Patent Publication Number: US-2002012979-A1

Title: Vitamin c production in microorganisms and plants

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     [0001] The present application claims priority under 35 U.S.C. §119(e) from U.S. Provisional Application Ser. No. 60/088,549, filed Jun. 8, 1998; from U.S. Provisional Application Ser. No. 60/125,073, filed Mar. 17, 1999; and from U.S. Provisional Application Ser. No. 60/125,054, filed Mar. 18, 1999. Each of U.S. Provisional Application Ser. Nos. 60/088,549, 60/125,073 and 60/125,054 is incorporated herein by reference in its entirety. 
    
    
     
       FIELD OF THE INVENTION  
       [0002] The present invention relates to vitamin C (L-ascorbic acid) production using genetically modified microorganisms and plants. In particular, the present invention relates to the use of nucleotide sugar epimerase enzymes for the biological production of ascorbic acid in plants and microorganisms.  
       BACKGROUND OF THE INVENTION  
       [0003] Nearly all forms of life, both plant and animal, either synthesize ascorbic acid (vitamin C) or require it as a nutrient. Ascorbic acid was first identified to be useful as a dietary supplement for humans and animals for the prevention of scurvy. Ascorbic acid, however, also affects human physiological functions such as the adsorption of iron, cold tolerance, the maintenance of the adrenal cortex, wound healing, the synthesis of polysaccharides and collagen, the formation of cartilage, dentine, bone and teeth, the maintenance of capillaries, and is useful as an antioxidant.  
       [0004] For use as a dietary supplement, ascorbic acid can be isolated from natural sources, such as rosehips, synthesized chemically through the oxidation of L-sorbose, or produced by the oxidative fermentation of calcium D-gluconate by Acetobacter suboxidans. Considine, “Ascorbic Acid,”  Van Nostrand&#39;s Scientific Encyclopedia,  Vol. 1, pp. 237-238, (1989). Ascorbic acid (predominantly intracellular) has also been obtained through the fermentation of strains of the microalga,  Chlorella pyrenoidosa.  See U.S. Pat. No. 5,001,059 by Skatrud, which is assigned to the assignee of the present application. It is believed that ascorbic acid is produced inside the chloroplasts of photosynthetic microorganisms and functions to neutralize energetic electrons produced during photosynthesis. Accordingly, ascorbic acid production is known in photosynthetic organisms as a protective mechanism.  
       [0005] Therefore, products and processes which improve the ability to biosynthetically produce ascorbic acid are desirable and beneficial for the improvement of human health.  
       SUMMARY OF THE INVENTION  
       [0006] One embodiment of the present invention relates to a method for producing ascorbic acid or esters thereof in a microorganism. The method includes the steps of: (a) culturing a microorganism having a genetic modification to increase the action of an enzyme selected from the group of hexokinase, glucose phosphate isomerase, phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase; and (b) recovering the ascorbic acid or esters produced by the microorganism. Preferably, the genetic modification is a genetic modification to increase the action of an enzyme selected from the group of GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. In one embodiment of the method of the present invention, the microorganism further includes a genetic modification to decrease the action of an enzyme having GDP-D-mannose as a substrate, other than GDP-D-mannose:GDP-L-galactose epimerase. Such a genetic modification can include, for example, a genetic modification to decrease the action of GDP-D-mannose-dehydrogenase.  
       [0007] In one embodiment, the genetic modification is a genetic modification to increase the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose, which can include GDP-D-mannose:GDP-L-galactose epimerase. In one embodiment, the epimerase binds NADPH. In one embodiment of this method, the genetic modification includes transformation of the microorganism with a recombinant nucleic acid molecule that expresses the epimerase. Such an epimerase can have a tertiary structure that substantially conforms to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Preferably, the epimerase has a structure having an average root mean square deviation of less than about 2.5 Å, and more preferably less than about 1 Å, over at least about 25% of Ca positions of the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws.  
       [0008] In one embodiment, the epimerase comprises a substrate binding site having a tertiary structure that substantially conforms to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Such a substrate binding site preferably has a tertiary structure with an average root mean square deviation of less than about 2.5 Å over at least about 25% of Cα positions of the tertiary structure of a substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws.  
       [0009] In another embodiment, the epimerase comprises a catalytic site having a tertiary structure that substantially conforms to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Such a catalytic site preferably has a tertiary structure with an average root mean square deviation of less than about 1 Å over at least about 25% of Cα positions of the tertiary structure of a catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. The catalytic site preferably includes the amino acid residues serine, tyrosine and lysine and in one embodiment, the tertiary structure positions of the amino acid residues serine, tyrosine and lysine substantially conform to tertiary structure positions of residues Ser107, Tyr136 and Lys140, respectively, as represented by atomic coordinates in Brookhaven Protein Data Bank Accession Code 1bws.  
       [0010] In yet another embodiment of this method, the epimerase comprises an amino acid sequence that aligns with SEQ ID NO:11 using a CLUSTAL alignment program, wherein amino acid residues in the amino acid sequence align with 100% identity with at least about 50%, and in another embodiment with at least about 75%, and in yet another embodiment with at least about 90% of non-Xaa residues in SEQ ID NO:11. In another embodiment, the epimerase comprises an amino acid sequence having at least 4 contiguous amino acid residues that are 100% identical to at least 4 contiguous amino acid residues of an amino acid sequence selected from the group of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. In yet another embodiment, the recombinant nucleic acid molecule comprises a nucleic acid sequence comprising at least about 12 contiguous nucleotides having 100% identity with at least about 12 contiguous nucleotides of a nucleic acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9.  
       [0011] In yet another embodiment of this method of the present invention, the epimerase comprises an amino acid sequence having a motif: Gly-Xaa-Xaa-Gly-Xaa-Xaa-Gly. In yet another embodiment, the recombinant nucleic acid molecule comprises a nucleic acid sequence that is at least about 15% identical, and in another embodiment, at least about 20% identical, and in another embodiment, at least about 25% identical, to a nucleic acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, as determined using a Lipman-Pearson method with Lipman-Pearson standard default parameters.  
       [0012] In yet another embodiment of this method of the present invention, the recombinant nucleic acid molecule comprises a nucleic acid sequence that hybridizes under stringent hybridization conditions to a nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase. The nucleic acid sequence encoding the GDP-4-keto-6-deoxy-D-mannose epimerase/reductase includes nucleic acid sequences selected from the group of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and the GDP-4-keto-6-deoxy-D-mannose epimerase/reductase can include an amino acid sequence selected from the group of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.  
       [0013] In one embodiment of the method of the present invention, the microorganism is selected from the group of bacteria, fungi and microalgae. In one embodiment, the microorganism is acid-tolerant. Preferred bacteria include, but are not limited to Azotobacter and Pseudomonas. Preferred fungi include, but are not limited to, yeast, including, but not limited to Saccharomyces yeast. Preferred microalgae include, but are not limited to, microalgae of the genera Prototheca and Chlorella, with microalgae of the genus Prototheca being particularly preferred.  
       [0014] In yet another embodiment of the method of the present invention, the microorganism is acid-tolerant and the step of culturing is conducted at a pH of less than about 6.0, and more preferably, at a pH of less than about 5.5, and even more preferably, at a pH of less than about 5.0. The step of culturing can be conducted in a fermentation medium that comprises a carbon source other than D-mannose in one embodiment, and in another embodiment, the step of culturing is conducted in a fermentation medium that comprises glucose as a carbon source.  
       [0015] In yet another embodiment of the present method, the step of culturing is conducted in a fermentation medium that is magnesium (Mg) limited. Preferably, the step of culturing is conducted in a fermentation medium that is Mg limited during a cell growth phase. In one embodiment, the fermentation medium includes less than about 0.5 g/L of Mg during a cell growth phase, and more preferably, less than about 0.2 g/L of Mg during a cell growth phase, and even more preferably, less than about 0.1 g/L of Mg during a cell growth phase.  
       [0016] Another embodiment of the present invention relates to a microorganism for producing ascorbic acid or esters thereof. The microorganism has a genetic modification to increase the action of an enzyme selected from the group of hexokinase, glucose phosphate isomerase, phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. Preferably, the genetic modification is a genetic modification to increase the action of an enzyme selected from the group of GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase, and even more preferably, to increase the action of GDP-D-mannose:GDP-L-galactose epimerase.  
       [0017] In one embodiment, the microorganism has been genetically modified to express a recombinant nucleic acid molecule encoding an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose, wherein the epimerase has a tertiary structure having an average root mean square deviation of less than about 2.5 A over at least about 25% of Cα positions of the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. In another embodiment, the microorganism has been genetically modified to express a recombinant nucleic acid molecule encoding an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose, wherein the epimerase comprises an amino acid sequence that aligns with SEQ ID NO:11 using a CLUSTAL alignment program, wherein amino acid residues in the amino acid sequence align with 100% identity with at least about 50% of non-Xaa residues in SEQ ID NO:11. Preferred microorganisms are disclosed as for the method discussed above.  
       [0018] Yet another embodiment of the present invention relates to a plant for producing ascorbic acid or esters thereof. Such a plant has a genetic modification to increase the action of an enzyme selected from the group of hexokinase, glucose phosphate isomerase, phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. In a preferred embodiment, the genetic modification is a genetic modification to increase the action of an enzyme selected from the group of GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase, and in a more preferred embodiment, the genetic modification is a genetic modification to increase the action of GDP-D-mannose:GDP-L-galactose epimerase.  
       [0019] In one embodiment, the plant further comprises a genetic modification to decrease the action of an enzyme having GDP-D-mannose as a substrate other than GDP-D-mannose:GDP-L-galactose epimerase. Such a genetic modification includes a genetic modification to decrease the action of GDP-D-mannose-dehydrogenase. Such a plant also includes a plant that has been genetically modified to express a recombinant nucleic acid molecule encoding an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose, wherein the epimerase has a tertiary structure having an average root mean square deviation of less than about 2.5 Å over at least about 25% of Cα positions of the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. In another embodiment, such a plant has been genetically modified to express a recombinant nucleic acid molecule encoding an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose, wherein the epimerase comprises an amino acid sequence that aligns with SEQ ID NO:11 using a CLUSTAL alignment program, wherein amino acid residues in the amino acid sequence align with 100% identity with at least about 50% of non-Xaa residues in SEQ ID NO:11.  
       [0020] In one embodiment, a plant for producing ascorbic acid or esters thereof according to the present invention is a microalga. Preferred microalgae include, but are not limited to microalgae of the genera Prototheca and Chlorella, with microalga of the genus Prototheca being particularly preferred. In another embodiment, the plant is a higher plant, with consumable higher plants being more preferred. 
     
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
     [0021]FIG. 1A is a schematic drawing of the pathway from glucose to GDP-D-mannose in plants.  
     [0022]FIG. 1B is a schematic drawing of the pathway from GDP-D-mannose to L-galactose-1-phosphate in plants.  
     [0023]FIG. 1C is a schematic drawing of the pathway from L-galactose to L-ascorbic acid in plants.  
     [0024]FIG. 2A is a schematic drawing of selected carbon flow from glucose in Prototheca.  
     [0025]FIG. 2B is a schematic drawing of selected carbon flow from glucose in Prototheca.  
     [0026]FIG. 3 is a schematic drawing that shows the lineage of mutants derived from  Prototheca moriformis  ATCC 75669, and their ability to produce L-ascorbic acid.  
     [0027]FIG. 4 is a bar graph illustrating the conversion of substrates by resting cells of strain NA45-3 following growth in media containing various magnesium concentrations and resuspension in media containing various magnesium concentrations.  
     [0028]FIG. 5 is a line graph showing the relationship between specific ascorbic acid formation in cultures of Prototheca strains and the specific activity of GDP-D-mannose:GDP-L-galactose epimerase in extracts prepared from cells harvested from the same cultures.  
     [0029]FIG. 6 is a line graph showing the relationship between specific epimerase activity and the degree of magnesium limitation in two strains, ATCC 75669 and EMS13-4.  
     [0030]FIG. 7 depicts the overall catalytic mechanism of GDP-D-mannose:GDP-L-galactose epimerase proposed by Barber (1979,  J. Biol. Chem.  254:7600-7603).  
     [0031]FIG. 8A depicts the catalytic mechanism of GDP-D-mannose-4,6-dehydratase (converts GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose).  
     [0032]FIG. 8B depicts the catalytic mechanism of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (converts GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose) (Chang, et al., 1988,  J. Biol. Chem.  263:1693-1697; Barber, 1980,  Plant Physiol.  66: 326-329).  
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0033] The present invention relates to a biosynthetic method and production microorganisms and plants for producing vitamin C (ascorbic acid, L-ascorbic acid, or AA). Such a method includes fermentation of a genetically modified microorganism to produce L-ascorbic acid. In particular, the present invention relates to the use of nucleotide sequences encoding epimerases, including the endogenous GDP-D-mannose:GDP-L-galactose epimerase from the L-ascorbic acid pathway, as well as epimerases having structural homology (e.g., by nucleotide/amino acid sequence and/or tertiary structure of the encoded protein) to GDP-4-keto-6-deoxy-D-mannose epimerase/reductases, or UDP-galactose 4-epimerases, for the purposes of improving the biosynthetic production of ascorbic acid. The present invention also relates to genetically modified microorganisms, such as strains of microalgae, bacteria and yeast useful for producing L-ascorbic acid, and to genetically modified plants, useful for producing consumable plant food products.  
     [0034] One embodiment of the present invention relates to a method to produce L-ascorbic acid by fermentation of a genetically modified microorganism. This method includes the steps of (a) culturing in a fermentation medium a microorganism having a genetic modification to increase the action of an enzyme selected from the group of hexokinase, glucose phosphate isomerase, phosphomannose isomerase, phosphomannomutase, GDP-mannose pyrophosphorylase, GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase; and (b) recovering L-ascorbic acid or esters thereof. The various enzymes in this list represent the enzymes involved in the vitamin C biosynthetic pathway in plants. It is uncertain at this time whether the enzyme represented by GDP-L-galactose phosphorylase is actually a phosphorylase or a pyrophosphorylase (i.e., GDP-L-galactose pyrophosphorylase). Therefore, use of the term “GDP-L-galactose phosphorylase” herein refers to either GDP-L-galactose phosphorylase or GDP-L-galactose pyrophosphorylase. In one aspect of the invention, this method includes the step of culturing in a fermentation medium a microorganism having a genetic modification to increase the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose. This aspect of the present invention is discussed in detail below.  
     [0035] Another embodiment of the present invention relates to a genetically modified microorganism for producing L-ascorbic acid or esters thereof. Another embodiment of the present invention relates to a genetically modified plant for producing L-ascorbic acid or esters thereof. Both genetically modified microorganisms (e.g., bacteria, yeast, microalgae) and plants (e.g., higher plants, microalgae) have a genetic modification to increase the action of an enzyme selected from the group of hexokinase, glucose phosphate isomerase, phosphomannose isomerase, phosphomannomutase, GDP-mannose pyrophosphorylase, GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. In a preferred embodiment, both genetically modified microorganisms (e.g., bacteria, yeast, microalgae) and plants (e.g., higher plants, microalgae) have a genetic modification to increase the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose. In one embodiment, the genetic modification includes the transformation of the microorganism or plant with the epimerase as described above.  
     [0036] To produce significantly high yields of L-ascorbic acid by the method of the present invention, a plant and/or microorganism is genetically modified to enhance production of L-ascorbic acid. As used herein, a genetically modified plant (such as a higher plant or microalgae) or microorganism, such as a microalga (Prototheca, Chlorella),  Escherichia coli,  or a yeast, is modified (i.e., mutated or changed) within its genome and/or by recombinant technology (i.e., genetic engineering) from its normal (i.e., wild-type or naturally occurring) form. In a preferred embodiment, a genetically modified plant or microorganism according to the present invention has been modified by recombinant technology. Genetic modification of a plant or microorganism can be accomplished using classical strain development and/or molecular genetic techniques, include genetic engineering techniques. Such techniques are generally disclosed herein and are additionally disclosed, for example, in Sambrook et al., 1989,  Molecular Cloning: A Laboratory Manual,  Cold Spring Harbor Labs Press; Roessler, 1995,  Plant Lipid Metabolism,  pp. 46-48; and Roessler et al., 1994, in Bioconversion for Fuels, Himmel et al. eds., American Chemical Society, Washington D.C., pp 255-70). These references are incorporated by reference herein in their entirety.  
     [0037] In some embodiments, a genetically modified plant or microorganism can include a natural genetic variant as well as a plant or microorganism in which nucleic acid molecules have been inserted, deleted or modified, including by mutation of endogenous genes (e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that the modifications provide the desired effect within the plant or microorganism. As discussed above, a genetically modified plant or microorganism includes a plant or microorganism that has been modified using recombinant technology.  
     [0038] As used herein, genetic modifications which result in a decrease in gene expression, an increase in inhibition of gene expression or inhibition of a gene product (i.e., the protein encoded by the gene), a decrease in the function of the gene, or a decrease in the function of the gene product can be referred to as inactivation (complete or partial), deletion, interruption, blockage, down-regulation, or decreased action of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such gene can be the result of a complete deletion of the gene encoding the protein (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene encoding the protein which results in incomplete or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity).  
     [0039] Genetic modifications which result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, up-regulation or increased action of a gene. Additionally, a genetic modification to a gene which modifies the expression, function, or activity of the gene can have an impact on the action of other genes and their expression products within a given metabolic pathway (e.g., by inhibition or competition). In this embodiment, the action (e.g., activity) of a particular gene and/or its product can be affected (i.e., upregulated or downregulated) by a genetic modification to another gene within the same metabolic pathway, or to a gene within a different metabolic pathway which impacts the pathway of interest by competition, inhibition, substrate formation, etc.  
     [0040] In general, a plant or microorganism having a genetic modification that affects L-ascorbic acid production has at least one genetic modification, as discussed above, which results in a change in the L-ascorbic acid production pathway as compared to a wild-type plant or microorganism grown or cultured under the same conditions. Such a modification in an L-ascorbic acid production pathway changes the ability of the plant or microorganism to produce L-ascorbic acid. According to the present invention, a genetically modified plant or microorganism preferably has an enhanced ability to produce L-ascorbic acid compared to a wild-type plant or microorganism cultured under the same conditions.  
     [0041] The present invention is based on the present inventors&#39; discovery of the biosynthetic pathway for L-ascorbic acid (vitamin C) in plants and microorganisms. Prior to the present invention, the metabolic pathway by which plants produce L-ascorbic acid, was not completely elucidated. The present inventors have demonstrated that L-ascorbic acid production in plants, including L-ascorbic acid-producing microorganisms (e.g., microalgae), is a pathway which uses GDP-D-mannose and involves sugar phosphates and NDP-sugars. In addition, the present inventors have made the surprising discovery that both L-galactose and L-galactono-γ-lactone can be rapidly converted into L-ascorbic acid in L-ascorbic acid-producing microalgae, including Prototheca and  Chlorella pyrenoidosa.  The entire pathway for L-ascorbic acid production in plants is set forth in FIGS.  1 A- 1 C. More particularly, FIG. 1A shows that the production of L-ascorbic acid in plants proceeds through the production of mannose intermediates to GDP-D-mannose, followed by the conversion of GDP-D-mannose to GDP-L-galactose by GDP-D-mannose:GDP-L-galactose epimerase (also known as GDP-D-mannose-3,5-epimerase) (FIG. 1B), and then by the subsequent progression to L-galactose-1-P, L-galactose, L-galactonic acid (optional), L-galactono-γ-lactone, and L-ascorbic acid (FIG. 1C). FIG. 1B also illustrates alternate pathways for the use of various intermediates, such as GDP-D-mannose. Certain aspects of this pathway have been independently described in a publication (Wheeler, et al., 1998,  Nature  393:365-369), incorporated herein by reference in its entirety.  
     [0042] Points within the L-ascorbic acid production pathway which can be targeted by genetic modification to affect the production of L-ascorbic acid can generally be categorized into at least one of the following pathways: (a) pathways affecting the production of GDP-D-mannose (e.g., pathways for converting a carbon source into GDP-D-mannose); (b) pathways for converting GDP-D-mannose into other compounds, (c) pathways associated with or downstream of the action of GDP-D-mannose:GDP-L-galactose epimerase, (d) pathways which compete for substrates involved in the production of any of the intermediates within the L-ascorbic acid production pathway, and in particular, with GDP-D-mannose, GDP-L-galactose, L-galactose-1-phosphate, L-galactose, L-galactono-γ-lactone, and/or L-ascorbic acid; and (e) pathways which inhibit production of any of the intermediates within the L-ascorbic acid production pathway, and in particular, with GDP-D-mannose, GDP-L-galactose, L-galactose-1-phosphate, L-galactose, L-galactono-γ-lactone, and/or L-ascorbic acid.  
     [0043] A genetically modified plant or microorganism useful in a method of the present invention typically has at least one genetic modification in the L-ascorbic acid production pathway which results in an enhanced production of L-ascorbic acid. In one embodiment, a genetically modified plant or microorganism has at least one genetic modification that results in: (a) an enhanced production of GDP-D-mannose; (b) an inhibition of pathways which convert GDP-D-mannose into compounds other than GDP-L-galactose; (c) an enhancement of action of the GDP-D-mannose:GDP-L-galactose epimerase; (d) an enhancement of the action of enzymes downstream of the GDP-D-mannose:GDP-L-galactose epimerase; (e) an inhibition of pathways which compete for substrates involved in the production of any of the intermediates within the L-ascorbic acid production pathway, and in particular, with GDP-D-mannose, GDP-L-galactose, L-galactose-1-phosphate, L-galactose, L-galactono-γ-lactone, and/or L-ascorbic acid; and (e) an inhibition of pathways which inhibit production of any of the intermediates within the L-ascorbic acid production pathway, and in particular, with GDP-D-mannose, GDP-L-galactose, L-galactose-1-phosphate, L-galactose, L-galactono-γ-lactone, and/or L-ascorbic acid.  
     [0044] An enhanced production of GDP-D-mannose by genetic modification of the plant or microorganism can be achieved by, for example, overexpression of enzymes such as hexokinase, glucose phosphate isomerase, phosphomannose isomerase (PMI), phosphomannomutase (PMM) and/or GDP-D-mannose pyrophosphorylase (GMP). Inhibition of pathways which convert GDP-D-mannose to compounds other than GDP-L-galactose can be achieved, for example, by modifications which inhibit polysaccharide synthesis, GDP-D-rhamnose synthesis, GDP-L-fucose synthesis and/or GDP-D-mannuronic acid synthesis. An increase in the action of the GDP-D-mannose:GDP-L-galactose epimerase and of enzymes downstream of the epimerase in the L-ascorbic acid production pathway can be achieved by genetic modifications which include, but are not limited to: overexpression of the epimerase gene (i.e, by overexpression of a recombinant nucleic acid molecule encoding the epimerase gene or a homologue thereof (discussed in detail below), and/or by mutation of the endogenous or recombinant gene to enhance expression of the gene) and/or overexpression of genes downstream of the epimerase which encode subsequent enzymes in the L-ascorbic acid pathway. Finally, metabolic pathways which compete with or inhibit the L-ascorbic acid production pathway can be inhibited by deleting or mutating enzymes, substrates or products which either inhibit or compete for an enzyme, substrate or product in the L-ascorbic acid pathway.  
     [0045] As discussed above, a genetically modified plant or microorganism useful in the method of the present invention can have at least one genetic modification (e.g., mutation in the endogenous gene or addition of a recombinant gene) in a gene encoding an enzyme involved in the L-ascorbic acid production pathway. Such genetic modifications preferably increase (i.e., enhance) the action of such enzymes such that L-ascorbic acid is preferentially produced as compared to other possible end products in related metabolic pathways. Such genetic modifications include, but are not limited to, overexpression of the gene encoding such enzyme, and deletion, mutation, or downregulation of genes encoding competitors or inhibitors of such enzyme. Preferred enzymes for which the action of the gene encoding such enzyme can be genetically modified include: hexokinase, glucose phosphate isomerase, phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. More preferably, a genetically modified plant or microorganism useful in the present invention has a genetic modification which increases the action of an enzyme selected from the group of GDP-D-mannose:GDP-L-galactose epimerase, GDP-L-galactose phosphorylase, L-galactose-1-P-phosphatase, L-galactose dehydrogenase, and/or L-galactono-γ-lactone dehydrogenase. Even more preferably, a genetically modified plant or microorganism useful in the present invention has a genetic modification which increases the action of GDP-D-mannose:GDP-L-galactose epimerase. These enzymes and the reactions catalyzed by such enzymes are illustrated in FIGS.  1 A- 1 C.  
     [0046] Prior to the present invention, without knowing the L-ascorbic acid biosynthetic (i.e., production) pathway, previous mutagenesis and screening efforts were limited in that only non-lethal mutations could be detected. One embodiment of the present invention relates to elimination of a key competing enzyme that diverts carbon flow from L-ascorbic acid synthesis. If such enzyme is absolutely required for growth on glucose, then mutants lacking the enzyme (and, therefore, having increased carbon flow to L-ascorbic acid) would have been nonviable and not have been detected during prior screening efforts. One such enzyme is phosphofructokinase (PFK) (See FIG. 2A). PFK is required for growth on glucose, and is the major step drawing carbon away from L-ascorbic acid biosynthesis (FIG. 2A). Elimination of PFK would render the cells nonviable on glucose-based media. Selection of a conditional mutant where PFK was inactivated by temperature shift, for example, may allow development of a L-ascorbic acid process where cell growth is achieved under permissive fermentation conditions, and L-ascorbic acid production (from glucose) is initiated by a shift to non-permissive condition. In this example, the temperature shift would eliminate carbon flow from glucose to glycolysis via PFK, thereby shunting carbon into the L-ascorbic acid branch of metabolism. This approach has application not only in natural L-ascorbic acid producing organisms, but also in L-ascorbic acid recombinant systems (genetically engineered plant or microorganisms) as discussed herein.  
     [0047] Knowing the identity and mechanism of the rate-limiting pathway enzymes in the L-ascorbic acid production pathway allows for design of specific inhibitors of the enzymes that are also growth inhibitory. Selection of mutants resistant to the inhibitors allows for the isolation of strains that contain L-ascorbic acid-pathway enzymes with more favorable kinetic properties. Therefore, one embodiment of the present invention is to identify inhibitors of the enzymes that are also growth inhibitory. These inhibitors are then used to select genetic mutants that overcome this inhibition and produce L-ascorbic acid at high levels. In this embodiment, the resultant plant or microorganism is a non-recombinant strain which can then be further modified by recombinant technology, if desired. In recombinant L-ascorbic acid producing strains, random mutagenesis and screening can be used as a final step to increase L-ascorbic acid production.  
