60 mg/mL.

10. The composition of claim 1 wherein said membrane protein is a G-protein coupled receptor.

11. The composition of claim 1, wherein said membrane protein comprises a β2AR protein.

12. The composition of claim 11, further comprising a ligand selected from the group consisting of carazolol, timolol, alprenolol, and clenbutorol.

13. A method of generating crystals of a membrane protein comprising:
mixing a lipid additive with a host lipid to form a lipid mixture, wherein said host lipid is a monoacylglycerol, and wherein said lipid additive is a sterol, and wherein said lipid additive is 5 to 20% w/w in said host lipid; and
combining said lipid mixture with a membrane protein solution to form a protein-lipid mixture, wherein said membrane protein is present in said protein-lipid mixture at a concentration of between 1 and 100 mg/ml; and
overlaying said protein-lipid mixture with a precipitant solution, wherein said precipitant solution comprises a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2000; 0.1 to 0.5 M of a salt; and a buffer, wherein said buffer has a pH between 4.5-8.0.

14. A method of screening a crystal of a GPCR present in a liquid cubic phase composition comprising:
preparing a lipidic cubic phase composition, wherein said composition comprises a G-protein coupled receptor (GPCR) protein, a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2,000; 0.1 to 0.5 M of a salt; a host lipid, wherein said host lipid is a monoacylglycerol; a lipid additive, wherein said lipid additive is a sterol and wherein said sterol is present at a concentration of 5-20% w/w in said host lipid; and a buffer, wherein said buffer has a pH between 4.5-8.0; and
exposing said composition to a first X-ray beam and determining a change in direction or intensity of said first X-ray beam;
exposing said composition to a second beam and determining a change in direction or intensity of said second X-ray beam;
identifying an area where said GPCR crystal is present in said composition; and
exposing said identified area to at least a third X-ray beam.