     [0048] In yet another embodiment genetic modifications are made to an L-ascorbic acid producing organism directly. This allows one to build upon a base of data acquired during prior classical strain improvement efforts, and perhaps more importantly, allows one to take advantage of undefined beneficial mutations that occurred during classical strain improvement. Furthermore, fewer problems are encountered when expressing native, rather than heterologous, genes. The most advanced system for development of genetic systems for microalgae has been developed for  Chlamydomonas reinhardtii.  Preferably, development of such a genetically modified production organism would include: isolation of mutant(s) with a specific nutritional requirement for use with a cloned selectable marker gene (similar to the ura3 mutants used in yeast and fungal systems); a cloned selectable marker such as URA3 or alternatively, identification and cloning of a gene that specifies resistance to a toxic compound (this would be analogous to the use of antibiotic resistance genes in bacterial systems, and, as is the case in yeast and other fungi, a means of inserting/removing the marker gene repeatedly would be required, unless several different selectable markers were developed); a transformation system for introducing DNA into the production organism and achieving stable transformation and expression; and, a promoter system (preferably several) for high-level expression of cloned genes in the organism.  
     [0049] Another embodiment of the present invention, discussed in detail below, is to place key genes or allelic variants and homologues thereof from L-ascorbic acid producing organisms (i.e., higher plants and microalgae) into a plant or microorganism that is more amenable to molecular genetic manipulation, including endogenous L-ascorbic acid producing microorganisms and suitable plants. For example, it is possible to identify a suitable non-pathogenic organism based on the requirement of growth (on glucose) at low pH (i.e., acid-tolerant organisms, discussed in detail below).  
     [0050] One suitable candidate for recombinant production in any suitable host organism is the gene (nucleic acid molecule) encoding GDP-D-mannose:GDP-L-galactose epimerase and homologues of the GDP-D-mannose:GDP-L-galactose epimerase, as well as any other epimerase that has structural homology at the primary (i.e., sequence) or tertiary (i.e., three dimensional) level, to a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, or to a UDP-galactose 4-epimerase. Many microorganisms produce GDP-D-mannose as a precursor to exopolysaccharide and glycoprotein production, even though such organisms may not make L-ascorbic acid. This aspect of the present invention is discussed in detail below.  
     [0051] Referring to FIGS.  1 A- 1 C, at least some of the enzymes from glucose-6-phosphate to GDP-D-mannose are present in many organisms. In fact, the entire sequence is present in bacteria such as  Azotobacter vinelandii  and  Pseudomonas aeruginosa,  and make up the early steps in the biosynthesis of the exopolysaccharide alginate. In this regard, it is possible that the only thing preventing these organisms from producing L-ascorbic acid could be the lack of GDP-D-mannose:GDP-L-galactose epimerase. The presence of PMI, PMM and GMP (see FIG. 1A) in so many organisms is important for two reasons. First, these organisms themselves could serve as alternate hosts for L-ascorbic acid production, by building on the existing early pathway enzymes and adding the required cloned genes (the epimerase and possibly others). Second, the genes encoding PMI, PMM and GMP can be cloned into a new organism where, together with the cloned epimerase, they would encode the overall pathway from glucose-6-phosphate to GDP-L-galactose.  
     [0052] In order to screen genomic DNA or cDNA libraries from different organisms and to isolate nucleic acid molecules encoding these enzymes such as the GDP-D-mannose:GDP-L-galactose epimerase, one can use any of a variety of standard molecular and biochemical techniques. For example, the GDP-D-mannose:GDP-L-galactose epimerase can be purified from an organism such as Prototheca, the N-terminal amino acid sequence can be determined (including, if necessary, the sequence of internal peptide fragments), and this information can be used to design degenerate primers for amplifying a gene fragment from the organism&#39;s DNA. This fragment would then be used to probe the library, and subsequently fragments that hybridize to the probe would be cloned in that organism or another suitable production organism. There is ample precedent for plant enzymes being expressed in an active form in bacteria, such as  E. coli.  Alternatively, yeast are also a suitable candidate for developing a heterologous system for L-ascorbic acid production.  
     [0053] It is to be understood that the present invention discloses a method comprising the use of a microorganism with an ability to produce commercially useful amounts of L-ascorbic acid in a fermentation process (i.e., preferably an enhanced ability to produce L-ascorbic acid compared to a wild-type microorganism cultured under the same conditions). This method is achieved by the genetic modification of one or more genes encoding a protein involved in an L-ascorbic acid pathway which results in the production (expression) of a protein having an altered (e.g., increased or decreased) function as compared to the corresponding wild-type protein. Preferably, such genetic modification is achieved by recombinant technology. It will be appreciated by those of skill in the art that production of genetically modified plants or microorganisms having a particular altered function as described elsewhere herein (e.g., an enhanced ability to produce GDP-D-mannose:GDP-L-galactose epimerase), such as by transformation of the plant or microorganism with a nucleic acid molecule which encodes a particular enzyme, can produce many organisms meeting the given functional requirement, albeit by virtue of a variety of different genetic modifications. For example, different random nucleotide deletions and/or substitutions in a given nucleic acid sequence may all give rise to the same phenotypic result (e.g., decreased enzymatic activity of the protein encoded by the sequence). The present invention contemplates any such genetic modification which results in the production of a plant or microorganism having the characteristics set forth herein.  
     [0054] A microorganism to be used in the fermentation method of the present invention is preferably a bacterium, a fungus, or a microalga which has been genetically modified according to the disclosure above. More preferably, a microorganism useful in the present invention is a microalga which is capable of producing L-ascorbic acid, although the present invention includes microorganisms which are genetically engineered to produce L-ascorbic acid using the knowledge of the key components of the pathway and the guidance provided herein. Even more preferably, a microorganism useful in the present invention is an acid-tolerant microorganism, such as microalgae of the genera Prototheca and Chlorella. Acid-tolerant yeast and bacteria are also known in the art. Acid-tolerant microorganisms are discussed in detail below. Particularly preferred microalgae include microalgae of the genera, Prototheca and Chlorella, with Prototheca being most preferred. All known species of Prototheca produce L-ascorbic acid. Production of ascorbic acid by microalgae of the genera Prototheca and Chlorella is described in detail in U.S. Pat. No. 5,792,631, issued Aug. 11, 1998, and in U.S. Pat. No. 5,900,370, issued May 4, 1999, both of which are incorporated herein by reference in their entirety. Preferred bacteria for use in the present invention include, but are not limited to, Azotobacter, Pseudomonas, and Escherichia, although acid-tolerant bacteria are more preferred. Preferred fungi for use in the present invention include yeast, and more preferably, yeast of the genus, Saccharomyces. A microorganism for use in the fermentation method of the present invention can also be referred to as a production organism. According to the present invention, microalgae can be referred to herein either as microorganisms or as plants.  
     [0055] A preferred plant to genetically modify according to the present invention is preferably a plant suitable for consumption by animals, including humans. More preferably, such a plant is a plant that naturally produces L-ascorbic acid, although other plants can be genetically modified to produce L-ascorbic acid using the guidance provided herein.  
     [0056] The L-ascorbic acid production pathways of the microalgae Prototheca and  Chlorella pyrenoidosa  will be addressed as specific embodiments of the present invention are described below. It will be appreciated that other plants and, in particular, other microorganisms, have similar L-ascorbic acid pathways and genes and proteins having similar structure and function within such pathways. It will also be appreciated that plants and microorganisms which do not naturally produce L-ascorbic acid can be modified according to the present invention to produce L-ascorbic acid. As such, the principles discussed below with regard to Prototheca and  Chlorella pyrenoidosa  are applicable to other plants and microorganisms, including genetically modified plants and microorganisms.  
     [0057] In one embodiment of the present invention, the action of an enzyme in the L-ascorbic acid production pathway is increased by amplification of the expression (i.e., overexpression) of an enzyme in the pathway, and particularly, the GDP-D-mannose:GDP-L-galactose epimerase, homologues of the epimerase, and/or enzymes downstream of the epimerase. Overexpression of an enzyme can be accomplished, for example, by introduction of a recombinant nucleic acid molecule encoding the enzyme. It is preferred that the gene encoding an enzyme in the L-ascorbic acid production pathway be cloned under control of an artificial promoter. The promoter can be any suitable promoter that will provide a level of enzyme expression required to maintain a sufficient level of L-ascorbic acid in the production organism. Preferred promoters are constitutive (rather than inducible) promoters, since the need for addition of expensive inducers is therefore obviated. The gene dosage (copy number) of a recombinant nucleic acid molecule according to the present invention can be varied according to the requirements for maximum product formation. In one embodiment, the recombinant nucleic acid molecule encoding a gene in the L-ascorbic acid production pathway is integrated into the chromosomes of the microorganism.  
     [0058] It is another embodiment of the present invention to provide a microorganism having one or more enzymes in the L-ascorbic acid production pathway with improved affinity for its substrates. An enzyme with improved affinity for its substrates can be produced by any suitable method of genetic modification or protein engineering. For example, computer-based protein engineering can be used to design an epimerase protein with greater stability and better affinity for its substrate. See for example, Maulik et al., 1997,  Molecular Biotechnology: Therapeutic Applications and Strategies,  Wiley-Liss, Inc., which is incorporated herein by reference in its entirety.  
     [0059] Recombinant nucleic acid molecules encoding proteins in the L-ascorbic acid production pathway can be modified to enhance or reduce the function (i.e., activity) of the protein, as desired to increase L-ascorbic acid production, by any suitable method of genetic modification. For example, a recombinant nucleic acid molecule encoding an enzyme can be modified by any method for inserting, deleting, and/or substituting nucleotides, such as by error-prone PCR. In this method, the gene is amplified under conditions that lead to a high frequency of misincorporation errors by the DNA polymerase used for the amplification. As a result, a high frequency of mutations are obtained in the PCR products. The resulting gene mutants can then be screened for enhanced substrate affinity, enhanced enzymatic activity, or reduced/increased inhibitory ability by testing the mutant genes for the ability to confer increased L-ascorbic acid production onto a test microorganism, as compared to a microorganism carrying the non-mutated recombinant nucleic acid molecule.  
     [0060] Another embodiment of the present invention includes a microorganism in which competitive side reactions are blocked, including all reactions for which GDP-D-mannose is a substrate other than the production of L-ascorbic acid. In a preferred embodiment, a microorganism having complete or partial inactivation (decrease in the action of) of genes encoding enzymes which compete with the GDP-D-mannose:GDP-L-galactose epimerase for the GDP-D-mannose substrate is provided. Such enzymes include GDP-D-mannase and/or GDP-D-mannose-dehydrogenase. As used herein, inactivation of a gene can refer to any modification of a gene which results in a decrease in the activity (i.e., expression or function) of such a gene, including attenuation of activity or complete deletion of activity.  
     [0061] As discussed above, a particularly preferred aspect of the method to produce L-ascorbic acid by fermentation of a genetically modified microorganism of the present invention includes the step of culturing in a fermentation medium a microorganism having a genetic modification to increase the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose. According to the present invention, such an epimerase can include the endogenous GDP-D-mannose:GDP-L-galactose epimerase from the L-ascorbic acid pathway, described above, as well as any other epimerase that has structural homology at the primary (i.e., sequence) or tertiary (i.e., three dimensional) level, to a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, or to a UDP-galactose 4-epimerase. Such structural homology is discussed in detail below. Preferably, such an epimerase is capable of catalyzing the conversion of GDP-D-mannose to GDP-L-galactose. In one embodiment, the genetic modification includes transformation of the microorganism with a recombinant nucleic acid molecule that expresses such an epimerase.  
     [0062] Therefore, the epimerase encompassed in the method and organisms of the present invention includes the endogenous epimerase which operates in the naturally occurring ascorbic acid biosynthetic pathway (referred to herein as GDP-D-mannose:GDP-L-galactose epimerase), GDP-4-keto-6-deoxy-D-mannose epimerase/reductases, and any other epimerase which is capable of catalyzing the conversion of GDP-D mannose to GDP-L-galactose and which is structurally homologous to a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase. An epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose according the present invention can be identified by biochemical and functional characteristics as well as structural characteristics. For example, an epimerase according to the present invention is capable of acting on GDP-D-mannose as a substrate, and more particularly, such an epimerase is capable of catalyzing the conversion of GDP-D-mannose to GDP-L-galactose. It is to be understood that such capabilities need not necessarily be the normal or natural function of the epimerase as it acts in its endogenous (i.e., natural) environment. For example, GDP-4-keto-6-deoxy-D-mannose epimerase/reductase in its natural environment under normal conditions, catalyzes the conversion of GDP-D-mannose to GDP-L-fucose and does not act directly on GDP-D-mannose (See FIG. 8A, B), however, such an epimerase is encompassed by the present invention for use in catalyzing the conversion of GDP-D-mannose to GDP-L-galactose for production of ascorbic acid, to the extent that it is capable of, or can be modified to be capable of, catalyzing the conversion of GDP-D-mannose to GDP-L-galactose. Therefore, the present invention includes epimerases which have the desired enzyme activity for use in production of ascorbic acid, are capable of having such desired enzyme activity, and/or are capable of being modified or induced to have such desired enzyme activity.  
     [0063] In one embodiment, an epimerase according to the present invention includes an epimerase that catalyzes the reaction depicted in FIG. 7. In another embodiment, an epimerase according to the present invention includes an epimerase that catalyzes the first of the reactions depicted in FIG. 8B. In one embodiment, an epimerase according to the present invention binds to NADPH. In another embodiment, an epimerase according to the present invention is NADPH-dependent for enzyme activity.  
     [0064] As discussed above, the present inventors have discovered that a key enzyme in L-ascorbic acid biosynthesis in plants and microorganisms is GDP-D-mannose:GDP-L-galactose epimerase (refer to FIGS.  1 A- 1 C). One embodiment of the invention described herein is directed to the manipulation of this enzyme and structural homologues of this enzyme to increase L-ascorbic acid production in genetically engineered plants and/or microorganisms. More particularly, the GDP-D-mannose:GDP-L-galactose epimerase of the L-ascorbic acid pathway and GDP-4-keto-6-deoxy-D-mannose epimerase/reductases are believed to be structurally homologous at both the sequence and tertiary structure level; a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is believed to be capable of functioning in the L-ascorbic acid biosynthetic pathway; and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or homologue thereof may be superior to a GDP-D-mannose-GDP-L-galactose epimerase for increasing L-ascorbic acid production in genetically engineered plants and/or microorganisms. Furthermore, the present inventors disclose the use of a nucleotide sequence encoding all or part of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase as a probe to identify the gene encoding GDP-D-mannose:GDP-L-galactose epimerase. Similarly, the present inventors disclose the use of a nucleotide sequence of the gene encoding GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to design oligonucleotide primers for use in a PCR-based strategy for identifying and cloning a gene encoding GDP-D-mannose:GDP-L-galactose epimerase.  
     [0065] Without being bound by theory, the present inventors believe that the following evidence supports the novel concept that the GDP-D-mannose:GDP-L-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductases have significant structural homology at the level of sequence and/or tertiary structure, and that the GDP-4-keto-6-deoxy-D-mannose epimerase/reductases and/or homologues thereof would be useful for production of ascorbic acid and/or for isolating the endogenous GDP-D-mannose:GDP-L-galactose epimerase.  
     [0066] Although prior to the present invention, it was not known that the GDP-D-mannose:GDP-L-galactose epimerase enzyme (also known as GDP-D-mannose-3,5-epimerase) plays a critical role in L-ascorbic acid biosynthesis, this enzyme was previously described to catalyze the overall reversible reaction between GDP-D-mannose and GDP-L-galactose (Barber, 1971,  Arch. Biochem. Biophys.  147:619-623; Barber, 1975,  Arch. Biochem. Biophys.  167:718-722; Barber, 1979,  J. Biol. Chem.  254:7600-7603; Hebda, et al., 1979,  Arch. Biochem. Biophys.  194:496-502; Barber and Hebda, 1982,  Meth. Enzymol.,  83:522-525). Despite these studies, GDP-D-mannose:GDP-L-galactose epimerase has never been well characterized nor has the gene encoding this enzyme been cloned and sequenced. Since the original work by Barber, GDP-D-mannose:GDP-L-galactose epimerase activity has been detected in the colorless microalga  Prototheca moriformis  by the assignee of the present application, and in  Arabidopsis thaliana  and pea embryonic axes (Wheeler, et al., 1998, ibid.).  
     [0067] Barber (1979,  J. Biol. Chem.  254:7600-7603) proposed a mechanism for GDP-D-mannose:GDP-L-galactose epimerase partially purified from the green microalga  Chlorella pyrenoidosa.  The overall conversion of GDP-D-mannose to GDP-L-galactose was proposed to proceed by oxidation of the hexosyl moiety at C-4 to a keto intermediate, ene-diol formation, and inversion of the configurations at C-3 and C-5 upon rehydration of the double bonds and stereospecific reduction of the keto group. The proposed mechanism is depicted in FIG. 7.  
     [0068] Based on Barber&#39;s work, Feingold and Avigad (1980, In  The Biochemistry of Plants,  Vol. 3: Carbohydrates; Structure and Function, P. K. Stompf and E. E. Conn, eds., Academic Press, NY) elaborated further on the proposed mechanism for GDP-D-mannose:GDP-L-galactose epimerase. This mechanism is based on the assumption that the epimerase contains tightly bound NAD + , and transfer of a hydride ion from C-4 of the substrate (GDP-D-mannose) to enzyme-associated NAD +  converts the enzyme to the reduced (NADH)form, generating enzyme-bound GDP-4-keto-D-mannose. The latter would then undergo epimerization by an ene-diol mechanism. The final product (GDP-L-galactose) would be released from the enzyme after stereospecific transfer of the hydride ion originally removed from C-4, simultaneously regenerating the oxidized form of the enzyme.  
     [0069] L-fucose (6-deoxy-L-galactose) is a component of bacterial lipopolysaccharides, mammalian and plant glycoproteins and polysaccharides of plant cell walls. L-fucose is synthesized de novo from GDP-D-mannose by the sequential action of GDP-D-mannose-4,6-dehydratase (an NAD(P)-dependent enzyme), and a bifunctional GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (NADPH-dependent), also referred to in scientific literature as GDP-fucose synthetase (Rizzi, et al., 1998,  Structure  6:1453-1465; Somers, et al., 1998,  Structure  6:1601-1612). This pathway for L-fucose biosynthesis appears to be ubiquitous (Rizzi, et al., 1998,  Structure  6:1453-1465). The mechanisms for GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are shown in FIG. 8A, B (Chang, et al., 1988,  J. Biol. Chem.  263:1693-1697; Barber, 1980,  Plant Physiol.  66:326-329).  
     [0070] Comparison of FIGS. 7 and 8A, B reveals that Barber&#39;s proposed mechanism for GDP-D-mannose:GDP-L-galactose epimerase is analogous to the reaction mechanism for GDP-4-keto-6-deoxy-D-mannose epimerase/reductase. The same mechanism has also been demonstrated for the epimerization reaction that occurs in the biosynthesis of two TDP-6-deoxy hexoses, TDP-L-rhamnose and TDP-6-deoxy-L-talose, from TDP-D-glucose (Liu and Thorson, 1994,  Ann. Rev. Microbiol.  48:223-256). In the latter cases, however, the final reduction at C-4 is catalyzed by NADPH-dependent reductases that are separate from the epimerase enzyme. These reductases have opposite stereospecificity, providing either TDP-L-rhamnose or TDP-6-deoxy-L-talose (Liu and Thorson, 1994,  Ann. Rev. Microbiol.  48:223-256).  
     [0071] In all of the mechanisms described above, NAD(P)H is required for the final reduction at C-4 (refer to FIG. 8B). In the work of Hebda, et al. (1979,  Arch. Biochem. Biophys.  194:496-502), it was reported that GDP-D-mannose:GDP-L-galactose epimerase from  C. pyrenoidosa  did not require NAD, NADP or NADH for activity. Strangely, NADPH was not tested. Based on the analogous mechanisms shown in FIGS. 7 and 8A, B, the present inventors believe that it is likely that GDP-D-mannose:GDP-L-galactose epimerase from  C. pyrenoidosa  requires NADPH for the final reduction step. Why activity was detected in vitro without NADPH addition is not known, but tight binding of NADPH to the enzyme could explain this observation. On the other hand, if the proposed mechanism of Feingold and Avigad (1980, in  The Biochemistry of Plants,  Vol. 3, p. 101-170: Carbohydrates; Structure and Function, P. K. Stompf and E. E. Conn, ed., Academic Press, NY) is correct, the reduced enzyme-bound cofactor generated in the first oxidation step of the epimerase reaction would serve as the source of electrons for the final reduction of the keto group at C-4 back to the alcohol. Thus no addition of exogenous reduced cofactor would be required for activity in vitro.  
     [0072] Recently, a human gene encoding the bifunctional GDP-4-keto-6-deoxy-D-mannose epimerase/reductase was cloned and sequenced (Tonetti, et al., 1996,  J. Biol. Chem.  271-27274-27279). This amino acid sequence of the human GDP-4-keto-6-deoxy-D-mannose epimerase/reductase shows significant homology (29% identity) to the  E. coli  GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (Tonetti, et al., 1998,  Acta Cryst.  D54:684-686; Somers, et al., 1998,  Structure  6:1601-1612, both of which are incorporated herein by reference in their entireties). Tonetti et al. and Somers et al. additionally disclosed the tertiary (three dimensional) structure of the  E. coli  GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (also known as GDP-fucose synthetase), and noted significant structural homology with another epimerase, UDP-galactose 4-epimerase (GalE). These epimerases also share significant homology at the sequence level. Since no gene encoding a GDP-D-mannose:GDP-L-galactose epimerase has been cloned and sequenced, homology with genes encoding GDP-4-keto-6-deoxy-D-mannose epimerase/reductases or with genes encoding a UDP-galactose 4-epimerase has not been demonstrated. However, based on the similarity of the reaction products for GDP-D-mannose:GDP-L-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (i.e., GDP-L-galactose and GDP-6-deoxy-L-galactose [i.e., GDP-L-fucose], respectively) and the common catalytic mechanisms (FIGS. 7 and 8A, B) the present inventors believe that the genes encoding the enzymes will have a high degree of sequence homology, as well as tertiary structural homology.  
     [0073] Significant structural homology between GDP-D-mannose:GDP-L-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductases may allow a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, or a homologue thereof, to function in the L-ascorbic acid biosynthetic pathway, and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase could potentially be even better than a GDP-D-mannose-GDP-L-galactose epimerase for increasing L-ascorbic acid production in genetically engineered plants and/or microorganisms. Furthermore, a nucleotide sequence encoding all or part of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase can be used as a probe to identify the gene encoding GDP-D-mannose:GDP-L-galactose epimerase. Likewise, the nucleotide sequence of the gene encoding GDP-4-keto-6-deoxy-D-mannose epimerase/reductase can be used to design oligonucleotide primers for use in a PCR-based strategy for identifying and cloning a gene encoding GDP-D-mannose:GDP-L-galactose epimerase.  
     [0074] The ability to substitute GDP-4-keto-6-D-mannose epimerase/reductase for GDP-D-mannose:GDP-L-galactose epimerase to enhance L-ascorbic acid biosynthesis in plants or microorganisms depends on the ability of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to act directly on GDP-D-mannose to form GDP-L-galactose. Evidence supporting this possibility already exists.  Arabidopsis thaliana  murl mutants are defective in GDP-D-mannose-4,6-dehydratase activity (Bonin, et al., 1997,  Proc. Natl. Acad. Sci.  94:2085-2090). These mutants are thus blocked in GDP-L-fucose biosynthesis, and consequently have less than 2% of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant (Zablackis, et al., 1996,  Science  272:1808-1810). The murl mutants are more brittle than wild-type plants, are slightly dwarfed and have an apparently normal life cycle (Zablackis, et al., 272:1808-1810). When murl mutants are grown in the presence of exogenous L-fucose, the L-fucose content in the plant is restored to the wild-type state (Bonin, et al., 1997,  Proc. Natl. Acad. Sci.  94:2085-2090). It was discovered (Zablackis, et al., 1996,  Science  272:1808-1810) that murl mutants contain, in the hemicellulose xyloglucan component of the primary cell wall, L-galactose in place of the normal L-fucose. L-galactose is not normally found in the xyloglucan component, but in murl mutants L-galactose partly replaces the terminal L-fucosyl residue. Bonin, et al. (1997,  Proc. Natl. Acad. Sci.  94:2085-2090) hypothesized that in the absence of a functional GDP-D-mannose-4,6-dehydratase in the murl mutants, the GDP-4-keto-6-deoxy-D-mannose epimerase/reductase normally involved in L-fucose synthesis may be able to use GDP-D-mannose directly, forming GDP-L-galactose. Another possibility, however, is that the enzymes involved in L-ascorbic acid biosynthesis in  A. thaliana  are responsible for forming GDP-L-galactose in the murl mutant. If this were true, it would suggest that in the wild-type plant, some mechanism exists that prevents GDP-L-galactose formed in the L-ascorbic acid pathway from entering cell wall biosynthesis and substituting for (competing with) GDP-L-fucose for incorporation into the xyloglucan component (since L-galactose is not present in the primary cell wall of the wild-type plant).  
     [0075] Because of the similar reaction mechanisms of GDP-D-mannose:GDP-L-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, and because of the evidence that GDP-4-keto-6-deoxy-D-mannose epimerase/reductase can act directly on GDP-D-mannose to form GDP-L-galactose, the present inventors believe that genes encoding all epimerases and epimerase/reductases that act on GDP-D-mannose have high homology. As such, one aspect of the present invention relates to the use of any epimerase (and nucleic acid sequences encoding such epimerase) having significant homology (at the primary, secondary and/or tertiary structure level) to a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or to a UDP-galactose 4-epimerase for the purpose of improving the biosynthetic production of L-ascorbic acid.  
     [0076] Therefore, as described above, one embodiment of the present invention relates to a method for producing ascorbic acid or esters thereof in a microorganism, which includes culturing a microorganism having a genetic modification to increase the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose. Also included in the present invention are genetically modified microorganisms and plants in which the genetic modification increases the action of an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose.  
     [0077] According to the present invention, an increase in the action of the GDP-D-mannose:GDP-L-galactose epimerase in the L-ascorbic acid production pathway can be achieved by genetic modifications which include, but are not limited to overexpression of the GDP-D-mannose:GDP-L-galactose epimerase gene, a homologue of such gene, or of any recombinant nucleic acid sequence encoding an epimerase that is homologous in primary (nucleic acid or amino acid sequence) or tertiary (three dimensional protein) structure to a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase, such as by overexpression of a recombinant nucleic acid molecule encoding the epimerase gene or a homologue thereof, and/or by mutation of the endogenous or recombinant gene to enhance expression of the gene.  
     [0078] According to the present invention, an epimerase that has a tertiary structure that is homologous to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is an epimerase that has a tertiary structure that substantially conforms to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws (Table 12). In another embodiment, an epimerase that has a tertiary structure that is homologous to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is an epimerase that has a tertiary structure that substantially conforms to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1GFS. As used herein, a “tertiary structure” or “three dimensional structure” of a protein, such terms being interchangeable, refers to the components and the manner of arrangement of the components in three dimensional space to constitute the protein. The use of the term “substantially conforms” refers to at least a portion of a tertiary structure of an epimerase which is sufficiently spatially similar to at least a portion of a specified three dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Brookhaven Protein Data Bank Accession Code 1bws) to allow the tertiary structure of at least said portion of the epimerase to be modeled or calculated (i.e., by molecular replacement) using the particular set of atomic coordinates as a basis for estimating the atomic coordinates defining the three dimensional configuration of the epimerase.  
     [0079] More particularly, a tertiary structure that substantially conforms to a given set of atomic coordinates is a structure having an average root-mean-square deviation (RMSD) of less than about 2.5 Å, and more preferably, less than about 2 Å, and, in increasing preference, less than about 1.5 Å, less than about 1 Å, less than about 0.5 Å, and most preferably, less than about 0.3 Å, over at least about 25% of the Cα positions as compared to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. In other embodiments, a structure that substantially conforms to a given set of atomic coordinates is a structure wherein such structure has the recited average root-mean-square deviation (RMSD) value over at least about 50% of the Cα positions as compared to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, such structure has the recited average root-mean-square deviation (RMSD) value over at least about 75% of the Cα positions as compared to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, such structure has the recited average root-mean-square deviation (RMSD) value over about 100% of the Cα positions as compared to the tertiary structure of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Methods to calculate RMSD values are well known in the art. Various software programs for determining the tertiary structural homology between one or more proteins are known in the art and are publicly available, such as QUANTA (Molecular Simulations Inc.).  
     [0080] A preferred epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose according to the method and genetically modified organisms of the present invention includes an epimerase that comprises a substrate binding site having a tertiary structure that substantially conforms to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Preferably, the tertiary structure of the substrate binding site of the epimerase has an average root-mean-square deviation (RMSD) of less than about 2.5 Å, and more preferably, less than about 2 Å, and, in increasing preference, less than about 1.5 Å, less than about 1 Å, less than about 0.5 Å, and most preferably, less than about 0.3 Å, over at least about 25% of the Cα positions as compared to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. In other embodiments, the tertiary structure of the substrate binding site of the epimerase has the recited average root-mean-square deviation (RMSD) value over at least about 50% of the Cα positions as compared to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, the tertiary structure of the substrate binding site of the epimerase has the recited average root-mean-square deviation (RMSD) value over at least about 75% of the Cα positions as compared to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, the tertiary structure of the substrate binding site of the epimerase has the recited average root-mean-square deviation (RMSD) value over about 100% of the Cα positions as compared to the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. The tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws is discussed in detail in Rizzi et al., 1998, ibid. Additionally, the tertiary structure of the substrate binding site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1GFS is discussed in detail in Somers et al., 1998, ibid.  
     [0081] Another preferred epimerase according to the present invention includes an epimerase that comprises a catalytic site having a tertiary structure that substantially conforms to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. Preferably, the tertiary structure of the catalytic site of the epimerase has an average root-mean-square deviation (RMSD) of less than about 2.5 Å, and more preferably, less than about 2 Å, and, in increasing preference, less than about 1.5 Å, less than about 1 Å, less than about 0.5 Å, and most preferably, less than about 0.3 Å, over at least about 25% of the Cα positions as compared to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws. In other embodiments, the tertiary structure of the catalytic site of the epimerase has the recited average root-mean-square deviation (RMSD) value over at least about 50% of the Cα positions as compared to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, the tertiary structure of the catalytic site of the epimerase has the recited average root-mean-square deviation (RMSD) value over at least about 75% of the Cα positions as compared to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws, and in another embodiment, the tertiary structure of the catalytic site of the epimerase has the recited average root-mean-square deviation (RMSD) value over 100% of the Cα positions as compared to the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws.  
     [0082] In one embodiment, an epimerase encompassed by the present invention includes an epimerase that has a catalytic site which includes amino acid residues: serine, tyrosine and lysine. In a preferred embodiment, the tertiary structure positions of the amino acid residues serine, tyrosine and lysine substantially conform to the tertiary structure position of residues Ser107, Tyr136 and Lys140, respectively, as represented by atomic coordinates in Brookhaven Protein Data Bank Accession Code 1bws. The tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1bws is discussed in detail in Rizzi et al., 1998, ibid. Additionally, the tertiary structure of the catalytic site of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase represented by the atomic coordinates having Brookhaven Protein Data Bank Accession Code 1GFS is discussed in detail in Somers et al., 1998, ibid.  
     [0083] In an even more preferred embodiment, the above definition of “substantially conforms” can be further defined to include atoms of amino acid side chains. As used herein, the phrase “common amino acid side chains” refers to amino acid side chains that are common to both the structures which substantially conforms to a given set of atomic coordinates and the structure that is actually represented by such atomic coordinates. Preferably, a tertiary structure that substantially conforms to a given set of atomic coordinates is a structure having an average root-mean-square deviation (RMSD) of less than about 2.5 Å, and more preferably, less than about 2 Å, and, in increasing preference, less than about 1.5 Å, less than about 1 Å, less than about 0.5 Å, and most preferably, less than about 0.3 Å over at least about 25% of the common amino acid side chains as compared to the tertiary structure represented by the given set of atomic coordinates. In another embodiment, a structure that substantially conforms to a given set of atomic coordinates is a structure having the recited average root-mean-square deviation (RMSD) value over at least about 50% of the common amino acid side chains as compared to the tertiary structure represented by the given set of atomic coordinates, and in another embodiment, such structure has the recited average root-mean-square deviation (RMSD) value over at least about 75% of the common amino acid side chains as compared to the tertiary structure represented by the given set of atomic coordinates, and in another embodiment, such a structure has the recited average root-mean-square deviation (RMSD) value over 100% of the common amino acid side chains as compared to the tertiary structure represented by the given set of atomic coordinates.  
     [0084] A tertiary structure of an epimerase which substantially conforms to a specified set of atomic coordinates can be modeled by a suitable modeling computer program such as MODELER (A. Sali and T. L. Blundell,  J. Mol. Biol.,  vol. 234:779-815, 1993 as implemented in the Insight II Homology software package (Insight II (97.0), MSI, San Diego)), using information, for example, derived from the following data: (1) the amino acid sequence of the epimerase; (2) the amino acid sequence of the related portion(s) of the protein represented by the specified set of atomic coordinates having a three dimensional configuration; and, (3) the atomic coordinates of the specified three dimensional configuration. Alternatively, a tertiary structure of an epimerase which substantially conforms to a specified set of atomic coordinates can be modeled using data generated from analysis of a crystallized structure of the epimerase. A tertiary structure of an epimerase which substantially conforms to a specified set of atomic coordinates can also be calculated by a method such as molecular replacement. Methods of molecular replacement are generally known by those of skill in the art (generally described in Brunger,  Meth. Enzym., vol.  276, pp. 558-580, 1997; Navaza and Saludjian,  Meth. Enzym.,  vol. 276, pp. 581-594, 1997; Tong and Rossmann,  Meth. Enzym.,  vol. 276, pp. 594-611, 1997; and Bentley,  Meth. Enzym.,  vol. 276, pp. 611-619, 1997, each of which are incorporated by this reference herein in their entirety) and are performed in a software program including, for example, XPLOR (Brunger, et al.,  Science,  vol. 235, p. 458, 1987). In addition, a structure can be modeled using techniques generally described by, for example, Sali,  Current opinions in Biotechnology,  vol. 6, pp. 437-451, 1995, and algorithms can be implemented in program packages such as Homology 95.0 (in the program Insight II, available from Biosym/MSI, San Diego, Calif.). Use of Homology 95.0 requires an alignment of an amino acid sequence of a known structure having a known three dimensional structure with an amino acid sequence of a target structure to be modeled. The alignment can be a pairwise alignment or a multiple sequence alignment including other related sequences (for example, using the method generally described by Rost,  Meth. Enzymol.,  vol. 266, pp. 525-539, 1996) to improve accuracy. Structurally conserved regions can be identified by comparing related structural features, or by examining the degree of sequence homology between the known structure and the target structure. Certain coordinates for the target structure are assigned using known structures from the known structure. Coordinates for other regions of the target structure can be generated from fragments obtained from known structures such as those found in the Protein Data Bank maintained by Brookhaven National Laboratory, Upton, N.Y. Conformation of side chains of the target structure can be assigned with reference to what is sterically allowable and using a library of rotamers and their frequency of occurrence (as generally described in Ponder and Richards,  J. Mol. Biol.,  vol. 193, pp. 775-791, 1987). The resulting model of the target structure, can be refined by molecular mechanics (such as embodied in the program Discover, available from Biosym/MSI) to ensure that the model is chemically and conformationally reasonable.  
     [0085] According to the present invention, an epimerase that has a nucleic acid sequence that is homologous at the primary structure level (i.e., is a homologue of) to a nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase includes any epimerase encoded by a nucleic acid sequence that is at least about 15%, and preferably at least about 20%, and more preferably at least about 25%, and even more preferably, at least about 30% identical to a nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase, and preferably to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. Similarly, an epimerase that has an amino acid sequence that is homologous to an amino acid sequence of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase includes any epimerase having an amino acid sequence that is at least about 15%, and preferably at least about 20%, and more preferably at least about 25%, and even more preferably, at least about 30% identical to an amino acid sequence of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase or a UDP-galactose 4-epimerase, and preferably to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.  
     [0086] According to one embodiment of the present invention, homology or percent identity between two or more nucleic acid or amino acid sequences is performed using methods known in the art for aligning and/or calculating percentage identity. To compare the homology/percent identity between two or more sequences as set forth above, for example, a module contained within DNASTAR (DNASTAR, Inc., Madison, Wis.) can be used. In particular, to calculate the percent identity between two nucleic acid or amino acid sequences, the Lipman-Pearson method, provided by the MegAlign module within the DNASTAR program, is preferably used, with the following parameters, also referred to herein as the Lipman-Pearson standard default parameters:  
     [0087] (1) Ktuple=2;  
     [0088] (2) Gap penalty=4;  
     [0089] (3) Gap length penalty=12.  
     [0090] Using the Lipman-Pearson method with these parameters, for example, the percent identity between the amino acid sequence for  E. coli  GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (SEQ ID NO:4) and human GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (FX) (SEQ ID NO:6) is 27.7%, which is comparable to the 27% identity described for these enzymes in Tonetti et al., 1998,  Acta Cryst. D 54:684-686.  
     [0091] According to another embodiment of the present invention, to align two or more nucleic acid or amino acid sequences, for example to generate a consensus sequence or evaluate the similarity at various positions between such sequences, a CLUSTAL alignment program (e.g., CLUSTAL, CLUSTAL V, CLUSTAL W), also available as a module within the DNASTAR program, can be used using the following parameters, also referred to herein as the CLUSTAL standard default parameters:  
     [0092] Multiple Alignment Parameters (i.e., for more than 2 sequences):  
     [0093] (1) Gap penalty=10;  
     [0094] (2) Gap length penalty=10;  
     [0095] Pairwise Alignment Parameters (i.e., for two sequences):  
     [0096] (1) Ktuple=1;  
     [0097] (2) Gap penalty=3;  
     [0098] (3) Window=5;  
     [0099] (4) Diagonals saved=5.  
     [0100] According to the present invention, a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase can be a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from any organism, including  Arabidopsis thaliana, Escherichia coli,  and human. A nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from  Arabidopsis thaliana  is represented herein by SEQ ID NO:1. SEQ ID NO:1 encodes a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase having an amino acid sequence represented herein as SEQ ID NO:2. A nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from  Escherichia coli  is represented herein by SEQ ID NO:3. SEQ ID NO:3 encodes a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase having an amino acid sequence represented herein as SEQ ID NO:4. A nucleic acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from homo sapiens is represented herein by SEQ ID NO:5. SEQ ID NO:5 encodes a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase having an amino acid sequence represented herein as SEQ ID NO:6.  
     [0101] According to the present invention, a UDP-galactose 4-epimerase can be a UDP-galactose 4-epimerase from any organism, including  Escherichia coli  and human. A nucleic acid sequence encoding a UDP-galactose 4-epimerase from  Escherichia coli  is represented herein by SEQ ID NO:7. SEQ ID NO:7 encodes a UDP-galactose 4-epimerase having an amino acid sequence represented herein as SEQ ID NO:8. A nucleic acid sequence encoding a UDP-galactose 4-epimerase from homo sapiens is represented herein by SEQ ID NO:9. SEQ ID NO:9 encodes a UDP-galactose 4-epimerase having an amino acid sequence represented herein as SEQ ID NO:10.  
     [0102] In a preferred embodiment, an epimerase encompassed by the present invention has an amino acid sequence that aligns with the amino acid sequence of SEQ ID NO:11, for example using a CLUSTAL alignment program, wherein amino acid residues in the amino acid sequence of the epimerase align with 100% identity with at least about 50% of non-Xaa residues in SEQ ID NO:11, and preferably at least about 75% of non-Xaa residues in SEQ ID NO:11, and more preferably, at least about 90% of non-Xaa residues in SEQ ID NO:11, and even more preferably 100% of non-Xaa residues in SEQ ID NO:11. The percent identity of residues aligning with 100% identity with non-Xaa residues can be simply calculated by dividing the number of 100% identical matches at non-Xaa residues in SEQ ID NO:11 by the total number of non-Xaa residues in SEQ ID NO:11. A preferred nucleic acid sequence encoding an epimerase encompassed by the present invention include a nucleic acid sequence encoding an epimerase having an amino acid sequence with the above described identity to SEQ ID NO:11. Such an alignment using a CLUSTAL alignment program is based on the same parameters as previously disclosed herein. SEQ ID NO:11 represents a consensus amino acid sequence of an epimerase which was derived by aligning at least portions of amino acid sequences SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, as described in Somers et al., 1998,  Structure  6:1601-1612, and can be approximately duplicated using CLUSTAL.  
     [0103] In another embodiment, an epimerase encompassed by the present invention includes an epimerase that has a catalytic site which includes amino acid residues: serine, tyrosine and lysine. Preferably, such serine, tyrosine and lysine residues are located at positions in the epimerase amino acid sequence which align using a CLUSTAL alignment program with positions Ser105, Tyr134 and Lys138 of consensus sequence SEQ ID NO:11, with positions Ser109, Tyr138 and Lys142 of sequence SEQ ID NO:2, with positions Ser107, Tyr136 and Lys140 of SEQ ID NO:4, with positions Ser114, Tyr143 and Lys147 of sequence SEQ ID NO:6, with positions Ser124, Tyr149 and Lys153 of sequence SEQ ID NO:8 or with positions Ser132, Tyr157 and Lys161 of sequence SEQ ID NO:10.  
     [0104] In another embodiment, an epimerase that has an amino acid sequence that is homologous to an amino acid sequence encoding a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase includes any epimerase that has an amino acid motif: Gly-Xaa-Xaa-Gly-Xaa-Xaa-Gly, which is found, for example in positions 8 through 14 of the consensus sequence SEQ ID NO:11, in positions 12 through 18 of SEQ ID NO:2, in positions 10 through 16 of SEQ ID NO:4, in positions 14 through 20 of SEQ ID NO:6, in positions 7 through 13 of SEQ ID NO:8, and in positions 9 through 15 of SEQ ID NO:10. Such a motif can be identified by its alignment with the same motif in the above-identified amino acid sequences using a CLUSTAL alignment program. Preferably, such motif is located within the first 25 N-terminal amino acids of the amino acid sequence of the epimerase.  
     [0105] In yet another embodiment, an epimerase encompassed by the present invention includes an epimerase that has a substrate binding site which includes amino acid residues that align using a CLUSTAL alignment program with at least 50% of amino acid positions Asn177, Ser178, Arg187, Arg209, Lys283, Asn165, Ser107, Ser108, Cys109, Asn133, Tyr136 and His179 of SEQ ID NO:4. Alignment with positions Ser107, Tyr136, Asn165, Arg209, is preferably with 100% identity (i.e., exact match of residue, under parameters for alignment).  
     [0106] In another embodiment of the present invention, an epimerase encompassed by the present invention comprises at least 4 contiguous amino acid residues having 100% identity with at least 4 contiguous amino acid residues of an amino acid sequence selected from the group of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, as determined using a Lipman-Pearson method with Lipman-Pearson standard default parameters or by comparing an alignment using a CLUSTAL program with CLUSTAL standard default parameters. According to the present invention, the term “contiguous” means to be connected in an unbroken sequence. For a first sequence to have “100% identity” with a second sequence means that the first sequence exactly matches the second sequence with no gaps between nucleotides or amino acids.  
     [0107] In another embodiment of the present invention, an epimerase encompassed by the present invention is encoded by a nucleic acid sequence that comprises at least 12 contiguous nucleic acid residues having 100% identity with at least 12 contiguous nucleic acid residues of a nucleic acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:10, as determined using a Lipman-Pearson method with Lipman-Pearson standard default parameters or by comparing an alignment using a CLUSTAL program with CLUSTAL standard default parameters.  
     [0108] In another embodiment of the present invention, an epimerase encompassed by the present invention is encoded by a nucleic acid sequence that hybridizes under stringent hybridization conditions to a nucleic acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984,  Anal. Biochem.  138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.  
     [0109] More particularly, stringent hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 75%, and most particularly at least about 80%. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 20° C. and about 35° C., more preferably, between about 28° C. and about 40° C., and even more preferably, between about 35° C. and about 45° C. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 30° C. and about 45° C., more preferably, between about 38° C. and about 50° C., and even more preferably, between about 45° C. and about 55° C. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G+C content of about 40%. Alternatively, T m  can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62.  
     [0110] In another embodiment of the present invention, an epimerase encompassed by the present invention is encoded by a nucleic acid sequence that comprises a nucleic acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 or a fragment thereof, wherein the fragment encodes a protein that is capable of catalyzing the conversion of GDP-D-mannose to GDP-L-galactose, such as under physiological conditions. In another embodiment, an epimerase encompassed by the present invention comprises an amino acid sequence selected from the group of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or a fragment thereof, wherein the fragment is capable of catalyzing the conversion of GDP-D-mannose to GDP-L-galactose. It is to be understood that the nucleic acid sequence encoding the amino acid sequences identified herein can vary due to degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be encoded by different nucleotide codons.  
     [0111] One embodiment of the present invention relates to a method to identify an epimerase that catalyzes conversion of GDP-D-mannose to GDP-L-galactose. Preferably, such a method is useful for identifying the GDP-D-mannose:GDP-L-galactose epimerase which catalyzes the conversion of GDP-D-mannose to GDP-L-galactose in the endogenous (i.e., naturally occurring L-ascorbic acid biosynthetic pathway of microorganisms and/or plants). Such a method can include the steps of: (a) contacting a source of nucleic acid molecules with an oligonucleotide at least about 12 nucleotides in length under stringent hybridization conditions, wherein the oligonucleotide is identified by its ability to hybridize under stringent hybridization conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5; and, (b) identifying nucleic acid molecules from the source of nucleic acid molecules which hybridize under stringent hybridization conditions to the oligonucleotide. Nucleic acid molecules identified by this method can then be isolated from the source using standard molecular biology techniques. Preferably, the source of nucleic acid molecules is obtained from a microorganism or plant that has an ascorbic acid production pathway. Such a source of nucleic acid molecules can be any source of nucleic acid molecules which can be isolated from an organism and/or which can be screened by hybridization with an oligonucleotide such as a probe or a PCR primer. Such sources include genomic and cDNA libraries and isolated RNA.  
     [0112] In order to screen cDNA libraries from different organisms and to isolate nucleic acid molecules encoding enzymes such as the GDP-D-mannose:GDP-L-galactose epimerase and related epimerases, one can use any of a variety of standard molecular and biochemical techniques. For example, oligonucleotide primers, preferably degenerate primers, can be designed using the most conserved regions of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase nucleic acid sequence, and such primers can be used in a polymerase chain reaction (PCR) protocol to amplify the same or related epimerases, including the GDP-D-mannose:GDP-L-galactose epimerase from the ascorbic acid pathway, from nucleic acids (e.g., genomic or cDNA libraries) isolated from a desired organism (e.g., a microorganism or plant having an L-ascorbic acid pathway). Similarly, oligonucleotide probes can be designed using the most conserved regions of a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase nucleic acid sequence and such probe can be used to identify and isolate nucleic acid molecules, such as from a genomic or cDNA library, that hybridize under conditions of low, moderate, or high stringency with the probe.  
     [0113] Alternatively, the GDP-D-mannose:GDP-L-galactose epimerase can be purified from an organism such as Prototheca, the N-terminal amino acid sequence can be determined (including the sequence of internal peptide fragments), and this information can be used to design degenerate primers for amplifying a gene fragment from the organism cDNA. This fragment would then be used to probe the cDNA library, and subsequently fragments that hybridize to the probe would be cloned in that organism or another suitable production organism. There is ample precedent for plant enzymes being expressed in an active form in bacteria, such as  E. coli.  Alternatively, yeast are also a suitable candidate for developing a heterologous system for L-ascorbic acid production.  
     [0114] As discussed above in general for increasing the action of an enzyme in the L-ascorbic acid pathway according to the present invention, in one embodiment of the present invention, the action of an epimerase that catalyzes the conversion of GDP-D-mannose to GDP-L-galactose is increased by amplification of the expression (i.e., overexpression) of such an epimerase. Overexpression of an epimerase can be accomplished, for example, by introduction of a recombinant nucleic acid molecule encoding the epimerase. It is preferred that the gene encoding an epimerase according to the present invention be cloned under control of an artificial promoter. The promoter can be any suitable promoter that will provide a level of epimerase expression required to maintain a sufficient level of L-ascorbic acid in the production organism. Preferred promoters are constitutive (rather than inducible) promoters, since the need for addition of expensive inducers is therefore obviated. The gene dosage (copy number) of a recombinant nucleic acid molecule according to the present invention can be varied according to the requirements for maximum product formation. In one embodiment, the recombinant nucleic acid molecule encoding an epimerase according to the present invention is integrated into the chromosome of the microorganism.  
     [0115] It is another embodiment of the present invention to provide a microorganism having one or more epimerases according to the present invention with improved affinity for its substrate. An epimerase with improved affinity for its substrate can be produced by any suitable method of genetic modification or protein engineering. For example, computer-based protein engineering can be used to design an epimerase protein with greater stability and better affinity for its substrate. See for example, Maulik et al., 1997,  Molecular Biotechnology: Therapeutic Applications and Strategies,  Wiley-Liss, Inc., which is incorporated herein by reference in its entirety.  
     [0116] As noted above, in the method for production of L-ascorbic acid of the present invention, a microorganism having a genetically modified L-ascorbic acid production pathway is cultured in a fermentation medium for production of L-ascorbic acid. An appropriate, or effective, fermentation medium refers to any medium in which a genetically modified microorganism of the present invention, when cultured, is capable of producing L-ascorbic acid. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients. One advantage of genetically modifying a microorganism as described herein is that although such genetic modifications can significantly alter the production of L-ascorbic acid, they can be designed such that they do not create any nutritional requirements for the production organism. Thus, a minimal-salts medium containing glucose as the sole carbon source can be used as the fermentation medium. The use of a minimal-salts-glucose medium for the L-ascorbic acid fermentation will also facilitate recovery and purification of the L-ascorbic acid product.  
     [0117] In one mode of operation of the present invention, the carbon source concentration, such as the glucose concentration, of the fermentation medium is monitored during fermentation. Glucose concentration of the fermentation medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the fermentation medium. As stated previously, the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L, and can be determined readily by trial. Accordingly, when glucose is used as a carbon source the glucose concentration in the fermentation medium is maintained in the range of from about 1 g/L to about 100 g/L, more preferably in the range of from about 2 g/L to about 50 g/L, and yet more preferably in the range of from about 5 g/L to about 20 g/L. Although the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is preferred to maintain the carbon source concentration of the fermentation medium by addition of aliquots of the original fermentation medium. The use of aliquots of the original fermentation medium are desirable because the concentrations of other nutrients in the medium (e.g. the nitrogen and phosphate sources) can be maintained simultaneously. Likewise, the trace metals concentrations can be maintained in the fermentation medium by addition of aliquots of the trace metals solution.  
     [0118] In an embodiment of the fermentation process of the present invention, a fermentation medium is prepared as described above. This fermentation medium is inoculated with an actively growing culture of genetically modified microorganisms of the present invention in an amount sufficient to produce, after a reasonable growth period, a high cell density. Typical inoculation cell densities are within the range of from about 0.1 g/L to about 15 g/L, preferably from about 0.5 g/L to about 10 g/L and more preferably from about 1 g/L to about 5 g/L, based on the dry weight of the cells. The cells are then grown to a cell density in the range of from about 10 g/L to about 100 g/L preferably from about 20 g/L to about 80 g/L, and more preferably from about 50 g/L to about 70 g/L. The residence times for the microorganisms to reach the desired cell densities during fermentation are typically less than about 200 hours, preferably less than about 120 hours, and more preferably less than about 96 hours.  
     [0119] The microorganisms useful in the method of the present invention can be cultured in conventional fermentation modes, which include, but are not limited to, batch, fed-batch, and continuous. It is preferred, however, that the fermentation be carried out in fed-batch mode. In such a case, during fermentation some of the components of the medium are depleted. It is possible to initiate fermentation with relatively high concentrations of such components so that growth is supported for a period of time before additions are required. The preferred ranges of these components are maintained throughout the fermentation by making additions as levels are depleted by fermentation. Levels of components in the fermentation medium can be monitored by, for example, sampling the fermentation medium periodically and assaying for concentrations. Alternatively, once a standard fermentation procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the fermentation. As will be recognized by those in the art, the rate of consumption of nutrient increases during fermentation as the cell density of the medium increases. Moreover, to avoid introduction of foreign microorganisms into the fermentation medium, addition is performed using aseptic addition methods, as are known in the art. In addition, a small amount of anti-foaming agent may be added during the fermentation.  
     [0120] The present inventors have determined that high levels of magnesium in the fermentation medium inhibits the production of L-ascorbic acid due to repression of enzymes early in the production pathway, although enzymes late in the pathway (i.e., from L-galactose to L-ascorbic acid) are not negatively affected (See Examples). Therefore, in a preferred embodiment of the method of the present invention, the step of culturing is carried out in a fermentation medium that is magnesium (Mg 2+ ) limited. Even more preferably, the fermentation is magnesium limited during the cell growth phase. Preferably, the fermentation medium comprises less than about 0.5 g/L of Mg 2+  during the cell growth phase of fermentation, and even more preferably, less than about 0.2 g/L of Mg 2+ , and even more preferably, less than about 0.1 g/L of Mg 2+ .  
     [0121] The temperature of the fermentation medium can be any temperature suitable for growth and ascorbic acid production, and may be modified according to the growth requirements of the production microorganism used. For example, prior to inoculation of the fermentation medium with an inoculum, the fermentation medium can be brought to and maintained at a temperature in the range of from about 20° C. to about 45° C., preferably to a temperature in the range of from about 25° C. to about 40° C., and more preferably in the range of from about 30° C. to about 38° C.  
     [0122] It is a further embodiment of the present invention to supplement and/or control other components and parameters of the fermentation medium, as necessary to maintain and/or enhance the production of L-ascorbic acid by a production organism. For example, in one embodiment, the pH of the fermentation medium is monitored for fluctuations in pH. In the fermentation method of the present invention, the pH is preferably maintained at a pH of from about pH 6.0 to about pH 8.0, and more preferably, at about pH 7.0. In the method of the present invention, if the starting pH of the fermentation medium is pH 7.0, the pH of the fermentation medium is monitored for significant variations from pH 7.0, and is adjusted accordingly, for example, by the addition of sodium hydroxide. In a preferred embodiment of the present invention, genetically modified microorganisms useful for production of L-ascorbic acid include acid-tolerant microorganisms. Such microorganisms include, for example, microalgae of the genera Prototheca and Chlorella (See U.S. Pat. No. 5,792,631, ibid. and U.S. Pat. No. 5,900,370, ibid.).  
     [0123] The production of ascorbic acid by culturing acid-tolerant microorganisms provides significant advantages over known ascorbic acid production methods. One such advantage is that such organisms are acidophilic, allowing fermentation to be carried out under low pH conditions, with the fermentation medium pH typically less than about 6. Below this pH, extracellular ascorbic acid produced by the microorganism during fermentation is relatively stable because the rate of oxidation of ascorbic acid in the fermentation medium by oxygen is reduced. Accordingly, high productivity levels can be obtained for producing L-ascorbic acid with acid-tolerant microorganisms according to the methods of the present invention. In addition, control of the dissolved oxygen content to very low levels to avoid oxidation of ascorbic acid is unnecessary. Moreover, this advantage allows for the use of continuous recovery methods because extracellular medium can be treated to recover the ascorbic acid product.  
     [0124] Thus, the present method can be conducted at low pH when acid-tolerant microorganisms are used as production organisms. The benefit of this process is that at low pH, extracellular ascorbic acid produced by the organism is degraded at a reduced rate than if the fermentation medium was at higher pH. For example, prior to inoculation of the fermentation medium with an inoculum, the pH of the fermentation medium can be adjusted, and further monitored during fermentation. Typically, the pH of the fermentation medium is brought to and maintained below about 6, preferably below 5.5, and more preferably below about 5. The pH of the fermentation medium can be controlled by the addition of ammonia to the fermentation medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the fermentation medium.  
     [0125] The fermentation medium can also be maintained to have a dissolved oxygen content during the course of fermentation to maintain cell growth and to maintain cell metabolism for L-ascorbic acid formation. The oxygen concentration of the fermentation medium can be monitored using known methods, such as through the use of an oxygen probe electrode. Oxygen can be added to the fermentation medium using methods known in the art, for example, through agitation and aeration of the medium by stirring or shaking. Preferably, the oxygen concentration in the fermentation medium is in the range of from about 20% to about 100% of the saturation value of oxygen in the medium based upon the solubility of oxygen in the fermentation medium at atmospheric pressure and at a temperature in the range of from about 30° C. to about 40° C. Periodic drops in the oxygen concentration below this range may occur during fermentation, however, without adversely affecting the fermentation.  
     [0126] The genetically modified microorganisms of the present invention are engineered to produce significant quantities of extracellular L-ascorbic acid. Extracellular L-ascorbic acid can be recovered from the fermentation medium using conventional separation and purification techniques. For example, the fermentation medium can be filtered or centrifuged to remove microorganisms, cell debris and other particulate matter, and L-ascorbic acid can be recovered from the cell-free supernate by conventional methods, such as, for example, ion exchange, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization.  
     [0127] One such example of L-ascorbic acid recovery is provided in U.S. Pat. No. 4,595,659 by Cayle, incorporated herein in its entirety be reference, which discloses the isolation of L-ascorbic acid from an aqueous fermentation medium by ion exchange resin adsorption and elution, which is followed by decoloration, evaporation and crystallization. Further, isolation of the structurally similar isoascorbic acid from fermentation medium by a continuous multi-bed extraction system of anion-exchange resins is described by K. Shimizu,  Agr. Biol. Chem.  31:346-353 (1967), which is incorporated herein in its entirety by reference.  
     [0128] Intracellular L-ascorbic acid produced in accordance with the present invention can also be recovered and used in a variety of applications. For example, cells from the microorganisms can be lysed and the ascorbic acid which is released can be recovered by a variety of known techniques. Alternatively, intracellular ascorbic acid can be recovered by washing the cells to extract the ascorbic acid, such as through diafiltration.  
     [0129] Development of a microorganism with enhanced ability to produce L-ascorbic acid by genetic modification can be accomplished using both classical strain development and molecular genetic techniques, and particularly, recombinant technology (genetic engineering). In general, the strategy for creating a microorganism with enhanced L-ascorbic acid production is to (1) inactivate or delete at least one, and preferably more than one of the competing or inhibitory pathways in which production of L-ascorbic acid is negatively affected (e.g., inhibited), and more significantly to (2) amplify the L-ascorbic acid production pathway by increasing the action of a gene(s) encoding an enzyme(s) involved in the pathway.  
     [0130] In one embodiment, the strategy for creating a microorganism with enhanced L-ascorbic acid production is to amplify the L-ascorbic acid production pathway by increasing the action of GDP-D-mannose:GDP-L-galactose epimerase, as discussed above. Such strategy includes genetically modifying the endogenous GDP-D-mannose:GDP-L-galactose epimerase such that L-ascorbic acid production is increased, and/or expressing/overexpressing a recombinant epimerase that catalyzes the conversion of GDP-D-mannose to GDP-L-galactose, which includes expression of recombinant GDP-D-mannose:GDP-L-galactose epimerase and/or homologues thereof, and of other recombinant epimerases such as GDP-4-keto-6-deoxy-D-mannose epimerase reductase and epimerases that share structural homology with such epimerase as discussed in detail above.  
     [0131] It is to be understood that a production organism can be genetically modified by recombinant technology in which a nucleic acid molecule encoding a protein involved in the L-ascorbic acid production pathway disclosed herein is transformed into a suitable host which is a different member of the plant kingdom from which the nucleic acid molecule was derived. For example, it is an embodiment of the present invention that a recombinant nucleic acid molecule encoding a GDP-D-mannose:GDP-L-galactose epimerase from a higher plant can be transformed into a microalgal host in order to overexpress the epimerase and enhance production of L-ascorbic acid in the microalgal production organism.  
     [0132] As previously discussed herein, in one embodiment, a genetically modified microorganism can be a microorganism in which nucleic acid molecules have been deleted, inserted or modified, such as by insertion, deletion, substitution, and/or inversion of nucleotides, in such a manner that such modifications provide the desired effect within the microorganism. A genetically modified microorganism is preferably modified by recombinant technology, such as by introduction of an isolated nucleic acid molecule into a microorganism. For example, a genetically modified microorganism can be transfected with a recombinant nucleic acid molecule encoding a protein of interest, such as a protein for which increased expression is desired. The transfected nucleic acid molecule can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transfected (i.e., recombinant) host cell in such a manner that its ability to be expressed is retained. Preferably, once a host cell of the present invention is transfected with a nucleic acid molecule, the nucleic acid molecule is integrated into the host cell genome. A significant advantage of integration is that the nucleic acid molecule is stably maintained in the cell. In a preferred embodiment, the integrated nucleic acid molecule is operatively linked to a transcription control sequence (described below) which can be induced to control expression of the nucleic acid molecule.  
     [0133] A nucleic acid molecule can be integrated into the genome of the host cell either by random or targeted integration. Such methods of integration are known in the art. For example, an  E. coli  strain ATCC 47002 contains mutations that confer upon it an inability to maintain plasmids which contain a ColE1 origin of replication. When such plasmids are transferred to this strain, selection for genetic markers contained on the plasmid results in integration of the plasmid into the chromosome. This strain can be transformed, for example, with plasmids containing the gene of interest and a selectable marker flanked by the 5′- and 3′-termini of the  E. coli  lacZ gene. The lacZ sequences target the incoming DNA to the lacZ gene contained in the chromosome. Integration at the lacZ locus replaces the intact lacZ gene, which encodes the enzyme β-galactosidase, with a partial lacZ gene interrupted by the gene of interest. Successful integrants can be selected for β-galactosidase negativity.  
     [0134] A genetically modified microorganism can also be produced by introducing nucleic acid molecules into a recipient cell genome by a method such as by using a transducing bacteriophage. The use of recombinant technology and transducing bacteriophage technology to produce several different genetically modified microorganism of the present invention is known in the art.  
     [0135] According to the present invention, a gene, for example the GDP-D-mannose:GDP-L-galactose epimerase gene, includes all nucleic acid sequences related to a natural epimerase gene such as regulatory regions that control production of the epimerase protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. In another embodiment, a gene, for example the GDP-D-mannose:GDP-L-galactose epimerase gene, can be an allelic variant that includes a similar but not identical sequence to the nucleic acid sequence encoding a given GDP-D-mannose:GDP-L-galactose epimerase gene. An allelic variant of a GDP-D-mannose:GDP-L-galactose epimerase gene which has a given nucleic acid sequence is a gene that occurs at essentially the same locus (or loci) in the genome as the gene having the given nucleic acid sequence, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5′ or 3′ untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given microorganism or plant and/or among a group of two or more microorganisms or plants.  
     [0136] In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation). As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof.  
     [0137] An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene. An isolated nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications provide the desired effect within the microorganism. A structural homologue of a nucleic acid sequence has been described in detail above. Preferably, a homologue of a nucleic acid sequence encodes a protein which has an amino acid sequence that is sufficiently similar to the natural protein amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) a nucleic acid molecule encoding the natural protein (i.e., to the complement of the nucleic acid strand encoding the natural protein amino acid sequence). A nucleic acid molecule homologue encodes a protein homologue. As used herein, a homologue protein includes proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol) in such a manner that such modifications provide the desired effect on the protein and/or within the microorganism (e.g., increased or decreased action of the protein).  
     [0138] A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene.  
     [0139] Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a gene involved in an L-ascorbic acid production pathway.  
     [0140] Knowing the nucleic acid sequences of certain nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules and/or (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions). Such nucleic acid molecules can be obtained in a variety of ways including traditional cloning techniques using oligonucleotide probes to screen appropriate libraries or DNA and PCR amplification of appropriate libraries or DNA using oligonucleotide primers. Preferred libraries to screen or from which to amplify nucleic acid molecule include bacterial and yeast genomic DNA libraries, and in particular, microalgal genomic DNA libraries. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.  
     [0141] The present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host microorganism of the present invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA and typically is a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecules. One type of recombinant vector, referred to herein as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules. Preferred recombinant vectors are capable of replicating in a transformed bacterial cells, yeast cells, and in particular, in microalgal cells.  
     [0142] Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection and biolistics.  
     [0143] A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules operatively linked to an expression vector containing one or more transcription control sequences. The phrase, operatively linked, refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. In the present invention, expression vectors are typically plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in a yeast host cell, a bacterial host cell, and preferably a microalgal host cell.  
     [0144] Nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in yeast or bacterial cells or preferably, in microalgal cells. A variety of such transcription control sequences are known to those skilled in the art.  
     [0145] It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into the host cell chromosome, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals, modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.  
     [0146] The following experimental results are provided for the purposes of illustration and are not intended to limit the scope of the invention.  
     EXAMPLES  
     Example 1  
     [0147] The present example describes the elucidation of the pathway from glucose to L-ascorbic acid through GDP-D-mannose in plants.  
     [0148] Since the present inventors have previously shown that Prototheca makes L-ascorbic acid (AA) from glucose, it was worthwhile to examine cultures for some of the early conversion products of glucose. In the past, the present inventors had concentrated on pathways from glucose to organic acids, based on the published pathway of L-ascorbic acid synthesis in animals and proposed pathways in plants. The present inventors demonstrate herein that the pathway from glucose to L-ascorbic acid involves not organic acids, but rather sugar phosphates and nucleotide diphosphate sugars (NDP-sugars).  
     [0149] Prior to the present invention, it was known that all cells synthesize polysaccharides by first forming NDP-sugars. The sugar moiety is then incorporated into polymer, while the cleaved NDP is recycled. A variety of polysaccharides are known, and are usually named based on the relative proportions of the sugar residues in the polymers. For example, a “galactomannan” contains mostly galactose, and to a lesser degree, mannose residues. The “biopolymer” from Prototheca strains isolated by the present inventors was analyzed and found to be 80% D-galactose, 18% rhamnose (D- or L-configuration not determined), and 2% L-arabinose. The present inventors provide evidence herein of how the respective NDP-sugars that make up the Prototheca biopolymer are formed, and what correlations exist between L-ascorbic acid synthesis and the formation of the NDP-sugar forms of the sugar residues found in the biopolymer.  
     [0150] The common NDP-sugar UDP-glucose is shown in FIG. 2B. This is formed in plants from glucose-1-P by the action of UDP-D-glucose pyrophosphorylase. UDP-glucose can be epimerized in plants to form UDP-D-galactose, using UDP-D-glucose-4-epimerase. UDP-D-galactose can also be formed by phosphorylation of D-galactose by galactokinase to form D-galactose-1-P, which can be converted to UDP-D-galactose by UDP-D-galactose pyrophosphorylase. These known routes were believed to account for the D-galactose in the Prototheca biopolymer. The UDP-L-arabinose can be formed by known reactions beginning with the oxidation of UDP-D-glucose to UDP-D-glucuronic acid (by UDP-D-glucose dehydrogenase), decarboxylation to UDP-D-xylose, and epimerization to UDP-L-arabinose. This accounts for the arabinose residues in the biopolymer. UDP-L-rhamnose is known to be formed from UDP-D-glucose, thus all three of the sugar moieties in the Prototheca biopolymer can be accounted for by a pathway through glucose-1-P and UDP-glucose. Alternatively, if the rhamnose in the biopolymer is D-rhamnose, it is not formed via UDP-D-glucose, but by oxidation of GDP-D-mannose (See FIG. 1).  
     [0151] GDP-D-rhamnose is formed by converting glucose, in turn, to D-glucose-6-P, D-fructose-6-P, D-mannose-6-P, D-mannose-1-P, GDP-D-mannose, and GDP-D-rhamnose. It was of interest to the present inventors that this route passes through GDP-D-mannose. Exogenous mannose is known to be converted to D-mannose-6-P in plants, and can enter the path above. D-mannose is converted to L-ascorbic acid by Prototheca cells cultured by the present inventors as well or better than glucose (see Example 4). The mechanism of conversion, in  Chlorella pyrenoidosa,  of GDP-D-mannose to GDP-L-galactose by GDP-D-mannose:GDP-L-galactose epimerase, has been known for years (See, Barber, 1971,  Arch. Biochem. Biophys.  147:619-623, incorporated herein by reference in its entirety). The present inventors have discovered herein that L-galactose and L-galactono-γ-lactone are rapidly converted to L-ascorbic acid by strains of Prototheca and  Chlorella pyrenoidosa.  Prior to the present invention, it was known that L-galactono-γ-lactone is converted to L-ascorbic acid in several plant systems, but the synthesis steps prior to this step were unknown. Based on the published literature and the present experimental evidence, the present inventors have determined that the L-ascorbic acid biosynthetic pathway in plants passes through GDP-D-mannose and involves sugar phosphates and NDP-sugars. The proposed pathway is shown in FIG. 1. Salient points relevant to the design and production of genetically modified microorganisms useful in the present method include:  
     [0152] 1. The enzymes leading from D-glucose to D-fructose-6-P are well known enzymes in the first, uncommitted steps of glycolysis.  
     [0153] 2. The enzymes involved in the conversion of D-fructose-6-P to GDP-D-mannose have been well characterized in plants, yeast, and bacteria, particularly  Azotobacter vinelandii  and  Pseudomonas aeruginosa,  which convert GDP-D-mannose to GDP-D-mannuronic acid, which is the precursor for alginate (See for example, Sa-Correia et al., 1987,  J. Bacteriol.  169:3224-3231; Koplin et al., 1992,  J. Bacteriol.  174:191-199; Oesterhelt et al., 1996,  Plant Science  121:19-27; Feingold et al., 1980,  The Biochemistry of Plants:  Vol 3: Carbohydrates, structure and function, P. K. Stampf &amp; E. E. Conn, eds., Academic Press, New York, pp. 101-170; Smith et al., 1992,  Mol. Cell Biol.  12:2924-2930; Boles et al., 1994,  Eur. J. Med.  220:83-96; Hashimoto et al., 1997,  J. Biol. Chem.  272:16308-16314, all of which are incorporated herein by reference in their entirety).  
     [0154] 3. Barber (1971, supra, and 1975) identified in  Chlorella pyrenoidosa  the enzyme activities for the conversion of GDP-D-mannose to GDP-L-galactose and L-galactose-1-P.  
     [0155] 4. The present inventors have shown herein the rapid conversion of L-galactose and L-galactono-γ-lactone to L-ascorbic acid by Prototheca cells.  
     [0156] 5. L-galactono-y-lactone and L-galactonic acid can be interconverted in solution by changing the pH of the solution; addition of base shifts the equilibrium to L-galactonic acid, while addition of acid shifts the equilibrium to the lactone. Cells may have an enzymatic means for this conversion in addition to this non-enzymatic route.  
     [0157] 6. In plants, GDP-L-fucose is also formed from GDP-D-mannose, presumably for incorporation into polysaccharide. Roberts (1971) fed labeled D-mannose to corn root tips and found the label in polysaccharide, specifically in the residues of D-mannose, L-galactose, and L-fucose. No label was detected in D-glucose, D-galactose, L-arabinose, or D-xylose. Prototheca and  C. pyrenoidosa  cells have the ability to convert L-fucose (6-deoxy-L-galactose) to a dipyridyl-positive product that was shown by HPLC not to be L-ascorbic acid. The present inventors believe that it is was the 6-deoxy analog of L-ascorbic acid.  
     Example 2  
     [0158] This example shows that in Prototheca, like other plants (Loewus, F. A. 1988. In: J. Priess (ed.), The Biochemistry of Plants, 14:85-107. New York, Academic Press) and the green microalga  Chlorella pyrenoidosa  (Renstrom, et al., 1983. Plant Sci. Lett. 28:299-305), ascorbic acid (AA) production from glucose proceeds by a biosynthetic pathway that allows retention of the configuration of the carbon skeleton of glucose.  
     [0159] Cultures of the strain UV77-247 were grown to moderate cell density in shake flasks with 1- 13 C-labeled glucose as 10% of the total glucose (40 g/L) . Incubation was as per the standard Mg-limited screen (see Example 3). The culture supernates were clarified, deionized to remove salts, lyophilized, and subjected to nuclear magnetic resonance (nmr) analysis to determine where in the AA molecule the  13 C was located. In each case, approximately 85% of the label was found at the C-1 position of AA, with most of the remaining label at the C-6 position. This strongly indicated that AA is synthesized from glucose by a pathway that retains the carbon chain configuration, i. e., C-1 of glucose becomes C-1 of AA. This has typically been observed in plants (Loewus, F. A. 1988. Ascorbic acid and its metabolic products. In: The Biochemistry of Plants, ed. J. Priess, 14:85-107. New York, Academic Press). Animals (Mapson, L. W. and F. A. Isherwood 1956. Biochem. J. 64:151-157; Loewus, F. A. 1960. J. Biol. Chem. 235(4) :937-939) and protists such as Euglena (Shigeoka, S., et al., 1979. J. Nutr. Sci. Vitaminol. 25:299-307), on the other hand, synthesize AA by a pathway that involves the inversion of configuration, i. e., C-1 of glucose becomes C-6 of AA. Demonstration of the inversion/non-inversion nature of the pathway was an important step in determining the pathway of AA biosynthesis since the two types of pathways require different types of enzymatic reactions. The label found at C-6 of AA is thought to be due to metabolism of glucose and subsequent gluconeogenesis. The metabolism of glucose in glycolysis proceeds through triose-phosphate intermediates. After this, the C-1 and C-6 carbons of glucose become biochemically equivalent. Hexose phosphates can be regenerated from the triose phosphates by gluconeogenesis, which is essentially a reversal of the degradative pathway. Consequently, metabolism of C-1-labeled glucose to triose phosphates with subsequent gluconeogenesis would result in the formation of hexose phosphate molecules labeled at either or both C-1 and C-6. If those hexose phosphates were precursors to AA, one would expect the AA to be similarly labeled. Consistent with this type of “isotopic mixing” is the observation that sucrose obtained from 1- 13 C-labeled glucose was labeled at positions 1, 6, 1′ and 6′.  
     [0160] Glucose can also be metabolized by the pentose phosphate pathway, the overall balanced equation for which is: 
     3Glucose-6-phosphate→2Fructose-6-phosphate+Glyceraldehyde-3-phosphate+3 CO 2   
     [0161] Based on the known biochemistry, it would then be expected that the label at each of the carbons in glucose (Table 1 left column) would appear at the positions for the other molecules shown, and that these patterns would be reflected in the AA formed from C-2- and C-3-labeled glucose.  
               TABLE 1                          Predicted Carbon Labeling of Metabolites of Glucose in the Pentose       Phosphate Pathway                     Labeled Glucose   Position of Labeled Carbon in:                                 Carbon   CO 2     F6P(1)   F6P(2)   G3P               1   +   −   −   −       2   −   1, 3   1   −       3   −   2   2, 3   −       4   −   4   4   1       5   −   5   5   2       6   −   6   6   3                  
 
     [0162] AA recovered from cultures fed glucose labeled at C-2 or C-3 was also analyzed for its labeling patterns (Table 2).  
               TABLE 2                          Labeling Pattern in AA after Cells were Fed 2- 13 C and 3- 13 C-glucose                         Carbon   Isotopic enhancement after growth on:                             Position in AA   C-2 labeled glucose   C-3 labeled glucose                                 1   1.0   0.4       2   10.0   0.9       3   0.5   9.9       4   0   2.8       5   2.2   0.2       6   0   0                  
 
     [0163] The data above again suggest a pathway from glucose to AA that proceeds by retention of configuration. As in the experiments with C-1 labeled glucose, approximately one-fifth of the label is present in “mirror image” position to the glucose label (C-5 for C-2 labeled glucose and C-4 for C-3 labeled glucose), indicating levels of gluconeogenesis consistent with those previously observed.  
     [0164] The small, but significant amount of enhancement observed in other positions is consistent with flux through the pentose phosphate pathway. As predicted above, carbon flux through this pathway would result in isotopic enhancement at positions 1 and 3 when cells were grown on 2- 13 C glucose and enhancement at position 2 when cells were grown on 3- 13 C glucose. This is indeed observed. That there is twice as much enhancement at C-1 as there is at C-3 after growth on 2- 13 C glucose is also predicted. These data indicate a small but measurable amount of carbon flux through the pentose phosphate pathway.  
     Example 3  
     [0165] This example shows the methods for generating, screening and isolating mutants of Prototheca with altered AA productivities compared to the starting strain ATCC 75669.  
     [0166] ATCC No. 75669, identified as  Prototheca moriformis  RSP1385 (unicellular green microalga), was deposited on Feb. 8, 1994, with the American Type Culture Collection (ATCC), Rockville, Md., 20852, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. Initial screening of Prototheca species and strains was reported in U.S. Pat. No. 5,900,370, ibid. Table 3 lists the formulations of the media for growth and maintenance of the strains. Glucose for fermentors was supplied as glucose monohydrate and calculated on an anhydrous basis. The recipe for the trace metals solution is given in Table 4. The standard growth temperature was 35° C. All organisms were cultured axenically.  
               TABLE 3                          Media for Growth and Maintenance of Prototheca Strains       All quantities are in g/L unless otherwise specified                                 Agar                                         Liquid           Ferro-   Stan-           Stand-   Mg-       zine   dard       Ingredient   ard   limiting   Slants   Plates   Plates               Potassium phosphate   1.3   1.3   2.0   0.27   2.0       monobasic       Potassium phosphate   3.8   3.8   2.0   1.4   2.0       dibasic       Trisodium citrate   7.7   7.7   2.6   1.3   2.6       dihydrate       Magnesium sulfate   0.40   0.02   0.4   0.01   0.4       heptahydrate       Ammonium sulfate   3.7   3.7   1.0   1.0   1.0       Trace Metals Solution   2 mL   2 mL   2 mL   2 mL   2 mL       Ferrous sulfate   1.5 mg   4.5 mg   1.5 mg   —   1.5 mg       heptahydrate       Calcium chloride   —   0.25   —   —   —       dihydrate       Manganous sulfate   —   0.08   —   —   —       monohydrate       Yeast extract   —   —   2.5   —   —       Agar   —   —   15   15   15                       (Noble)       pH before autoclaving   7.2   7.2   7.2   7.2   7.2                 Autoclave, then add                                     Copper sulfate, penta-   —   —   —   2 mL   —       hydrate, 100 g/L       40 g/L Ferrozine   —   —   —   8.8 mL   —       in 5 mM phosphate       (pH 7.5 final)       Ferric ammonium sulfate   —   —   —   3.8 mL   —       dodecahydrate, 40 g/L       50% glucose with   40 mL   60 mL   10 mL   10 mL   10 mL       25 mg/L thiamine HCl                  
 
     [0167]               TABLE 4                          Trace Metals Solution                                         mL Indiv. Stock           Molecular   Conc. of Individ.   per liter       Compound   Weight   Solutions, g/L   of Working Stock                                         Distilled Water   —   —   823           Hydrochloric Acid   —   Conc.   20       Cobalt Chloride   237.9   24.0   6.5       hexahydrate       Boric acid    61.8   38.1   24       Zinc sulfate   287.5   35.3   50       heptahydrate       Manganous sulfate   169.0   24.6   50       monohydrate       Sodium molybdate   242.0   23.8   2.0       dihydrate       Calcium chloride   147.0   —   11.4 g       dihydrate       Vanadyl sulfate   199.0   10.0   8.0       dihydrate       Nickel nitrate   290.8    5.0   8.0       hexahydrate       Sodium selenite   173.0    5.0   8.0                    
     [0168] Mutant isolates were generated by treatment with one or more of the following agents: nitrous acid (NA); ethyl methane sulfonate (EMS); or ultraviolet light (UV). Typically, glucose-depleted cells grown in standard liquid medium were washed and resuspended in 25 mM phosphate buffer, pH 7.2, diluted to approximately 10 7  colony-forming units per mL (cfu/mL), exposed to the mutagen to achieve about 99% kill, incubated 4-8 hours in the dark, and spread onto standard agar medium, or agar media containing differential agents.  
     [0169] Some mutant colonies on standard agar medium were picked randomly and subcultured to master plates. Other isolation plates were inverted over chloroform to lyse cells on the surface of the colonies and allow them to release AA. Released AA was detected by spraying the treated plates with a solution of 2,6-dichrorophenol-indophenol (1.25 g/L in 70% EtOH). The ability of AA to reduce this blue redox dye to its colorless form is the basis for a standard assay of AA (Omaye, et al., 1979.  Meth. Enzymol.  62:3-11.). Colonies derived from mutagenized cells were saved to master plates for further evaluation if their clear halos were significantly larger than the halos typical of the other mutants in that group. Other mutagenized cells were spread onto plates containing an AA detection system incorporated directly into the agar. This system is based on the ability of AA to reduce ferric iron to ferrous iron. The compound ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine) was present in the agar to complex with the ferrous iron and give a violet color reaction. The ferrozine agar formulation is shown in Table 3. Colonies giving the darkest color reactions were master-plated. When screening for non-AA-producing strains (blocked mutants), white colonies were chosen against a background of relatively dark colonies.  
     [0170] For primary screening of tube cultures, cells were inoculated from master plates into 4 mL of Mg-limiting medium in 16×125 mm test tubes, and tubes were shaken in a slanted position on a rotary shaker at 300 rpm for four days. After both three and four days of incubation aliquots were removed for AA assay and cell density determination. Those for AA assay were centrifuged at 1500×g for 5 min and the resulting supernates were removed for either calorimetric assay or high pressure liquid chromatography (HPLC). Promising isolates were retested in tube culture. Those passing the tube screen were tested in shake flasks.  
     [0171] For secondary screening of flask cultures, cells were inoculated into 50 mL of standard flask medium in 250 mL baffled shake flasks, and incubated on a rotary shaker at 180 rpm until glucose depletion (24-48 hours). A second series of flasks of Mg-sufficient standard medium was inoculated from the first set to a cell density of 0.15 A 620 , and incubated for 24 hours. A third series of Mg-limiting flask medium was inoculated from the second set by a 1/50 dilution and incubated for 96 hours. Flasks were sampled for AA analysis and cell density measurements during this time as required. Aliquots for supernatant AA analysis were centrifuged at 5000×g for 5 min. Aliquots for total whole broth AA analysis were first extracted for 15 min with an equal volume of 5% trichloroacetic acid (TCA) before centrifugation. Aliquots of the resulting supernates were removed for either colorimetric assay or HPLC analysis.  
     [0172] For colorimetric assay of AA, a modification of the method of Omaye, et al. (1979. Meth. Enzymol. 62:3-11) was used. Twenty-five μL aliquots of culture supernates were added to wells of 96-well microplates, and 125 μL of color reagent was added. The color reagent consisted of four parts 0.5% aqueous 2,2′-dipyridyl and one part 8.3 mM ferric ammonium sulfate in 27% (v/v) o-phosphoric acid, the two components being mixed immediately before use. After one hour, the absorbance at 520 nm was read. AA concentration was calculated by comparison of the absorbances of AA standards.  
     [0173] HPLC analysis was based on that of Running, et al., (1994). Supernates were chromatographed on a Bio-Rad HPX-87H organic acid column (Bio-Rad Laboratories, Richmond, Calif.) with 13 mM nitric acid as solvent, at a flow rate of 0.7 mL/min at room temperature. Detection was at either 254 nm using a Waters 441 detector (Millipore Corp., Milford, Mass.), or at 245 nm using a Waters 481 detector. This system can distinguish between the L- and D-isomers of AA.  
     [0174] For dry weight determinations of cell density, 5 mL whole broth samples were centrifuged at 5000×g for 5 min, washed once with distilled water, and the pellet was washed into a tared aluminum weighing pan. Cells were dried for 8-24 h at 105° C. Cell weight was calculated by difference.  
     [0175] Table 5 shows the abilities of various mutants of Prototheca to synthesize AA.  
               TABLE 5                          AA Synthesizing Ability of Various Prototheca Mutants in Flask Screen                         Specific AA Formation, mg AA per L/Culture A 620 ,           during Mg-limited Incubation                         Strain   2 Days Incubation   4 Days Incubation                                 ATCC 75669   22   35       EMS13-4   79   166       UV213-1   0   0       UV218-1   0   0       UV244-1   0   0       UV244-15   58   68       UV77-247   56   83       UV140-1   67   100       UV164-6   91   131       NA21-14   27   78       UV82-21   0   0       UV127-10   50   95       SP2-3   3   4                  
 
     [0176] The genealogy of these isolates is presented graphically in the “family tree” of FIG. 3. ATCC No. ______, identified as  Prototheca moriformis  EMS13-4 (unicellular green microalga), was deposited on May 25, 1999, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. ATCC No. ______, identified as  Prototheca moriformis  UV127-10 (unicellular green microalga), was deposited on May 25, 1999, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. ATCC No. ______, identified as  Prototheca moriformis  SP2-3 (unicellular green microalga), was deposited on May 25, 1999, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.  
     Example 4  
     [0177] The following example shows that both growing and resting cells of Prototheca can rapidly convert L-galactose and L-galactono-γ-lactone to AA, and that conversion of D-mannose to AA by Prototheca is more rapid than conversion of D-glucose.  
     [0178] Shake flask cultures of the mutant strain UV77-247 were grown to glucose depletion in standard liquid medium (Table 3). Cells were washed twice and resuspended in complete medium with the glucose substituted by one of the compounds listed below. Cell suspensions were incubated for 24 hours at 35° C. with shaking, and the entire suspension was extracted with TCA as above and assayed for AA.  
     [0179] Tables 6-8 show that both growing and resting cells of strain UV77-247 can rapidly convert L-galactose and L-galactono-γ-lactone to AA. In these experiments, D-fructose and D-galactose were converted to AA at the same rate as D-glucose, suggesting that they are metabolized to AA through the same route as D-glucose. None of the organic acids suggested in the literature to be intermediates in the biosynthesis of AA were converted to AA, including sorbosone, which has been proposed as an intermediate by Saito et al. (1990 Plant Physiol. 94:1496-1500).  
               TABLE 6                          Conversion of Compounds by Resting Cells of Strain UV77-247                                 AA Relative to No       Substrate (50 mM)   Total AA, mg/L   Substrate Control                                 L-galactose   965   623       L-galactono-γ-lactone   818   476       D-fructose   590   248       D-glucosone   589   247       D-glucose   584   242       D-galactose   542   200       D-glucose (10 mM)   388   46       D-gluconolactone   382   40       D-gulono-γ-lactone   366   24       D-glucuronate   364   22       L-sorbosone   342   0       None   342   0       2-keto-D-gluconic acid   341   −1       D-isoascorbic acid (10 mM)   330   −12       D-glucuronolactone   329   −13       D-gluconic acid   309   −33       D-galacturonic acid   297   −45       L-idonate   296   −46                  
 
     [0180] Since strain UV77-247 converted L-galactose and L-galactono-γ-lactone to AA much more rapidly than it did glucose, it suggests that these compounds are intermediates in the AA biosynthetic pathway and that they are “downstream” from glucose.  
     [0181] The data in Tables 7 and 8 also show that growing and resting cells of UV77-247 consistently convert D-mannose to AA at a rate greater than that of glucose.  
               TABLE 7                          Conversion of Compounds to AA by Resting Cells of Strain UV77-247                                 Ascorbic Acid, mg/L                                     Compound   25.5 h   30 h   47 h                       L-galactose   667   718   620           L-galactono-γ-lactone   644   681   749           D-glucosone   465   462   354           D-mannose   448   462   399           D-fructose   402   408   367           d-glucose   395   404   351           D-galactose   352   361   337           none   287   288   258                      
 
     [0182]               TABLE 8                          Conversion of Compounds to AA by Growing Cells of Strain UV77-247                                 Ascorbic Acid,                   mg/L   A 620     AA/A 620                           Compound   25.5h   44h                                         L-galactose   249   506   4.5   112       D-mannose   228   488   5.6   87       L-galactono-γ-lactone   214   342   5.0   68       D-glucose   178   398   5.9   67       D-fructose   181   383   5.9   65       D-glucosone   176   362   5.7   64       D-galactose   185   380   5.9   64       none   182   249   4.4   57       D-gluconic acid (K)   178   262   5.0   52       L-idonate (Na)   182   232   4.7   49       2-keto-D-gluconic acid   182   255   5.3   48       2-deoxy-D-glucose   181   227   4.8   47       D-glucuronic acid lactone   165   218   5.0   44       D-glucuronic acid (Na)   173   241   5.6   43       L-gulono-γ-lactone   152   195   5.0   39       L-sorbosone   178   160   4.7   34       D-glucono-δ-lactone   130   190   5.7   33       D-galacturonic acid   130   180   6.0   30                    
     [0183] These cells converted L-galactose, L-galactono-γ-lactone and D-mannose to AA more rapidly than they did glucose, suggesting that mannose exerts its effect in the biosynthetic pathway “downstream” from glucose.  
     Example 5  
     [0184] Using the methods described above, a collection of mutants was assembled. The specific AA formation for representative mutants are shown in Table 5. The genealogy of these isolates is presented graphically in the “family tree” of FIG. 3.  
     [0185] These isolates were tested for their ability to convert compounds which could be converted to AA by strain UV77-247. Testing was done as in Example 4. Results are shown in Table 9.  
               TABLE 9                          Conversion of Compounds to AA by Resting Cells       of Mutant Strains of Prototheca of Varying Abilities to Synthesize AA                         Absolute AA, mg/L                                                     L-   L-gal-       Fruc-       Strain   Buffer   Glucose   galactose   γ-lact.   Mannose   tose                                                 EMS13-4   53   97   191   173   139   ND       UV127-10   45   140   213   140   128   143        SP2-3   19   19   204   146   24   27       NA21-14   61   80   147   158   118   115        UV82-21   15   16   183   175   18   17       UV213-1   16   15   170   135   17   16       UV218-1   16   18   136   176   19   21       UV244-1   16   16   164   162   16   16       UV244-15   26   77   30   21   94   89       UV244-16   28   64   53   53   53   66                          
 
     [0186] These data suggest that the mutational blocks in those strains which convert fructose and mannose to AA poorly are before (“upstream” from) L-galactose and L-galactono-γ-lactone in the pathway.  
     Example 6  
     [0187] The following example shows that magnesium inhibits early steps in the production of AA.  
     [0188] To address the question of whether magnesium actually inhibits AA synthesis, strain NA45-3 (ATCC 209681) was grown in magnesium (Mg)-limited and Mg-sufficient medium. ATCC No. 209681, identified as  Prototheca moriformis  NA45-3 (Source: repeated mutagenesis of ATCC No. 75669; Eucaryotic alga. Division Chlorophyta, Class Chlorophyceae, Order Chlorococcales), was deposited on Mar. 13, 1998, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. Cells from both cultures were harvested and resuspended in the cell-free supernate from the Mg-limited culture, and to half of each cell suspension additional magnesium was added in order to bring the level in the suspension to the Mg-sufficient level. The four conditions under which assays were run were as follows.  
               TABLE 10                          Conditions Used to Test the Effect of Magnesium on AA Production                                     Magnesium concentration, g/L, during:                                     Condition   Growth   Assay                                              1 Mg &gt; 1 Mg   0.02   0.02            1 Mg &gt; 10 Mg   0.02   0.2           10 Mg &gt; 1 Mg   0.2   0.02           10 Mg &gt; 10 Mg   0.2   0.2                      
 
     [0189] Substrates previously shown to lead to the formation of AA, namely D-glucose, D-glucosone, D-fructose. D-galactose, D-mannose, and L-galactono-γ-lactone, were added at 20 g/L to the four cell suspensions. Accumulation of AA after 24 hours was measured and compared to a control in which no substrate was added. The results of this study are shown graphically in FIG. 4.  
     [0190] When cells growing under magnesium-limited conditions were incubated with substrates in low-magnesium broth (1 Mg&gt;1 Mg condition), all showed significant and similar accumulation of AA over the control condition. When the same cells were incubated in high magnesium broth (1 Mg&gt;10 Mg condition), the accumulation of AA was reduced about 40% for all substrates except D-mannose and L-galactono-γ-lactone, suggesting that 1) the rate-limiting step in the conversion of D-glucose, D-glucosone, D-fructose, and D-galactose to AA is inhibited by magnesium or 2) magnesium stimulates an enzyme which results in the conversion of these compounds to some other compound(s), reducing the amount of substrate available for AA synthesis. On the other hand, conversion of D-mannose and L-galactono-γ-lactone appeared to be unaffected by the presence of magnesium in the resuspension buffer, indicating that either 1) magnesium-inhibited enzymes are not involved in the conversion of these substrates to AA or 2) D-mannose and L-galactono-γ-lactone enter the pathway far enough downstream from the point where they can be siphoned off by side reactions involving Mg-requiring enzymes.  
     [0191] When cells were grown under magnesium-sufficient conditions, very little AA accumulation from any of the D-sugars was observed, regardless of the level of magnesium in the resuspension broth. Accumulation of AA from L-galactono-γ-lactone, however, was enhanced over that observed when cells are grown in Mg-limited conditions. This suggests that enzymes early in the pathway are repressed under Mg-sufficient conditions. Thus, the D-substrates all behaved similarly, with the exception of the apparent lack of magnesium inhibition of D-mannose conversion to AA. This would suggest that D-mannose enters the AA biosynthetic pathway at a point other than the other D-sugars.  
     [0192]FIGS. 2A and 2B represent some of the fates of glucose in plants. The first enzymatic step in this scheme which commits carbon to glycolysis is the conversion of fructose-6-P to fructose-1,6-diP by phosphofructokinase (PFK). This reaction is essentially irreversible, and leads to the well known TCA cycle and oxidative phosphorylation, with concomitant ATP and NADH/NADPH generation. PFK has an absolute requirement for magnesium. If magnesium is limiting, this reaction could slow and eventually stop, blocking the flow of carbon through glycolysis and beyond, and would result in cessation of cell division even in the presence of excess glucose. One would expect fructose-6-P to accumulate under these conditions, fueling AA synthesis by the pathway shown in FIGS. 1 and 2.  
     Example 7  
     [0193] The following example shows the correlation in Prototheca between AA production and the activity levels of the enzymes in the AA pathway.  
     Phosphomannose Isomerase (PMI) Assay  
     [0194] PMI activity was first assayed (See FIG. 1). Ten strains representing a range of AA productivities were grown according to the standard protocol to measure AA-synthesizing ability. Cells were harvested 96 hours into magnesium-limited incubation, washed and resuspended in buffer containing 50 mM Tris/10 mM MgCl 2 , pH 7.5. The suspended cells were broken in a French press, spun at 30,000×g for 30 minutes, and desalted through Sephadex G-25 (Pharmacia PD-10 columns). Reactions were carried out in the reverse direction by adding various volumes of extracts to solutions of Tris/Mg buffer containing 0.15 U phosphoglucose isomerase (EC 5.3.1.9), 0.5 U glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and 1.0 mm NADP. Reactions were initiated by addition of 3 mM (final) mannose-6-phosphate. Final reaction volume was 1.0 mL. All components were dissolved in Tris/Mg buffer. Activities were taken as the change in A 340 /min. From these activities was subtracted the activities measured in identical reaction mixtures lacking the M-6-P substrate. Specific activities were calculated by normalizing the activities for protein concentration in the reactions. Protein in the original extracts was determined by the method of Bradford, using a kit from Bio-Rad Laboratories (Hercules, Calif.). All enzymes and nucleotides were purchased from Sigma Chemical Co. (St. Louis, Mo.).  
     Phosphomannomutase (PMM) Assay  
     [0195] Phosphomannomutase was measured in a similar manner in the same strains, but these assay reaction mixtures also contained 0.25 mM glucose-1,6-diphosphate, 0.5 U commercially available PMI, and the reactions were started with the addition of 3.0 mM (final) mannose-1-phosphate rather than mannose-6-phosphate.  
     Phosphofructokinase (PFK) Assay  
     [0196] To shed light on the possibility that the enhancement of AA concentration in cultures which were limited for magnesium was due to a diversion of carbon from normal metabolism by a reduced activity of the first committed step in glycolysis (PFK) the strains were also assayed to confirm the presence of this enzyme activity. Cells were cultured, washed and broken as above. Extracts were centrifuged at 100,000×g for 90 min before desalting. Reactions were carried out in the forward direction by adding various volumes of extracts to solutions of Tris/Mg buffer containing 1.5 mM dithiothreitol, 0.86 U aldolase (EC 4.1.2.13), 1.4 U α-glycerophosphate dehydrogenase (EC 1.1.1.8), 14 U triosephosphate isomerase (EC 5.3.1.1), 0.11 mM NADH, and 1.0 mM ATP. Reactions were initiated by addition of 5 mM (final) fructose-6-phosphate. Final reaction volume was 1.0 mL. All components were dissolved in Tris/Mg buffer. Activities were taken as the change in A 340 /min. From these activities were subtracted the activities measured in identical reaction mixtures lacking the F-6-P substrate. Specific activities were calculated by normalizing the activities for protein concentration in the reaction. Protein in the original extracts was determined as above.  
     GDP-D-mannose Pyrophosphorylase (GMP) Assay  
     [0197] These same mutant strains were assayed for the next enzyme in the proposed pathway, GMP. Strains were grown both according to the standard Mg-limiting protocol (harvested 43-48 hours into magnesium-limited incubation) and in standard Mg-sufficient medium (harvesting all cells before glucose depletion). Washed cell pellets were resuspended in 50 mM phosphate buffer, pH 7.0, containing 20% (v/v) glycerol and 0.1 M sodium chloride (3 mL buffer/g wet cells), and broken in a French press. Crude extracts were spun at 15,000×g for 15 minutes. Reactions were carried out in the forward direction by adding various volumes of extracts to solutions of 50 mM phosphate/4 mM MgCl 2  buffer, pH 7.0, containing 1 mM GTP. Reactions were initiated by addition of 1 mM (final) mannose-1-phosphate. Final reaction volume was 0.1 mL. Reaction mixtures were incubated at 30° C. for 10 min, filtered through a 0.45 μm PVDF syringe filter, and analyzed for GDP-mannose by HPLC. A Supelcosil SAX1 column (4.6×250 mm) was used with a solvent gradient (1 mL/min) of: A—6 mM potassium phosphate, pH 3.6; B—500 mM potassium phosphate, pH 4.5. The gradient was: 0-3 min, 100% A; 3-10 min, 79% A; 10-15 min, 29% A. Column temperature was 30° C. Two assays that showed enzyme activity proportional to the amount of protein were averaged. Control no-substrate and no-extract reactions were also run. Specific activity was calculated by normalizing the activity for protein concentration in the reaction. Protein in the original extracts was determined as above.  
     GDP-D-mannose:GDP-L-galactose Epimerase Assay  
     [0198] Further tests measured the activities of the next enzyme in the proposed pathway, GDP-D-mannose:GDP-L-galactose epimerase. Strains were grown according to the standard protocol, harvested 43-48 hours into magnesium-limited incubation, washed, and resuspended in buffer containing 50 mM MOPS/S mM EDTA, pH 7.2. Washed pellets were broken in a French press, and spun at 20,000×g for 20 min. Protein determinations were made as above and a dilution series of each was made, ranging from 0.4 to 2.2 mg protein/mL. 50 μL aliquots of these dilutions were added to 10 μL aliquots of 6.3 mM GDP-D-mannose in which a portion of this substrate was universally labeled with  14 C in the mannose moiety. This substrate had an activity of 16 μCi/mL before dilution into the reaction mixture. Reactions were stopped after 10 min by transferring 20 μL of the mixture into microfuge tubes containing 20 μL of 250 mM trifluoroacetic acid (TFA) containing 1.0 g/L each D-mannose and L-galactose. These tubes were sealed and boiled for 10 min, cooled, spun for 60 sec in a Beckman Microfuge E, and 5 μL of each hydrolysate was spotted on 20×20 cm plastic-backed EM Science Silica gel 60 thin-layer chromatography plates (#5748/7), with 1 cm lanes created by scoring with a blunt stylus. After drying, plates were twice chromatographed for 2.5 hours in ethyl acetate:isopropanol:water, 65:22.3:12.7 (plates were dried between runs). Spots of free sugars were visualized by spraying dried plates with 0.5% p-anisaldehyde in a 62% ethanolic solution of 0.89 M sulfuric acid and 0.17 mM glacial acetic acid, and heating at 105° C. for about 15 min. Spots of L-galactose and D-mannose were cut from the plates and counted in a scintillation counter (Beckman model 2800). For time-zero control counts, 16.7 μL of each extract dilution was added to 23.3 μL of the labeled substrate above, which had been diluted 1:7 with the TFA/mannose/galactose solution.  
     [0199] Table 11 summarizes the results of the five enzyme assays for the strains tested, along with their specific AA formations.  
               TABLE 11                          Specific Enzyme Activities (mU)* of Selected Mutant Prototheca Strains                             GMP                                                     AA Specific                   Mg-           Strain   Form, mg/g   PMI   PMM   PFK   Mg-limited   sufficient   Epimerase                                                     UVI 64-6   78.4                       0.79       EMS13-4   73.7   10.8   69.6   13.5   2.6   6.8   0.78       UV140-1   69.9                       0.78       NA45-3   61.4                       0.58       UV77-247   44.4                       0.52       UV127-10   40.1   11.1   45.8   24.4   4.3   5.9   0.39       UV244-15   24.5   14.3   41.5       3.1   5.3   0.42       NA21-14   23.6   12.1   60.3   47.4   2.4   7.6   0.27       ATCC 75669   21.9                       0.28       UV244-16   5.0   16.5   85.6   4.3   5.2       SP2-3   2.0   17.7   47.0   64.5   2.0   7.5   0.03       UV218-1   0.4   15.9   72.1       2.7   7.0   0.83       UV213-1   0.1   19.7   47.7   32.6   3.2   6.7   0.60       UV82-21   0.0   14.6   70.6   30.4   4.1   7.5   0.15       UV244-1   0.0   18.2   51.1       5.5   12   0.15                          
 
     [0200] The only enzyme which showed a strong correlation between activity and the ability to synthesize AA was the GDP-D-mannose:GDP-L-galactose epimerase. This correlation is depicted in FIG. 5. All of the strains which produced measurable amounts of AA had measurable amounts of epimerase activity. The converse was not true: four of the strains which synthesize little or no AA had significant epimerase activities. These strains are candidates for having mutations which affect enzymatic steps downstream from the epimerase. Since all of the strains tested can synthesize AA from L-galactose and L-galactono-γ-lactone (see Examples 4 and 5), the genetic lesion(s) in these four mutants must lie between GDP-L-galactose and free L-galactose.  
     Example 8  
     [0201] The next example shows the relationship between GDP-D-mannose:GDP-L-galactose epimerase activity and the degree of magnesium limitation in two strains, the original unmutagenized parent strain ATCC 75669, and one of the best AA producers, EMS13-4 (ATCC ______).  
     [0202] Four flasks of each strain were grown according to the standard protocol. One culture of each was harvested 24 hours into magnesium-limited incubation, and every 24 hours thereafter for a total of four days. One flask of each strain was also harvested 24 hours into magnesium sufficient incubation. All cultures had glucose remaining when harvested. FIG. 6 shows graphically the AA productivity and epimerase activity in EMS13-4 and ATCC 75669 as the cultures became Mg-limited. Epimerase activity in EMS13-4 was significantly greater than that in ATCC 75669 at all time points. There was also a concurrent rapid rise in both AA productivity and epimerase activity in EMS13-4 as the cultures became increasingly Mg-limited. While there was a moderate increase in AA productivity in ATCC 75669 as Mg became more limiting, there was no effect on epimerase activity.  
     Example 9  
     [0203] The following example shows the results of epimerase assays performed with extracts of two  E. coli  strains into which were cloned the  E. coli  gene for GDP-4-keto-6-deoxy-D-mannose epimerase/reductase.  
     [0204] The  E. coli  K12 wca gene cluster is responsible for cholanic acid production; wcaG encodes a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase.  
     [0205] The  E. coli  wcaG sequence (nucleotides 4 through 966 of SEQ ID NO:3) was amplified by PCR from  E. coli  W3110 genomic DNA using primers WG EcoRI 5 (5′ TAGAATTCAGTAAACAACGAGTTTTTATTGCTGG 3′; SEQ ID NO:12) and WG Xhol 3 (5′ AACTCGAGTTACCCCCAAAGCGGTCTTGATTC 3′; SEQ ID NO:13). The 973-bp PCR product was ligated into the vector pPCR-Script SK(+) (Stratagene, LaJolla, Calif.). The 973-bp ExoRII/XhoI fragment was moved from this plasmid into the ExoRII/XhoI sites of pGEX-5X-1 (Amersham Pharmacia Biotech, Piscataway, N.J.), creating plasmid pSW67-1. Plasmid pGEX-5X-1 is a GST gene fusion vector which adds a 26-kDa GST moiety onto the N-terminal end of the protein of interest.  E. coli  BL21(DE3) was transformed with pSW67-1 and pGEX-5X-1, resulting in strains BL21(DE3)/pSW67-1 and BL21(DE3)/pGEX-5X-1.  
     [0206] The  E. coli  wcaG sequence (nucleotides 1 through 966 of SEQ ID NO:3) was also amplified by PCR from  E. coli  W3110 genomic DNA using primers WG EcoRI 5-2 (5′ CTGGAGTCGAATTCATGAGTAAACAACGAG 3′; SEQ ID NO:14) and WG PstI 3 (5′ AACTGCAGTTACCCCCGAAAGCGGTCTTGATTC 3′; SEQ ID NO:15). The 976-bp PCR product was ligated into a pPCR-Script (Stratagene). The 976-bp ExoRII/PstI fragment was moved from this plasmid into the ExoRII/PstI sites of expression vector pKK223-3 (Amersham Pharmacia Biotech), creating plasmid pSW75-2.  E. coli  JM105 was transformed with pKK223-3 and pSW75-2, resulting in strains JM105/pKK223-3 and JM105/pSW75-2.  
     [0207] All six strains were grown in duplicate at 37° C. with shaking in 2X YTA medium until an optical density of 0.8-1.0 at 600 nm was reached (about three hours). 2X YTA contains 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride and 100 mg/L ampicillin. One of each culture was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to 1 mM final concentration. All 12 cultures were incubated for an additional four hours, washed in 0.9% NaCl, and the cells were frozen at −80° C. Prior to pelleting the cells for preparation of extracts, a portion of each culture was used for a plasmid DNA miniprep to confirm the presence of the appropriate plasmids in these strains. A protein preparation of each culture was also run on SDS gels to confirm expression of a protein of the appropriate size where expected. Frozen pellets were thawed, resuspended in 2.5 mL MOPS/EDTA buffer, pH 7.2, broken in a French Press (10,000 psi), spun for 20 min at 20,000×g, assayed for protein as above and diluted to 0.01, 0.1, 1.0 and 3 mg/mL protein.  
     [0208] Induction of the strain BL21(DE3)/pGEX-5X-1 resulted in high-level expression of a 26-kDa protein indicating the synthesis of the native GST protein. Induction of strain BL21(DE3)/pSW67-1 resulted in high-level expression of a 62-kDa protein, indicating the synthesis of the native GST protein (26K) fused to the wcaG gene product (36K). An aliquot of the fusion protein was treated with the protease Factor Xa (New England Biolabs, Beverly, Mass.), which cleaves near the GST/wcaG junction. Induction of the strain JM105/pSW75-2 resulted in high level expression of a 36-kDa protein, indicating the synthesis of the wcaG gene product. No such protein was detected in JM105/pKK223-3 (vector only).  
     [0209] Next, it was of interest to test extracts in the standard epimerase assay described in Example 7 to determine if any of the extracts containing the wcaG product could bring about the conversion of GDP-D-mannose to GDP-L-galactose. The extracts to be assayed are:  
     [0210] BL21(DE3) Group  
     [0211] 1. BL21(DE3) uninduced  
     [0212] 2. BL21(DE3) induced with 1 mM IPTG  
     [0213] 3. BL21(DE3)/pGEX-5X-1 uninduced  
     [0214] 4. BL21(DE3)/pGEX-5X-1 induced with 1 mM IPTG  
     [0215] 5. BL21(DE3)/pSW67-1 uninduced  
     [0216] 6. BL21(DE3)/pSW67-1 induced with 1 mM IPTG; fusion protein intact  
     [0217] 7. BL21(DE3)/pSW67-1 induced with 1 mM IPTG; GST moiety cleaved  
     [0218] JM105 Group  
     [0219] 1. JM105 uninduced  
     [0220] 2. JM105 induced with 1 mM IPTG  
     [0221] 3. JM105/pKK223-3 uninduced  
     [0222] 4. JM105/pKK223-3 induced with 1 mM IPTG  
     [0223] 5. JM105/pSW75-2 uninduced  
     [0224] 6. JM105/pSW75-2 induced with 1 mM IPTG  
     [0225] Extracts 1 and 7 from the BL21(DE3) group and extracts 1 and 6 from the JM105 group were tested for GDP-D-mannose:GDP-L-galactose epimerase-like activity in a pilot experiment. In this initial experiment, no epimerase activity was detected in any of the extracts. At this time, such a result can be attributed to a number of possibilities. First, it is possible that the wcaG gene product is incapable of catalyzing the conversion of GDP-D-mannose to GDP-L-galactose, although this conclusion can not be reached until several other parameters are tested. Second, it is possible that under the assay conditions which are satisfactory to measure activity for the endogenous GDP-D-mannose:GDP-L-galactose epimerase, the wcaG gene product does not have GDP-D-mannose:GDP-L-galactose epimerase-like activity. Therefore, alternate conditions should be tested. Additionally, confirmation experiments should be performed to confirm the accuracy of the pilot conditions. Third, although the BL21(DE3) and the JM105 clones produce proteins of the expected size, the constructs have not been sequenced to confirm the proper coding sequence for the wcaG gene product and thereby rule out PCR or cloning errors which may render the wcaG gene product inactive. Fourth, the protein formed from the cloned sequence is full-length, but inactive, for example, due to incorrect tertiary structure (folding). Fifth, the gene is overexpressed, resulting in accumulation of insoluble and inactive protein products (inclusion bodies). Future experiments will attempt to determine whether the constructs have or can be induced to have the ability to catalyze the conversion of GDP-D-mannose to GDP-L-galactose, and to use the sequences to isolate the endogenous GDP-D-mannose:GDP-L-galactose epimerase.  
     [0226] Table 12 provides the atomic coordinates for Brookhaven Protein Data Bank Accession Code 1bws:  
                           TABLE 12                          HEADER   EPIMERASE/REDUCTASE   27-SEP-98   1BWS                             TITLE       CRYSTAL STRUCTURE OF GDP-4-KETO-6-DEOXY-D-MANNOSE           TITLE   2   EPIMERASE/REDUCTASE FROM  ESCHERICHIA COLI  A KEY ENZYME IN       TITLE   3   THE BIOSYNTHESIS OF GDP-L-FUCOSE       COMPND       MOL ID: 1;       COMPND   2   MOLECULE: GDP-4-KETO-6-DEOXY-D-MANNOSE EPIMERASE/REDUCTASE;       COMPND   3   CHAIN: A;       COMPND   4   ENGINEERED: YES;       COMPND   5   BIOLOGICAL UNIT: HOMODIMER       SOURCE       MOL ID: 1;       SOURCE   2   ORGANISM SCIENTIFIC:  ESCHERICHIA COLI;         SOURCE   3   EXPRESSION SYSTEM:  ESCHERICHIA COLI         KEYWDS       EPIMERASE/REDUCTASE, GDP-L-FUCOSE BIOSYNTHESIS       EXPDTA       X-RAY DIFFRACTION       AUTHOR       DE M. RIZZITONETTIFLORA       REVDAT   1   13-JAN-99 1BWS 0       JRNL       AUTH DE D .RIZZITONETTIVIGEVANISTURLABISSOFLORA       JRNL       TITL GDP-4-KETO-6-DEOXYD-MANNOSE EPIMERASE/REDUCTASE       JRNL       TITL 2 FROM  ESCHERICHIA COLI , A KEY ENZYME IN THE       JRNL       TITL 3 BIOSYNTHESIS OF GDP-L-FUCOSE, DISPLAYS THE       JRNL       TITL 4 STRUCTURAL CHARACTERISTICS OF THE RED PROTEIN       JRNL       TITL 5 HOMOLOGY SUPERFAMILY       JRNL       REF STRUCTURE (LONDON)   1998       JRNL       REFN     9999       REMARK   1       REMARK   2       REMARK   2   RESOLUTION. 2.2 ANGSTROMS       REMARK   3       REMARK   3   REFINEMENT.       REMARK   3   PROGRAM   : TNT       REMARK   3   AUTHORS   : TRONRUD, TEN EYCK, MATTHEWS       REMARK   3       REMARK   3   DATA USED IN REFINEMENT.                             REMARK   3   RESOLUTION RANGE HIGH (ANGSTROMS)   : 2.2       REMARK   3   RESOLUTION RANGE LOW (ANGSTROMS)   : 15.0       REMARK   3   DATA CUTOFF (SIGMA(F))   : 0.0       REMARK   3   COMPLETENESS FOR RANGE (%)   : 99.7       REMARK   3   NUMBER OF REFLECTIONS   : 24481       REMARK   3       REMARK   3   USING DATA ABOVE SIGMA CUTOFF.       REMARK   3   CROSS-VALIDATION METHOD   : NONE       REMARK   3   FREE R VALUE TEST SET SELECTION   : NULL       REMARK   3   R VALUE (WORKING + TEST SET)   : NULL       REMARK   3   R VALUE (WORKING SET)   : NONE       REMARK   3   FREE R VALUE   : NULL       REMARK   3   FREE R VALUE TEST SET SIZE (%)   : NONE       REMARK   3   FREE R VALUE TEST SET COUNT   : NULL       REMARK   3       REMARK   3   USING ALL DATA, NO SIGMA CUTOFF.                             REMARK   3   R VALUE (WORKING + TEST SET, NO CUTOFF)   : NULL       REMARK   3   R VALUE (WORKING SET, NO CUTOFF)   : 0.202       REMARK   3   FREE R VALUE (NO CUTOFF)   : 0.287       REMARK   3   FREE R VALUE TEST SET SIZE (%, NO CUTOFF)   : NULL       REMARK   3   FREE R VALUE TEST SET COUNT (NO CUTOFF)   : NULL       REMARK   3   TOTAL NUMBER OF REFLECTIONS (NO CUTOFF)   : NULL       REMARK   3                         REMARK   3   NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.                             REMARK   3   PROTEIN ATOMS   : 2527       REMARK   3   NUCLEIC ACID ATOMS   : NULL       REMARK   3   OTHER ATOMS   : 109       REMARK   3                             REMARK   3   WILSON B VALUE (FROM FCALC, A**2)   : NULL       REMARK   3                                     REMARK   3   EMS DEVIATIONS FROMIDEAL VALUES.   EMS   WEIGHT   COUNT       REMARK   3   BOND LENGTHS (A)   : 0.016   ; NULL   ; NULL       REMARK   3   BOND ANGLES (DEGREES)   : 1.65   ; NULL   ; NULL       REMARK   3   TORSION ANGLES (DEGREES)   : NULL   ; NULL   ; NULL       REMARK   3   PSEUDOROTATION ANGLES (DEGREES)   : NULL   ; NULL   ; NULL       REMARK   3   TRIGONAL CARBON PLANES (A)   : NULL   ; NULL   ; NULL       REMARK   3   GENERAL PLANES (A)   : NULL   ; NULL   ; NULL       REMARK   3   ISOTROPIC THERMAL FACTORS (A**2)   : NULL   ; NULL   ; NULL       REMARK   3   NON-BONDED CONTACTS (A)   : NULL   ; NULL   ; NULL       REMARK   3       REMARK   3   INCORRECT CHIRAL-CENTERS (COUNT)   : NULL       REMARK   3       REMARK   3   BULK SOLVENT MODELING.                             REMARK   3   METHOD USED   : NULL       REMARK   3   KSOL   : NULL       REMARK   3   ESOL   : NULL       REMARK   3       REMARK   3   RESTRAINT LIBRARIES.                         REMARK   3   STEREOCHEMISTRY : NULL       REMARK   3   ISOTROPIC THERMAL FACTOR RESTRAINTS : NULL       REMARK   3       REMARK   3   OTHER REFINEMENT REMARKS: NULL       REMARK   4       REMARK   4   1BWS COMPLIES WITH FORMAT V. 2.2, 16-DEC-1996       REMARK   5       REMARK   5   WARNING       REMARK   5   1BWS: THIS IS LAYER 1 RELFASE.       REMARK   5       REMARK   5   PLEASE NOTE THAT THIS ENTRY WAS RELEASED AFTER DEPOSITOR       REMARK   5   CHECKING AND APPROVAL BUT WITHOUT PDB STAFF INTERVENTION.       REMARK   5   AN AUXILIARY FILE, AUX1BWS.RPT, IS AVAILABLE FROM THE       REMARK   5   PDB FTP SERVER AND IS ACCESSIBLE THROUGH THE 3DB BROWSER.       REMARK   5   THE FILE CONTAINS THE OUTPUT OF THE PROGRAM WHAT CHECK AND       REMARK   5   OTHER DIAGNOSTICS.       REMARK   5       REMARK   5   NOMENCLATURE IN THIS ENTRY, INCLUDING HET RESIDUE NAMES       REMARK   5   AND HET ATOM NAMES, HAS NOT BEEN STANDARDIZED BY THE PDB       REMARK   5   PROCESSING STAFF.   A LAYER 2 ENTRY WILL BE RELEASED SHORTLY       REMARK   5   AFTER THIS STANDARDIZATION IS COMPLETED AND APPROVED BY THE       REMARK   5   DEPOSITOR. THE LAYER 2 ENTRY WILL BE TREATED AS A       REMARK   5   CORRECTION TO THIS ONE, WITH THE APPROPRIATE REVDAT RECORD.       REMARK   5       REMARK   5   FURTHER INFORMATION INCLUDING VALIDATION CRITERIA USED IN       REMARK   5   CHECKING THIS ENTRY AND A LIST OF MANDATORY DATA FIELDS       REMARK   5   ARE AVAILABLE FROM THE PDB WEB SITE AT       REMARK   5   HTTP://WWW.PDB.BNL.GOV/.       REMARK   200       REMARK   200   EXPERIMENTAL DETAILS                             REMARK   200   EXPERIMENT TYPE   : X-RAY DIFFRACTION       REMARK   200   DATE OF DATA COLLECTION   : AUG-1997       REMARK   200   TEMPERATURE (KELVIN)   : 120       REMARK   200   PH   : 6.5       REMARK   200   NUMBER OF CRYSTALS USED   :1       REMARK   200       REMARK   200   SYNCHROTRON (Y/N)   N       REMARK   200   RADIATION SOURCE   : NONE       REMARK   200   BEAMLINE   : NULL       REMARK   200   X-RAY GENERATOR MODEL   : RIGAKU RU200       REMARK   200   MONOCHROMATIC OR LAUE (M/L)   : M       REMARK   200   WAVELENGTH OR RANGE (A)   : 1.5418       REMARK   200   MONOCHROMATOR   : NULL       REMARK   200   OPTICS   : NULL       REMARK   200       REMARK   200   DETECTOR TYPE   : IMAGE PLATE       REMARK   200   DETECTOR MANUFACTURER   : RAXIS       REMARK   200   INTENSITY-INTEGRATION SOFTWARE   : MOSFLM       REMARK   200   DATA SCALING SOFTWARE   : SCALA       REMARK   200       REMARK   200   NUMBER OF UNIQUE REFLECTIONS   : 24481       REMARK   200   RESOLUTION RANGE HIGH (A)   : 2.2       REMARK   200   RESOLUTION RANGE LOW (A)   : 15.0       REMARK   200   REJECTION CRITERIA (SIGMA(I))   : NONE       REMARK   200       REMARK   200   OVERALL.       REMARK   200   COMPLETENESS FOR RANGE (%)   : 99.7       REMARK   200   DATA REDUNDANCY   : 4.3       REMARK   200   R MERGE (I)   : 0.057       REMARK   200   R SYM (I)   : NONE       REMARK   200   &lt;I/SIGMA(I)&gt; FOR THE DATA SET   : 13.6       REMARK   200       REMARK   200   IN THE HIGHEST RESOLUTION SHELL.                         REMARK   200   HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : NULL       REMARK   200   HIGHEST RESOLUTION SHELL, RANGE LOW (A) : NULL                             REMARK   200   COMPLETENESS FOR SHELL (%)   : NULL       REMARK   200   DATA REDUNDANCY IN SHELL   : NULL       REMARK   200   R MERGE FOR SHELL (I)   : NULL       REMARK   200   R SYM FOR SHELL (I)   : NULL       REMARK   200   &lt;I/SIGMA(I)&gt; FOR SHELL   : NULL       REMARK   200                         REMARK   200   DIFFRACTION PROTOCOL: NULL       REMARK   200   METHOD USED TO DETERMINE THE STRUCTURE: MIR       REMARK   200   SOFTWARE USED: NULL       REMARK   200   STARTING MODEL: NULL       REMARK   200       REMARK   200   REMARK: NULL       REMARK   280       REMARK   280   CRYSTAL       REMARK   280   SOLVENT CONTENT, VS (%): NULL       REMARK   280   MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): NULL       REMARK   280       REMARK   280   CRYSTALLIZATION CONDITIONS: NULL       REMARK   290       REMARK   290   CRYSTALLOGRAPHIC SYNMETRY       REMARK   290   SYMMETRY OPERATORS FOR SPACE GROUP: P 32 2 1       REMARK   290                                 REMARK   290       SYMOP   SYMMETRY       REMARK   290       NNWHMM   OPERATOR       REMARK   290       1555   X,Y,Z       REMARK   290       2555   −Y,X−Y,Z+2/3       REMARK   290       3555   Y−X,−X,Z+1/3       REMARK   290       4555   Y,X,−Z       REMARK   290       5555   X−Y,−Y,1/3−Z       REMARK   290       6555   −X,Y−X,2/3−Z       REMARK   290                             REMARK   290       WHERE NNN —&gt; OPERATOR NUMBER                             REMARK   290       MMM —&gt; 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   −0.500045   −0.865974   0.000000   0.00000       REMARK   290   SMTRY2   2   0.866077   −0.499955   0.000000   0.00000       REMARK   290   SMTRY3   2   0.000000   0.000000   1.000000   50.58553       REMARK   290   SMTRY1   3   −0.499955   0.865974   0.000000   0.00000       REMARK   290   SMTRY2   3   −0.866077   −0.500045   0.000000   0.00000       REMARK   290   SMTRY3   3   0.000000   0.000000   1.000000   25.29276       REMARK   290   SMTRY1   4   −0.500045   0.865922   0.000000   0.00000       REMARK   290   SMTRY2   4   0.866077   0.500045   0.000000   0.00000       REMARK   290   SMTRY3   4   0.000000   0.000000   1.000000   0.00000       REMARK   290   SMTRY1   5   1.000000   0.000104   0.000000   0.00000       REMARK   290   SMTRY2   5   0.000000   1.000000   0.000000   0.00000       REMARK   290   SMTRY3   5   0.000000   0.000000   1.000000   25.29276       REMARK   290   SMTRY1   6   −0.499955   0.866026   0.000000   0.00000       REMARK   290   SMTRY2   6   −0.866077   0.499955   0.000000   0.00000       REMARK   290   SMTRY3   6   0.000000   0.000000   1.000000   50.58553       REMARK   290                         REMARK   290   REMARK: NULL       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; SSSEQ = SEQUENCE NUMBER; I = INSERTION CODE):       REMARK   465       REMARK   465   M RES C SSSEQI                                 REMARK   465   MET A   1           REMARK   465   SER A   2       REMARK   465   ASP A   317       REMARK   465   ARG A   318       REMARK   465   PHE A   319       REMARK   465   ARG A   320       REMARK   465   GLY A   321       REMARK   800                         REMARK   800   SITE       REMARK   800   SITE IDENTIFIER: CAT       REMARK   800   SITE DESCRIPTION:       REMARK   800   CATALYTIC RESIDUE       REMARK   800       REMARK   800   SITE IDENTIFIER: CAT       REMARK   800   SITE DESCRIPTION:       REMARK   800   CATALYTIC RESIDUE       REMARK   800       REMARK   800   SITE IDENTIFIER: CAT       REMARK   800   SITE DESCRIPTION:       REMARK   800   CATALYTIC RESIDUE       REMARK   800                                         DBREF   1BWS A   3   316   SWS   P32055   FCL  ECOLI                                                                                   SEQRES   1   A   321   MET   SER   LYS   GLN   ARG   VAL   PHE   ILE   ALA   GLY   HIS   ARG   GLY       SEQRES   2   A   321   MET   VAL   GLY   SER   ALA   ILE   ARG   ARG   GLN   LEU   GLU   GLN   ARS       SEQRES   3   A   321   GLY   ASP   VAL   GLU   LEU   VAL   LEU   ARS   THR   ARG   ASP   GLU   LEO       SEQRES   4   A   321   ASN   LEU   LEU   ASP   SER   ARG   ALA   VAL   HIS   ASP   PHE   PHE   ALA       SEQRES   5   A   321   SER   GLU   ARS   ILE   ASP   GLN   VAL   TYR   LEU   ALA   ALA   ALA   LYS       SEQRES   6   A   321   VAL   GLY   GLY   ILE   VAL   ALA   ASN   ASN   THR   TYR   PRO   ALA   ASP       SEQRES   7   A   321   PHE   ILE   TYR   GLN   ASN   MET   MET   ILE   GLU   SER   ASN   ILE   ILE       SEQRES   8   A   321   HIS   ALA   ALA   HIS   GLN   ASN   ASP   VAL   ASN   LYS   LEU   LEU   PHE       SEQRES   9   A   321   LEU   SLY   SER   SER   CYS   ILE   TYR   PRO   LYS   LEU   ALA   LYS   GLN       SEQRES   10   A   321   PRO   MET   ALA   GLU   SER   GLU   LEU   LEU   GLN   GLY   THR   LEU   GLU       SEQRES   11   A   321   PRO   TER   ASN   GLU   PRO   TYR   ALA   ILE   ALA   LYS   ILE   ALA   SLY       SEQRES   12   A   321   ILE   LYS   LEU   CYS   GLU   SER   TYR   ASN   ARG   GLN   TYR   GLY   ARG       SEQRES   13   A   321   ASP   TYR   ARS   SER   VAL   MET   PRO   THE   ASN   LEU   TYR   GLY   PRO       SEQRES   14   A   321   HIS   ASP   ASN   PHE   HIS   PRO   SER   ASN   SER   HIS   VAL   ILE   PRO       SEQRES   15   A   321   ALA   LEU   LEU   ARG   ARG   PHE   HIS   GLU   ALA   THR   ALA   GLN   ASN       SEQRES   16   A   321   ALA   PRO   ASP   VAL   VAL   VAL   TRP   GLY   SER   GLY   THR   PRO   MET       SEQRES   17   A   321   ARS   GLU   PHE   LEU   HIS   VAL   ASP   ASP   MET   ALA   ALA   ALA   SER       SEQRES   18   A   321   ILE   HIS   VAL   MET   GLU   LEU   ALA   HIS   GLU   VAL   TRP   LEU   GLU       SEQRES   19   A   321   ASN   THR   GLN   PRO   MET   LEU   SER   HIS   ILE   ASN   VAL   GLY   THR       SEQRES   20   A   321   SLY   VAL   ASP   CYS   THR   ILE   ARG   ASP   VAL   ALA   GLN   THR   ILE       SEQRES   21   A   321   ALA   LYS   VAL   VAL   GLY   TYR   LYS   GLY   ARG   VAL   VAL   PHE   ASP       SEQRES   22   A   321   ALA   SER   LYS   PRO   ASP   GLY   THR   PRO   ARG   LYS   LEU   LEU   ASP       SEQRES   23   A   321   VAL   THR   ARG   LEU   HIS   GLN   LEU   GLY   TRP   TYR   HIS   GLU   ILE       SEQRES   24   A   321   SER   LEU   GLU   ALA   GLY   LEU   ALA   SER   THR   TYR   GLN   TRP   PHE       SEQRES   25   A   321   LEU   GLU   ASN   GLN   ASP   ARG   PHE   ARG   GLY                                     HET   NDP    1       0                         HETNAM   NDP NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE       HETSYN   NDP NADP                             FORMUL   2   NDP   C21 H23 N7 O17 P3 3-       FORMUL   3   HOH   *109(E2 O1)                                                                         HELIX   1   1   MET A   14   GLN A   25   1                           12       HELIX   2   2   SER A   44   GLU A   54   1                           11       HELIX   3   3   ILE A   69   THR A   74   1                           6       HELIX   4   4   PRO A   76   ASN A   97   1                           22       HELIX   5   5   SER A   108   ILE A   110   5                           3       HELIX   6   6   GLU A   121   GLU A   123   5                           3       HELIX   7   7   GLU A   134   TYR A   154   1                           21       HELIX   8   8   VAL A   180   ALA A   193   1                           14       HELIX   9   9   VAL A   214   GLU A   226   1                           13       HELIX   10   10   HIS A   229   GLU   A   234   1                           6       HELIX   11   11   ILE A   253   VAL A   264   1                           12       HELIX   12   12   THR A   288   GLN A   292   1                           5       HELIX   13   13   LEU A   301   GLU A   314   1                           14       SHEET   1   A   6 VAL A   29   VAL A   32   0                               SHEET   2   A   6 GLN A   4   ALA A   9   1   N   GLN A   4   O   GLU A   30       SHEET   3   A   6 GLN A   58   LEU A   61   1   N   GLN A   58   O   PHE A   7       SHEET   4   A   6 LYS A   101   LEU A   105   1   N   LYS A   101   O   VAL A   59       SHEET   5   A   6 ASP A   157   PRO A   163   1   N   ASP A   157   O   LEU A   102       SHEET   6   A   6 ILE A   243   VAL A   245   1   N   ILE A   243   O   MET A   162       SHEET   1   B   2 ASN A   165   TYR A   167   0       SHEET   2   B   2 PHE A   211   HIS A   213   1   N   LEU A   212   O   ASN A   165       SHEET   1   C   2 ASP A   198   TRP A   202   0       SHEET   2   C   2 ARG A   269   ASP A   273   1   N   ARG A   269   O   VAL A   199                                 SITE   1   CAT   1 TYR   136       SITE   2   CAT   1 LYS   140       SITE   3   CAT   1 SER   107                                                             CRYST1   104.200   104.200   75.880   90.00   90.00   120.00   P   32   2   1   6                                 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   0.009597   0.005541   0.000000   0.00000       SCALE2   0.000000   0.011081   0.000000   0.00000       SCALE3   0.000000   0.000000   0.013179   0.00000                                                             HETATM   1   O   HOH       1   55.652   −16.806   22.535   1.00   8.73   O       HETATM   2   O   HOH       3   58.494   −10.639   18.740   1.00   13.17   O       HETATM   3   O   HOH       4   58.230   −11.715   27.770   1.00   19.07   O       HETATM   4   O   HOH       5   57.252   −3.759   30.107   1.00   11.21   O       HETATM   5   O   HOH       6   58.298   −10.011   25.527   1.00   15.74   O       HETATM   6   O   HOH       7   49.321   6.583   38.815   1.00   19.33   O       HETATM   7   O   HOH       8   53.785   −4.262   22.464   1.00   10.94   O       HETATM   8   O   HOH       10   74.652   2.888   9.141   1.00   17.80   O       HETATM   9   O   HOH       11   49.761   0.826   32.896   1.00   22.02   O       HETATM   10   O   HOH       12   55.530   −11.162   28.526   1.00   11.39   O       HETATM   11   O   HOH       13   75.027   7.034   27.353   1.00   16.30   O       HETATM   12   O   HOH       14   49.994   −2.314   11.032   1.00   21.33   O       HETATM   13   O   HOH       15   61.323   −8.959   29.657   1.00   22.84   O       HETATM   14   O   HOH       16   61.029   −11.560   29.131   1.00   21.24   O       HETATM   15   O   HOH       17   50.684   5.881   10.130   1.00   15.88   O       HETATM   16   O   HOH       18   64.506   −6.302   32.989   1.00   21.05   O       HETATM   17   O   HOH       19   57.856   −16.398   25.085   1.00   22.86   O       HETATM   18   O   HOH       20   38.979   26.536   19.070   1.00   21.08   O       HETATM   19   O   HOH       21   38.042   33.487   21.909   1.00   19.01   O       HETATM   20   O   HOH       24   38.172   35.775   20.827   1.00   33.46   O       HETATM   21   O   HOH       25   70.916   −11.128   15.244   1.00   31.37   O       HETATM   22   O   HOH       26   54.205   19.360   28.396   1.00   35.76   O       HETATM   23   O   HOH       27   50.436   2.654   16.783   1.00   12.25   O       HETATM   24   O   HOH       28   69.692   19.108   38.979   1.00   49.77   O       HETATM   25   O   HOH       29   56.432   −8.877   19.303   1.00   22.52   O       HETATM   26   O   HOH       30   60.832   3.415   42.349   1.00   17.39   O       HETATM   27   O   HOH       31   53.889   −12.706   29.764   1.00   22.40   O       HETATM   28   O   HOH       32   37.887   26.373   28.058   1.00   18.09   O       HETATM   29   O   HOH       33   49.201   11.173   26.867   1.00   33.95   O       HETATM   30   O   HOH       34   46.762   −0.278   31.394   1.00   20.63   O       HETATM   31   O   HOH       35   41.731   27.568   43.302   1.00   27.39   O       HETATM   32   O   HOH       36   66.827   11.202   28.929   1.00   13.23   O       HETATM   33   O   HOH       37   46.834   14.396   40.819   1.00   46.02   O       HETATM   34   O   HOH       38   61.342   1.064   43.868   1.00   26.68   O       HETATM   35   O   HOH       42   70.597   16.422   37.837   1.00   19.26   O       HETATM   36   O   HOH       44   72.275   −9.089   33.407   1.00   22.11   O       HETATM   37   O   HOH       45   42.685   34.461   33.955   1.00   17.32   O       HETATM   38   O   HOH       46   53.480   13.394   38.364   1.00   20.19   O       HETATM   39   O   HOH       47   56.085   21.757   44.744   1.00   33.50   O       HETATM   40   O   HOH       48   35.741   32.691   23.517   1.00   19.49   O       HETATM   41   O   HOH       49   40.458   36.700   34.312   1.00   34.53   O       HETATM   42   O   HOH       50   75.440   7.267   29.948   1.00   18.07   O       HETATM   43   O   HOH       51   47.476   18.347   20.851   1.00   34.16   O       HETATM   44   O   HOH       53   52.837   −16.344   19.587   1.00   25.92   O       HETATM   45   O   HOH       55   46.415   9.073   20.108   1.00   31.91   O       HETATM   46   O   HOH       57   45.912   35.170   36.133   1.00   35.55   O       HETATM   47   O   HOH       58   60.247   −2.880   41.919   1.00   16.85   O       HETATM   48   O   HOH       60   64.974   6.086   24.501   1.00   32.16   O       HETATM   49   O   HOH       61   52.103   4.683   4.978   1.00   35.72   O       HETATM   50   O   HOH       62   50.888   40.154   36.463   1.00   38.35   O       HETATM   51   O   HOH       63   44.373   31.233   37.336   1.00   20.07   O       HETATM   52   O   HOH       64   57.280   27.757   42.451   1.00   21.74   O       HETATM   53   O   HOH       65   58.409   23.769   45.517   1.00   58.42   O       HETATM   54   O   HOH       66   68.690   −11.764   35.335   1.00   57.07   O       HETATM   55   O   HOH       67   42.746   25.153   23.465   1.00   27.05   O       HETATM   56   O   HOH       68   53.638   −16.457   32.292   1.00   31.71   O       HETATM   57   O   HOH       69   33.390   41.716   31.408   1.00   29.92   O       HETATM   58   O   HOH       70   57.768   17.897   42.434   1.00   25.75   O       HETATM   59   O   HOH       71   75.647   9.164   11.766   1.00   35.13   O       HETATM   60   O   HOH       72   62.032   33.292   44.749   1.00   46.18   O       HETATM   61   O   HOH       73   47.310   14.312   34.285   1.00   31.18   O       HETATM   62   O   HOH       74   79.660   −3.947   15.913   1.00   34.63   O       HETATM   63   O   HOH       75   46.929   5.343   4.550   1.00   23.14   O       HETATM   64   O   HOH       76   73.475   12.039   28.412   1.00   27.26   O       HETATM   65   O   HOH       77   46.297   −6.982   30.032   1.00   43.41   O       HETATM   66   O   HOH       78   68.528   −3.422   40.869   1.00   38.47   O       HETATM   67   O   HOH       79   62.080   −1.448   42.803   1.00   24.60   O       HETATM   68   O   HOH       80   65.330   18.150   40.726   1.00   41.00   O       HETATM   69   O   HOH       81   51.775   16.128   37.607   1.00   25.11   O       HETATM   70   O   HOH       83   54.266   28.682   43.313   1.00   27.61   O       HETATM   71   O   HOH       85   73.291   −15.479   20.603   1.00   37.54   O       HETATM   72   O   HOH       86   34.760   21.479   28.544   1.00   43.87   O       HETATM   73   O   HOH       87   37.326   24.131   29.677   1.00   24.47   O       HETATM   74   O   HOH       88   65.168   20.148   6.735   1.00   26.10   O       HETATM   75   O   HOH       89   59.196   12.089   13.630   1.00   25.24   O       HETATM   76   O   HOH       91   66.576   −6.235   40.279   1.00   43.11   O       HETATM   77   O   HOH       93   37.339   29.394   25.515   1.00   27.56   O       HETATM   78   O   HOH       94   52.339   -17.014   42.271   1.00   48.96   O       HETATM   79   O   HOH       95   40.511   32.927   31.717   1.00   22.46   O       HETATM   80   O   HOH       96   78.580   13.121   34.138   1.00   27.98   O       HETATM   81   O   HOH       97   65.090   15.704   34.876   1.00   18.96   O       HETATM   82   O   HOH       99   84.562   2.951   27.181   1.00   35.92   O       HETATM   83   O   HOH       100   50.386   9.761   9.646   1.00   23.18   O       HETATM   84   O   HOH       101   67.649   −0.851   38.764   1.00   24.99   O       HETATM   85   O   HOH       102   44.001   4.293   34.315   1.00   31.13   O       HETATM   86   O   HOH       103   59.386   −5.071   26.211   1.00   29.10   O       HETATM   87   O   HOH       104   77.364   4.745   41.506   1.00   35.32   O       HETATM   88   O   HOH       105   59.034   21.201   32.414   1.00   23.43   O       HETATM   89   O   HOH       106   42.463   34.698   14.327   1.00   38.86   O       HETATM   90   O   HOH       107   70.217   14.292   20.864   1.00   42.39   O       HETATM   91   O   HOH       108   76.999   8.130   25.862   1.00   32.91   O       HETATM   92   O   HOH       109   49.766   29.937   22.173   1.00   42.52   O       HETATM   93   O   HOH       110   72.473   13.536   38.823   1.00   33.32   O       HETATM   94   O   HOH       111   64.328   −12.084   38.608   1.00   37.99   O       HETATM   95   O   HOH       112   60.161   16.382   42.682   1.00   35.68   O       HETATM   96   O   HOH       113   47.602   13.639   27.016   1.00   26.01   O       HETATM   97   O   HOH       115   64.606   11.644   40.107   1.00   30.33   O       HETATM   98   O   HOH       116   61.231   −15.137   27.255   1.00   38.76   O       HETATM   99   O   HOH       117   65.324   −11.223   35.098   1.00   30.45   O       HETATM   100   O   HOH       119   56.602   17.219   44.932   1.00   36.53   O       HETATM   101   O   HOH       120   37.564   19.860   23.135   1.00   31.27   O       HETATM   102   O   HOH       121   64.845   5.057   21.132   1.00   45.57   O       HETATM   103   O   HOH       123   63.391   16.801   26.898   1.00   38.46   O       HETATM   104   O   HOH       124   42.567   6.134   32.635   1.00   31.56   O       HETATM   105   O   HOH       125   72.485   13.236   35.059   1.00   29.61   O       HETATM   106   O   HOH       126   65.229   3.650   44.032   1.00   36.86   O       HETATM   107   O   HOH       127   37.089   7.148   31.083   1.00   39.58   O       HETATM   108   O   HOH       128   73.327   10.546   12.123   1.00   34.97   O       HETATM   109   O   HOH       129   74.450   10.299   26.598   1.00   30.80   O       HETATM   110   AO5*   NDP   A   1   67.524   13.055   26.692   1.00   36.42   O       HETATM   111   AC5*   NDP   A   1   68.089   12.297   25.614   1.00   9.30   C       HETATM   112   AC4*   NDP   A   1   69.601   12.124   25.858   1.00   27.73   C       HETATM   113   AO4*   NDP   A   1   70.193   11.258   24.848   1.00   22.87   O       HETATM   114   AC3*   NDP   A   1   70.484   13.390   25.873   1.00   17.83   C       HETATM   115   AO3*   NDP   A   1   71.192   13.436   27.066   1.00   16.11   O       HETATM   116   AC2*   NDP   A   1   71.373   13.220   24.626   1.00   11.46   C       HETATM   117   AO2*   NDP   A   1   72.623   13.886   24.655   1.00   31.96   O       HETATM   118   AC1*   NDP   A   1   71.510   11.702   24.656   1.00   19.02   C       HETATM   119   O3   NDP   A   1   65.336   13.590   26.129   1.00   20.59   O       HETATM   120   NO5*   NDP   A   1   63.536   11.943   26.448   1.00   28.99   O       HETATM   121   NC5*   NDP   A   1   64.328   10.843   25.957   1.00   24.89   C       HETATM   122   NC4*   NDP   A   1   63.467   9.646   25.686   1.00   31.79   C       HETATM   123   NO4*   NDP   A   1   62.837   9.337   26.908   1.00   28.82   O       HETATM   124   NC3*   NDP   A   1   62.340   9.837   24.665   1.00   11.50   C       HHTATM   125   NO3*   NDP   A   1   62.891   9.402   23.461   1.00   28.60   O       HETATM   126   NC2*   NDP   A   1   61.152   8.996   25.138   1.00   28.11   C       HETATM   127   NO2*   NDP   A   1   60.881   7.662   24.715   1.00   24.30   O       HETATM   128   NC1*   NDP   A   1   61.547   8.875   26.580   1.00   35.35   C       HETATM   129   AP2*   NDP   A   1   73.104   15.069   23.823   1.00   32.96   P       HETATM   130   AOP1   NDP   A   1   74.500   15.308   24.308   1.00   37.84   O       HETATM   131   AOP2   NDP   A   1   72.797   14.925   22.348   1.00   36.66   O       HETATM   132   AOP3   NDP   A   1   72.163   16.217   23.958   1.00   31.97   O       HETATM   133   AP   NDP   A   1   66.660   14.257   26.393   1.00   26.17   XX       HETATM   134   AO1   NDP   A   1   66.886   14.795   25.047   1.00   15.31   XX       HETATM   135   AO2   NDP   A   1   66.439   15.207   27.521   1.00   34.39   XX       HETATM   136   AN9   NDP   A   1   71.820   11.224   23.353   1.00   13.63   XX       HETATM   137   AC8   NDP   A   1   71.104   11.316   22.200   1.00   12.41   XX       HETATM   138   AN7   NDP   A   1   71.758   10.835   21.161   1.00   15.71   XX       HETATM   139   AC5   NDP   A   1   72.933   10.313   21.710   1.00   16.17   XX       HETATM   140   AC6   NDP   A   1   74.053   9.657   21.140   1.00   31.35   XX       HETATM   141   AN6   NDP   A   1   74.165   9.464   19.819   1.00   12.59   XX       HETATM   142   AN1   NDP   A   1   75.078   9.280   21.942   1.00   17.56   XX       HETATM   143   AC2   NDP   A   1   74.971   9.578   23.251   1.00   15.44   XX       HETATM   144   AN3   NDP   A   1   74.027   10.302   23.889   1.00   24.82   XX       HETATM   145   AC4   NDP   A   1   73.036   10.653   23.047   1.00   17.48   XX       HETATM   146   NP   NDP   A   1   64.183   13.106   27.191   1.00   25.47   N       HETATM   147   NO1   NDP   A   1   63.142   14.169   27.253   1.00   28.69   N       HETATM   148   NO2   NDP   A   1   64.837   12.643   28.492   1.00   24.32   N       HETATM   149   NN1   NDP   A   1   60.598   9.775   27.109   1.00   23.63   N       HETATM   150   NC2   NDP   A   1   60.143   10.905   26.442   −99.00   78.36   N       HETATM   151   NC3   NDP   A   1   59.070   11.648   27.007   −99.00   100.00   N       HETATM   152   NC7   NDP   A   1   58.497   13.017   26.528   −99.00   100.00   N       HETATM   153   NO7   NDP   A   1   59.358   13.703   25.972   −99.00   100.00   N       HETATM   154   NN7   NDP   A   1   57.207   13.400   26.912   −99.00   84.38   N       HETATM   155   NC4   NDP   A   1   58.442   11.146   28.137   −99.00   100.00   N       HETATM   156   NC5   NDP   A   1   58.912   9.963   28.754   −99.00   100.00   N       HETATM   157   NC6   NDP   A   1   59.951   9.266   28.147   −99.00   100.00   N       ATOM   158   N   LYS   A   3   76.227   −5.632   44.315   1.00   61.49   N       ATOM   159   CA   LYS   A   3   76.152   −4.302   43.684   1.00   58.00   C       ATOM   160   C   LYS   A   3   75.985   −4.421   42.171   1.00   52.79   C       ATOM   161   O   LYS   A   3   76.921   −4.737   41.419   1.00   44.76   O       ATOM   162   CE   LYS   A   3   77.359   −3.417   44.030   1.00   59.74   C       ATOM   163   CG   LYS   A   3   77.011   −1.944   44.314   1.00   50.87   C       ATOM   164   CD   LYS   A   3   78.208   −1.161   44.894   1.00   61.21   C       ATOM   165   CE   LYS   A   3   77.855   −0.377   46.186   1.00   100.00   C       ATOM   166   NZ   LYS   A   3   78.857   −0.401   47.343   1.00   70.61   N       ATOM   167   N   GLN   A   4   74.746   −4.242   41.747   1.00   45.15   N       ATOM   168   CA   GLN   A   4   74.408   −4.326   40.347   1.00   37.18   C       ATOM   169   C   GLN   A   4   74.983   −3.166   39.561   1.00   34.93   C       ATOM   170   O   GLN   A   4   75.127   −2.050   40.087   1.00   28.48   O       ATOM   171   CE   GLN   A   4   72.915   −4.445   40.221   1.00   34.65   C       ATOM   172   CG   GLN   A   4   72.456   −5.854   40.584   1.00   31.82   C       ATOM   173   CD   GLN   A   4   72.570   −6.788   39.405   1.00   79.25   C       ATOM   174   OE1   GLN   A   4   72.165   −6.452   38.286   1.00   100.00   O       ATOM   175   NE2   GLN   A   4   73.206   −7.925   39.623   1.00   80.24   N       ATOM   176   N   ARG   A   5   75.475   −3.495   38.375   1.00   27.16   N       ATOM   177   CA   ARG   A   5   76.146   −2.546   37.483   1.00   39.16   C       ATOM   178   C   ARG   A   5   75.191   +321 2.018   36.433   1.00   38.22   C       ATOM   179   O   ARG   A   5   74.938   −2.698   35.438   1.00   32.44   O       ATOM   180   CB   ARG   A   5   77.398   −3.163   36.826   1.00   41.76   C       ATOM   181   CG   ARG   A   5   78.692   −2.954   37.663   1.00   37.34   C       ATOM   182   CD   ARG   A   5   80.015   −3.236   36.876   1.00   32.99   C       ATOM   183   NE   ARG   A   5   81.036   −2.203   37.125   1.00   25.71   N       ATOM   184   CZ   ARG   A   5   81.617   −1.488   36.169   1.00   32.53   C       ATOM   185   NE1   ARG   A   5   81.293   −1.704   34.904   1.00   40.07   N       ATOM   186   NH2   ARG   A   5   82.516   −0.551   36.474   1.00   100.00   N       ATOM   187   N   VAL   A   6   74.743   −0.773   36.659   1.00   32.08   N       ATOM   188   CA   VAL   A   6   73.715   −0.082   35.881   1.00   28.89   C       ATOM   189   C   VAL   A   6   74.161   1.021   34.897   1.00   29.37   C       ATOM   190   O   VAL   A   6   74.745   2.041   35.274   1.00   22.50   O       ATOM   191   CB   VAL   A   6   72.577   0.378   36.813   1.00   23.52   C       ATOM   192   CG1   VAL   A   6   71.366   0.960   36.006   1.00   20.29   C       ATOM   193   CG2   VAL   A   6   72.108   −0.852   37.644   1.00   18.45   C       ATOM   194   N   PHE   A   7   73.948   0.749   33.615   1.00   22.92   N       ATOM   195   CA   PHE   A   7   74.267   1.710   32.573   1.00   27.15   C       ATOM   196   C   PHE   A   7   72.975   2.423   32.192   1.00   20.24   C       ATOM   197   O   PHE   A   7   71.994   1.788   31.815   1.00   20.71   O       ATOM   198   CB   PHE   A   7   74.864   1.004   31.374   1.00   18.98   C       ATOM   199   CG   PHE   A   7   74.916   1.836   30.115   1.00   21.83   C       ATOM   200   CD1   PHE   A   7   75.521   3.087   30.108   1.00   19.36   C       ATOM   201   CD2   PHE   A   7   74.483   1.284   28.886   1.00   23.50   C       ATOM   202   CE1   PHE   A   7   75.614   3.828   28.902   1.00   27.52   C       ATOM   203   CE2   PHE   A   7   74.548   1.996   27.685   1.00   19.33   C       ATOM   204   CZ   PHE   A   7   75.128   3.255   27.673   1.00   18.59   C       ATOM   205   N   ILE   A   8   72.959   3.727   32.454   1.00   18.75   N       ATOM   206   CA   ILE   A   8   71.844   4.588   32.112   1.00   14.25   C       ATOM   207   C   ILE   A   8   72.337   5.351   30.909   1.00   11.22   C       ATOM   208   O   ILE   A   8   73.259   6.165   30.998   1.00   17.76   O       ATOM   209   CE   ILE   A   8   71.507   5.605   33.212   1.00   14.15   C       ATOM   210   CG1   ILE   A   8   71.356   4.949   34.582   1.00   8.24   C       ATOM   211   CG2   ILE   A   8   70.183   6.342   32.874   1.00   16.85   C       ATOM   212   CD1   ILE   A   8   71.091   5.961   35.707   1.00   10.32   C       ATOM   213   N   ALA   A   9   71.896   4.906   29.752   1.00   16.42   N       ATOM   214   CA   ALA   A   9   72.256   5.559   28.513   1.00   18.74   C       ATOM   215   C   ALA   A   9   71.530   6.913   28.511   1.00   28.45   C       ATOM   216   O   ALA   A   9   70.411   7.032   29.045   1.00   22.39   O       ATOM   217   CB   ALA   A   9   71.808   4.731   27.311   1.00   14.43   C       ATOM   218   N   GLY   A   10   72.199   7.922   27.940   1.00   20.06   N       ATOM   219   CA   GLY   A   10   71.706   9.284   27.911   1.00   18.62   C       ATOM   220   C   GLY   A   10   71.407   9.819   29.305   1.00   16.40   C       ATOM   221   O   GLY   A   10   70.379   10.448   29.481   1.00   17.36   O       ATOM   222   N   HIS   A   11   72.295   9.581   30.272   1.00   10.32   N       ATOM   223   CA   HIS   A   11   72.068   9.966   31.688   1.00   13.90   C       ATOM   224   C   HIS   A   11   72.008   11.504   31.916   1.00   21.52   C       ATOM   225   O   HIS   A   11   71.700   11.994   32.983   1.00   13.22   O       ATOM   226   CE   HIS   A   11   73.153   9.350   32.581   1.00   14.88   C       ATOM   227   CG   HIS   A   11   74.502   9.948   32.326   1.00   23.73   C       ATOM   228   ND1   HIS   A   11   75.239   9.648   31.197   1.00   24.90   N       ATOM   229   CD2   HIS   A   11   75.167   10.952   32.956   1.00   16.35   C       ATOM   230   CE1   HIS   A   11   76.317   10.407   31.170   1.00   22.54   C       ATOM   231   NE2   HIS   A   11   76.271   11.240   32.197   1.00   17.56   N       ATOM   232   N   ARG   A   12   72.310   12.288   30.908   1.00   22.31   N       ATOM   233   CA   ARG   A   12   72.147   13.693   31.122   1.00   18.90   C       ATOM   234   C   ARG   A   12   70.851   14.244   30.495   1.00   26.34   C       ATOM   235   O   ARG   A   12   70.572   15.426   30.604   1.00   25.37   O       ATOM   236   CE   ARG   A   12   73.352   14.418   30.587   1.00   25.93   C       ATOM   237   CG   ARG   A   12   74.582   13.943   31.279   1.00   53.87   C       ATOM   238   CD   ARG   A   12   75.757   14.619   30.699   1.00   32.53   C       ATOM   239   NE   ARG   A   12   76.359   15.576   31.605   1.00   69.90   N       ATOM   240   CZ   ARG   A   12   76.971   16.675   31.178   1.00   100.00   C       ATOM   241   NH1   ARG   A   12   77.001   16.948   29.867   1.00   100.00   N       ATOM   242   NH2   ARG   A   12   77.526   17.508   32.056   1.00   100.00   N       ATOM   243   N   GLY   A   13   70.078   13.420   29.800   1.00   18.25   N       ATOM   244   CA   GLY   A   13   68.802   13.904   29.258   1.00   16.50   C       ATOM   245   C   GLY   A   13   67.849   14.144   30.428   1.00   18.88   C       ATOM   246   O   GLY   A   13   68.202   13.902   31.624   1.00   14.04   O       ATOM   247   N   MET   A   14   66.653   14.632   30.103   1.00   16.00   N       ATOM   248   CA   MET   A   14   65.688   14.981   31.128   1.00   13.49   C       ATOM   249   C   MET   A   14   65.293   13.760   31.901   1.00   14.02   C       ATOM   250   O   MET   A   14   65.408   13.713   33.145   1.00   17.06   O       ATOM   251   CE   MET   A   14   64.442   15.605   30.524   1.00   11.57   C       ATOM   252   CG   MET   A   14   63.320   15.628   31.559   1.00   20.77   C       ATOM   253   SD   MET   A   14   61.926   16.766   31.110   1.00   29.16   5       ATOM   254   CE   MET   A   14   62.527   17.108   29.574   1.00   30.68   C       ATOM   255   N   VAL   A   15   64.798   12.769   31.158   1.00   25.23   N       ATOM   256   CA   VAL   A   15   64.439   11.468   31.738   1.00   20.90   C       ATOM   257   C   VAL   A   15   65.654   10.713   32.378   1.00   17.26   C       ATOM   258   O   VAL   A   15   65.590   10.239   33.524   1.00   18.41   O       ATOM   259   CB   VAL   A   15   63.752   10.550   30.680   1.00   23.25   C       ATOM   260   CG1   VAL   A   15   63.330   9.253   31.310   1.00   15.71   C       ATOM   261   CG2   VAL   A   15   62.528   11.193   30.183   1.00   13.40   C       ATOM   262   N   GLY   A   16   66.784   10.642   31.665   1.00   20.39   N       ATOM   263   CA   GLY   A   16   67.941   9.904   32.186   1.00   19.54   C       ATOM   264   C   GLY   A   16   68.522   10.432   33.492   1.00   29.29   C       ATOM   265   O   GLY   A   16   68.896   9.659   34.434   1.00   16.91   O       ATOM   266   N   SER   A   17   68.642   11.755   33.499   1.00   12.53   N       ATOM   267   CA   SER   A   17   69.154   12.460   34.650   1.00   21.93   C       ATOM   268   C   SER   A   17   68.209   12.214   35.818   1.00   13.35   C       ATOM   269   O   SER   A   17   68.677   11.957   36.915   1.00   24.19   O       ATOM   270   CB   SER   A   17   69.378   13.942   34.333   1.00   15.52   C       ATOM   271   OG   SER   A   17   68.153   14.619   34.372   1.00   22.95   O       ATOM   272   N   ALA   A   18   66.896   12.143   35.590   1.00   17.52   N       ATOM   273   CA   ALA   A   18   65.991   11.828   36.729   1.00   13.14   C       ATOM   274   C   ALA   A   18   66.220   10.393   37.307   1.00   19.29   C       ATOM   275   O   ALA   A   18   66.149   10.150   38.522   1.00   16.94   O       ATOM   276   CB   ALA   A   18   64.460   12.046   36.334   1.00   14.33   C       ATOM   277   N   ILE   A   19   66.484   9.432   36.430   1.00   20.80   N       ATOM   278   CA   ILE   A   19   66.705   8.078   36.900   1.00   18.08   C       ATOM   279   C   ILE   A   19   67.975   8.090   37.730   1.00   16.09   C       ATOM   280   O   ILE   A   19   68.018   7.530   38.820   1.00   20.73   O       ATOM   281   CB   ILE   A   19   66.804   7.079   35.710   1.00   17.58   C       ATOM   282   CG1   ILE   A   19   65.444   6.812   35.162   1.00   10.09   C       ATOM   283   CG2   ILE   A   19   67.309   5.666   36.133   1.00   21.60   C       ATOM   284   CD1   ILE   A   19   65.528   6.361   33.741   1.00   19.05   C       ATOM   285   N   ARG   A   20   68.984   8.771   37.198   1.00   18.13   N       ATOM   286   CA   ARG   A   20   70.286   8.897   37.836   1.00   20.25   C       ATOM   287   C   ARG   A   20   70.231   9.491   39.242   1.00   30.62   C       ATOM   288   O   ARG   A   20   70.957   9.091   40.129   1.00   33.00   O       ATOM   289   CB   ARG   A   20   71.201   9.743   36.957   1.00   11.71   C       ATOM   290   CG   ARG   A   20   72.610   9.781   37.449   1.00   23.79   C       ATOM   291   CD   ARG   A   20   72.881   11.107   38.060   1.00   36.76   C       ATOM   292   NE   ARG   A   20   74.297   11.443   38.062   1.00   48.34   N       ATOM   293   CZ   ARG   A   20   74.990   11.841   36.988   1.00   100.00   C       ATOM   294   NH1   ARG   A   20   74.393   11.931   35.808   1.00   100.00   N       ATOM   295   NH2   ARG   A   20   76.289   12.139   37.076   1.00   100.00   N       ATOM   296   N   ARG   A   21   69.368   10.461   39.439   1.00   22.10   N       ATOM   297   CA   ARG   A   21   69.216   11.052   40.750   1.00   17.45   C       ATOM   298   C   ARG   A   21   68.721   10.007   41.730   1.00   26.71   C       ATOM   299   O   ARG   A   21   69.147   10.001   42.885   1.00   30.27   O       ATOM   300   CB   ARG   A   21   68.142   12.144   40.708   1.00   17.93   C       ATOM   301   CG   ARG   A   21   68.682   13.522   40.321   1.00   27.57   C       ATOM   302   CD   ARG   A   21   67.586   14.599   40.130   1.00   23.02   C       ATOM   303   NE   ARG   A   21   67.619   15.000   38.743   1.00   55.12   N       ATOM   304   CZ   ARG   A   21   66.538   15.103   37.995   1.00   10.55   C       ATOM   305   NH1   ARG   A   21   65.343   14.974   38.552   1.00   29.80   N       ATOM   306   NH2   ARG   A   21   66.665   15.435   36.715   1.00   61.45   N       ATOM   307   N   GLN   A   22   67.713   9.223   41.345   1.00   27.48   N       ATOM   308   CA   GLN   A   22   67.167   8.257   42.313   1.00   24.79   C       ATOM   309   C   GLN   A   22   68.137   7.127   42.547   1.00   31.37   C       ATOM   310   O   GLN   A   22   68.394   6.724   43.685   1.00   27.47   O       ATOM   311   CB   GLN   A   22   65.818   7.706   41.894   1.00   17.11   C       ATOM   312   CG   GLN   A   22   64.921   8.745   41.243   1.00   66.14   C       ATOM   313   CD   GLN   A   22   63.425   8.456   41.397   1.00   41.27   C       ATOM   314   OE1   GLN   A   22   63.002   7.329   41.762   1.00   29.34   O       ATOM   315   NE2   GLN   A   22   62.610   9.464   41.046   1.00   20.12   N       ATOM   316   N   LEU   A   23   68.697   6.652   41.448   1.00   27.99   N       ATOM   317   CA   LEU   A   23   69.649   5.575   41.500   1.00   24.48   C       ATOM   318   C   LEU   A   23   70.828   5.971   42.334   1.00   28.87   C       ATOM   319   O   LEU   A   23   71.288   5.218   43.165   1.00   30.79   O       ATOM   320   CB   LEU   A   23   70.036   5.107   40.089   1.00   22.72   C       ATOM   321   CG   LEU   A   23   68.966   4.0723   9.658   1.00   26.16   C       ATOM   322   CD1   LEU   A   23   69.271   3.083   38.481   1.00   24.80   C       ATOM   323   CD2   LEU   A   23   68.427   3.284   40.835   1.00   22.91   C       ATOM   324   N   GLU   A   24   71.279   7.192   42.153   1.00   28.77   N       ATOM   325   CA   GLU   A   24   72.419   7.675   42.909   1.00   33.79   C       ATOM   326   C   GLU   A   24   72.363   7.388   44.412   1.00   35.94   C       ATOM   327   O   GLU   A   24   73.381   7.140   45.031   1.00   39.07   O       ATOM   328   CB   GLU   A   24   72.647   9.165   42.653   1.00   36.21   C       ATOM   329   CG   GLU   A   24   74.068   9.482   42.243   1.00   42.54   C       ATOM   330   CD   GLU   A   24   74.158   10.689   41.333   1.00   89.51   C       ATOM   331   OE1   GLU   A   24   73.386   11.663   41.549   1.00   43.21   O       ATOM   332   OE2   GLU   A   24   74.994   10.646   40.398   1.00   66.28   O       ATOM   333   N   GLN   A   25   71.182   7.422   45.000   1.00   45.70   N       ATOM   334   CA   GLN   A   25   71.039   7.152   46.432   1.00   47.57   C       ATOM   335   C   GLN   A   25   70.887   5.669   46.740   1.00   67.34   C       ATOM   336   O   GLN   A   25   70.285   5.286   47.726   1.00   74.06   O       ATOM   337   CB   GLN   A   25   69.783   7.842   46.905   1.00   51.85   C       ATOM   338   CG   GLN   A   25   69.500   9.084   46.109   1.00   44.91   C       ATOM   339   CD   GLN   A   25   68.419   9.913   46.742   1.00   100.00   C       ATOM   340   OE1   GLN   A   25   68.271   9.947   47.972   1.00   100.00   O       ATOM   341   NE2   GLN   A   25   67.624   10.602   45.911   1.00   100.00   N       ATOM   342   N   ARG   A   26   71.322   4.831   45.825   1.00   75.37   N       ATOM   343   CA   ARG   A   26   71.182   3.4074   6.026   1.00   74.87   C       ATOM   344   C   ARG   A   26   72.568   2.791   46.147   1.00   74.08   C       ATOM   345   O   ARG   A   26   73.440   2.997   45.289   1.00   77.00   O       ATOM   346   CB   ARG   A   26   70.390   2.790   44.885   1.00   52.44   C       ATOM   347   CG   ARG   A   26   68.916   2.927   45.070   1.00   43.51   C       ATOM   348   CD   ARG   A   26   68.428   1.752   45.864   1.00   40.70   C       ATOM   349   NE   ARG   A   26   67.200   1.176   45.338   1.00   42.33   N       ATOM   350   CZ   ARG   A   26   67.126   0.508   44.196   1.00   32.07   C       ATOM   351   NH1   ARG   A   26   68.215   0.324   43.486   1.00   44.02   N       ATOM   352   NH2   ARG   A   26   65.968   0.017   43.771   1.00   77.32   N       ATOM   353   N   GLY   A   27   72.778   2.114   47.266   1.00   46.30   N       ATOM   354   CA   GLY   A   27   74.060   1.531   47.549   1.00   46.82   C       ATOM   355   C   GLY   A   27   74.140   0.165   46.923   1.00   55.45   C       ATOM   356   O   GLY   A   27   75.204   −0.453   46.877   1.00   64.43   O       ATOM   357   N   ASP   A   28   73.017   −0.315   46.428   1.00   40.98   N       ATOM   358   CA   ASP   A   28   73.016   −1.647   45.861   1.00   40.35   C       ATOM   359   C   ASP   A   28   73.266   −1.536   44.400   1.00   39.55   C       ATOM   360   O   ASP   A   28   73.109   −2.518   43.654   1.00   48.80   O       ATOM   361   CB   ASP   A   28   71.680   −2.335   46.127   1.00   47.80   C       ATOM   362   CG   ASP   A   28   70.503   −1.373   46.064   1.00   35.34   C       ATOM   363   OD1   ASP   A   28   70.705   −0.140   46.095   1.00   39.23   O       ATOM   364   OD2   ASP   A   28   69.383   −1.870   45.872   1.00   69.86   O       ATOM   365   N   VAL   A   29   73.651   −0.329   43.996   1.00   31.03   N       ATOM   366   CA   VAL   A   29   73.881   −0.050   42.591   1.00   28.44   C       ATOM   367   C   VAL   A   29   75.166   0.676   42.281   1.00   28.00   C       ATOM   368   O   VAL   A   29   75.505   1.699   42.892   1.00   34.83   O       ATOM   369   CB   VAL   A   29   72.696   0.760   42.000   1.00   30.68   C       ATOM   370   CG1   VAL   A   29   72.935   1.088   40.549   1.00   23.65   C       ATOM   371   CG2   VAL   A   29   71.416   −0.028   42.156   1.00   27.95   C       ATOM   372   N   GLU   A   30   75.824   0.219   41.230   1.00   30.76   N       ATOM   373   CA   GLU   A   30   76.995   0.924   40.736   1.00   28.38   C       ATOM   374   C   GLU   A   30   76.678   1.471   39.332   1.00   31.03   C       ATOM   375   O   GLU   A   30   76.368   0.720   38.397   1.00   26.64   O       ATOM   376   CB   GLU   A   30   78.199   0.006   40.722   1.00   31.84   C       ATOM   377   CG   GLU   A   30   79.355   0.539   41.533   1.00   89.26   C       ATOM   378   CD   GLU   A   30   80.667   0.264   40.858   1.00   100.00   C       ATOM   379   OE1   GLU   A   30   81.082   −0.922   40.872   1.00   88.94   O       ATOM   380   OE2   GLU   A   30   81.202   1.206   40.219   1.00   100.00   O       ATOM   381   N   LEU   A   31   76.665   2.789   39.207   1.00   22.24   N       ATOM   382   CA   LEU   A   31   76.269   3.391   37.945   1.00   29.37   C       ATOM   383   C   LEU   A   31   77.404   3.507   36.941   1.00   25.79   C       ATOM   384   O   LEU   A   31   78.485   3.969   37.256   1.00   29.41   O       ATOM   385   CB   LEU   A   31   75.632   4.760   38.191   1.00   30.20   C       ATOM   386   CG   LEU   A   31   74.329   4.763   38.994   1.00   29.37   C       ATOM   387   CD1   LEU   A   31   73.841   6.143   39.240   1.00   23.43   C       ATOM   388   CD2   LEU   A   31   73.275   3.962   38.281   1.00   23.04   C       ATOM   389   N   VAL   A   32   77.146   3.100   35.711   1.00   21.94   N       ATOM   390   CA   VAL   A   32   78.143   3.265   34.685   1.00   25.48   C       ATOM   391   C   VAL   A   32   77.535   4.242   33.669   1.00   38.76   C       ATOM   392   O   VAL   A   32   76.429   3.999   33.180   1.00   29.70   O       ATOM   393   CB   VAL   A   32   78.517   1.902   34.055   1.00   34.25   C       ATOM   394   CG1   VAL   A   32   79.587   2.079   32.970   1.00   30.56   C       ATOM   395   CG2   VAL   A   32   79.003   0.950   35.139   1.00   25.27   C       ATOM   396   N   LEU   A   33   78.219   5.375   33.457   1.00   30.19   N       ATOM   397   CA   LEU   A   33   77.732   6.463   32.621   1.00   22.71   C       ATOM   398   C   LEU   A   33   78.727   6.979   31.645   1.00   29.55   C       ATOM   399   O   LEU   A   33   79.896   7.152   31.988   1.00   30.09   O       ATOM   400   CB   LEU   A   33   77.423   7.635   33.514   1.00   19.75   C       ATOM   401   CG   LEU   A   33   76.729   7.200   34.779   1.00   19.38   C       ATOM   402   CD1   LEU   A   33   76.814   8.344   35.762   1.00   27.24   C       ATOM   403   CD2   LEU   A   33   75.271   6.913   34.444   1.00   22.07   C       ATOM   404   N   ARG   A   34   78.239   7.4213   0.496   1.00   15.09   N       ATOM   405   CA   ARG   A   34   79.154   8.008   29.541   1.00   26.04   C       ATOM   406   CA   ARG   A   34   78.469   9.173   28.916   1.00   36.57   C       ATOM   407   O   ARG   A   34   77.288   9.130   28.651   1.00   38.59   O       ATOM   408   CB   ARG   A   34   79.486   7.048   28.398   1.00   22.89   C       ATOM   409   CG   ARG   A   34   80.579   6.081   28.706   1.00   23.29   C       ATOM   410   CD   ARG   A   34   81.370   6.575   29.860   1.00   52.06   C       ATOM   411   NE   ARG   A   34   81.783   5.458   30.711   1.00   80.25   N       ATOM   412   CZ   ARG   A   34   82.646   4.530   30.323   1.00   41.94   C       ATOM   413   NH1   ARG   A   34   83.173   4.596   29.104   1.00   53.02   N       ATOM   414   NH2   ARG   A   34   82.983   3.547   31.148   1.00   25.56   N       ATOM   415   N   THR   A   35   79.248   10.156   28.539   1.00   31.58   N       ATOM   416   CA   THR   A   35   78.703   11.282   27.833   1.00   29.33   C       ATOM   417   C   THR   A   35   78.719   10.951   26.340   1.00   32.53   C       ATOM   418   O   THR   A   35   79.350   9.944   25.962   1.00   28.08   O       ATOM   419   CB   THR   A   35   79.527   12.527   28.145   1.00   37.49   C       ATOM   420   OG1   THR   A   35   80.644   12.429   27.560   1.00   31.91   O       ATOM   421   CG2   THR   A   35   79.627   12.642   29.651   1.00   19.38   C       ATOM   422   N   ARG   A   36   78.032   11.780   25.529   1.00   30.02   N       ATOM   423   CA   ARG   A   36   78.002   11.639   24.056   1.00   29.37   C       ATOM   424   C   ARG   A   36   79.406   11.765   23.503   1.00   31.46   C       ATOM   425   O   ARG   A   36   79.772   11.012   22.591   1.00   36.56   O       ATOM   426   CB   ARG   A   36   77.054   12.650   23.354   1.00   37.34   C       ATOM   427   CG   ARG   A   36   76.937   12.465   21.846   −99.00   49.47   C       ATOM   428   CD   ARG   A   36   76.020   13.515   21.232   −99.00   63.09   C       ATOM   429   NE   ARG   A   36   75.528   13.124   19.915   −99.00   75.23   N       ATOM   430   CZ   ARG   A   36   74.381   13.549   19.391   −99.00   91.44   C       ATOM   431   NH1   ARG   A   36   73.605   14.375   20.079   −99.00   79.32   N       ATOM   432   NH2   ARG   A   36   74.009   13.144   18.185   −99.00   78.73   N       ATOM   433   N   ASP   A   37   80.217   12.677   24.063   1.00   41.30   N       ATOM   434   CA   ASP   A   37   81.606   12.710   23.601   1.00   44.91   C       ATOM   435   C   ASP   A   37   82.410   11.481   24.043   1.00   24.99   C       ATOM   436   O   ASP   A   37   83.211   10.978   23.261   1.00   42.22   O       ATOM   437   CB   ASP   A   37   82.347   14.048   23.718   −99.00   47.07   C       ATOM   438   CG   ASP   A   37   81.881   14.887   24.876   −99.00   62.99   C       ATOM   439   OD1   ASP   A   37   80.679   14.839   25.204   −99.00   64.45   O       ATOM   440   OD2   ASP   A   37   82.711   15.638   25.429   −99.00   69.84   O       ATOM   441   N   GLU   A   38   82.129   10.950   25.235   1.00   19.39   N       ATOM   442   CA   GLU   A   38   82.790   9.717   25.682   1.00   27.84   C       ATOM   443   C   GLU   A   38   82.203   8.527   24.901   1.00   37.14   C       ATOM   444   O   GLU   A   38   82.873   7.511   24.699   1.00   35.04   O       ATOM   445   CB   GLU   A   38   82.691   9.435   27.207   1.00   25.18   C       ATOM   446   CG   GLU   A   38   83.116   10.549   28.183   1.00   37.45   C       ATOM   447   CD   GLU   A   38   82.807   10.212   29.655   1.00   21.13   C       ATOM   448   OE1   GLU   A   38   81.623   9.997   30.014   1.00   55.97   O       ATOM   449   OE2   GLU   A   38   83.757   9.978   30.419   1.00   98.78   O       ATOM   450   N   LEU   A   39   80.948   8.610   24.478   1.00   25.52   N       ATOM   451   CA   LEU   A   39   80.440   7.483   23.739   1.00   18.17   C       ATOM   452   C   LEU   A   39   79.291   7.764   22.825   1.00   20.34   C       ATOM   453   O   LEU   A   39   78.152   7.810   23.259   1.00   26.35   O       ATOM   454   CB   LEU   A   39   80.123   6.313   24.657   1.00   14.56   C       ATOM   455   CG   LEU   A   39   79.410   5.075   24.058   1.00   19.52   C       ATOM   456   CD1   LEU   A   39   80.205   4.392   22.994   1.00   18.84   C       ATOM   457   CD2   LEU   A   39   78.890   4.051   25.084   1.00   17.41   C       ATOM   458   N   ASN   A   40   79.598   7.860   21.543   1.00   16.73   N       ATOM   459   CA   ASN   A   40   78.548   7.971   20.540   1.00   21.55   C       ATOM   460   C   ASN   A   40   77.798   6.649   20.308   1.00   24.53   C       ATOM   461   O   ASN   A   40   78.328   5.720   19.688   1.00   19.96   O       ATOM   462   CB   ASN   A   40   79.130   8.367   19.216   1.00   18.45   C       ATOM   463   CG   ASN   A   40   78.054   8.727   18.225   1.00   42.19   C       ATOM   464   OD1   ASN   A   40   78.327   9.093   17.080   1.00   38.89   O       ATOM   465   ND2   ASN   A   40   76.827   8.730   18.697   1.00   23.71   N       ATOM   466   N   LEU   A   41   76.543   6.622   20.754   1.00   21.08   N       ATOM   467   CA   LEU   A   41   75.649   5.465   20.650   1.00   15.03   C       ATOM   468   C   LEU   A   41   75.225   5.068   19.213   1.00   18.22   C       ATOM   469   O   LEU   A   41   74.681   3.971   18.980   1.00   25.72   O       ATOM   470   CE   LEU   A   41   74.426   5.705   21.532   1.00   15.65   C       ATOM   471   CG   LEU   A   41   74.822   6.029   22.974   1.00   21.90   C       ATOM   472   CD1   LEU   A   41   73.604   6.413   23.749   1.00   20.59   C       ATOM   473   CD2   LEU   A   41   75.481   4.796   23.609   1.00   17.97   C       ATOM   474   N   LEU   A   42   75.542   5.916   18.238   1.00   12.45   N       ATOM   475   CA   LEU   A   42   75.256   5.607   16.831   1.00   15.99   C       ATOM   476   C   LEU   A   42   76.290   4.680   16.280   1.00   26.18   C       ATOM   477   O   LEU   A   42   76.066   4.039   15.257   1.00   22.41   O       ATOM   478   CE   LEU   A   42   75.282   6.873   15.984   1.00   17.85   C       ATOM   479   CG   LEU   A   42   74.180   7.854   16.399   1.00   30.70   C       ATOM   480   CD1   LEU   A   42   74.318   9.184   15.704   1.00   24.31   C       ATOM   481   CD2   LEU   A   42   72.764   7.241   16.208   1.00   31.13   C       ATOM   482   N   ASP   A   43   77.462   4.705   16.911   1.00   26.87   N       ATOM   483   CA   ASP   A   43   78.579   3.875   16.486   1.00   19.29   C       ATOM   484   C   ASP   A   43   78.583   2.519   17.163   1.00   13.33   C       ATOM   485   O   ASP   A   43   79.051   2.348   18.297   1.00   18.75   O       ATOM   486   CB   ASP   A   43   79.870   4.580   16.776   1.00   31.06   C       ATOM   487   CG   ASP   A   43   81.083   3.758   16.380   1.00   30.68   C       ATOM   488   OD1   ASP   A   43   80.971   2.551   16.082   1.00   32.36   O       ATOM   489   OD2   ASP   A   43   82.187   4.308   16.499   1.00   37.83   O       ATOM   490   N   SER   A   44   78.139   1.544   16.377   1.00   16.89   N       ATOM   491   CA   SER   A   44   77.978   0.173   16.789   1.00   17.67   C       ATOM   492   C   SER   A   44   79.237   −0.463   17.392   1.00   20.40   C       ATOM   493   O   SER   A   44   79.206   −1.126   18.444   1.00   26.27   O       ATOM   494   CE   SER   A   44   77.504   −0.617   15.581   1.00   13.85   C       ATOM   495   OG   SER   A   44   76.800   −1.740   16.063   1.00   43.83   O       ATOM   496   N   ARG   A   45   80.335   −0.301   16.682   1.00   15.63   N       ATOM   497   CA   ARG   A   45   81.616   −0.788   17.154   1.00   19.94   C       ATOM   498   C   ARG   A   45   81.910   −0.225   18.521   1.00   29.48   C       ATOM   499   O   ARG   A   45   82.244   −0.937   19.457   1.00   27.65   O       ATOM   500   CB   ARG   A   45   82.684   −0.261   16.203   1.00   27.46   C       ATOM   501   CG   ARG   A   45   83.463   −1.338   15.495   1.00   92.03   C       ATOM   502   CD   ARG   A   45   84.854   −1.418   16.077   1.00   100.00   C       ATOM   503   NE   ARG   A   45   85.636   −2.533   15.527   1.00   100.00   N       ATOM   504   CZ   ARG   A   45   86.092   −3.570   16.236   1.00   100.00   C       ATOM   505   NH1   ARG   A   45   85.791   −3.695   17.547   1.00   100.00   N       ATOM   506   NH2   ARG   A   45   86.773   −4.544   15.642   1.00   100.00   N       ATOM   507   N   ALA   A   46   81.772   1.090   18.629   1.00   31.04   N       ATOM   508   CA   ALA   A   46   82.045   1.743   19.881   1.00   24.72   C       ATOM   509   C   ALA   A   46   81.111   1.176   20.899   1.00   17.73   C       ATOM   510   O   ALA   A   46   81.512   0.825   22.027   1.00   22.73   O       ATOM   511   CB   ALA   A   46   81.839   3.221   19.751   1.00   27.16   C       ATOM   512   N   VAL   A   47   79.835   1.119   20.531   1.00   17.54   N       ATOM   513   CA   VAL   A   47   78.878   0.608   21.508   1.00   21.41   C       ATOM   514   C   VAL   A   47   79.262   −0.812   21.914   1.00   30.25   C       ATOM   515   C   VAL   A   47   79.192   −1.202   23.097   1.00   15.85   O       ATOM   516   CB   VAL   A   47   77.470   0.668   20.989   1.00   18.59   C       ATOM   517   CG1   VAL   A   47   76.503   0.042   22.012   1.00   16.88   C       ATOM   518   CG2   VAL   A   47   77.115   2.096   20.756   1.00   16.28   C       ATOM   519   N   HIS   A   48   79.692   −1.585   20.920   1.00   21.00   N       ATOM   520   CA   HIS   A   48   80.028   −2.969   21.192   1.00   20.17   C       ATOM   521   C   HIS   A   48   81.268   −3.079   22.117   1.00   32.98   C       ATOM   522   O   HIS   A   48   81.289   −3.850   23.102   1.00   28.20   O       ATOM   523   CB   HIS   A   48   80.063   −3.801   19.855   1.00   14.93   C       ATOM   524   CG   HIS   A   48   78.686   −4.172   19.338   1.00   26.67   C       ATOM   525   ND1   HIS   A   48   78.085   −5.394   19.600   1.00   28.83   N       ATOM   526   CD2   HIS   A   48   77.758   −3.448   18.659   1.00   25.56   C       ATOM   527   CE1   HIS   A   48   76.887   −5.430   19.043   1.00   20.08   C       ATOM   528   NE2   HIS   A   48   76.660   −4.260   18.475   1.00   25.22   N       ATOM   529   N   ASP   A   49   82.217   −2.170   21.902   1.00   22.62   N       ATOM   530   CA   ASP   A   49   83.455   −2.169   22.674   1.00   24.23   C       ATOM   531   C   ASP   A   49   83.171   −1.899   24.122   1.00   38.72   C       ATOM   532   O   ASP   A   49   83.708   −2.551   25.027   1.00   35.44   O       ATOM   533   CB   ASP   A   49   84.396   −1.112   22.127   1.00   30.29   C       ATOM   534   CG   ASP   A   49   84.991   −1.503   20.775   1.00   52.45   C       ATOM   535   OD1   ASP   A   49   85.007   −2.726   20.449   1.00   42.67   O       ATOM   536   OD2   ASP   A   49   85.416   −0.587   20.029   1.00   73.76   O       ATOM   537   N   PHE   A   50   82.294   −0.929   24.324   1.00   32.19   N       ATOM   538   CA   PHE   A   50   81.902   −0.550   25.649   1.00   29.76   C       ATOM   539   C   PHE   A   50   81.299   −1.765   26.359   1.00   30.31   C       ATOM   540   O   PHE   A   50   81.715   −2.124   27.449   1.00   29.22   O       ATOM   541   CB   PHE   A   50   80.892   0.610   25.576   1.00   23.82   C       ATOM   542   CG   PHE   A   50   80.137   0.843   26.859   1.00   19.13   C       ATOM   543   CD1   PHE   A   50   80.740   1.515   27.931   1.00   20.14   C       ATOM   544   CD2   PHE   A   50   78.835   0.360   27.018   1.00   13.99   C       ATOM   545   CE1   PHE   A   50   80.034   1.742   29.129   1.00   25.81   C       ATOM   546   CE2   PHE   A   50   78.114   0.553   28.212   1.00   22.84   C       ATOM   547   CZ   PHE   A   50   78.698   1.276   29.259   1.00   23.40   C       ATOM   548   N   PHE   A   51   80.280   −2.367   25.768   1.00   21.75   N       ATOM   549   CA   PHE   A   51   79.655   −3.451   26.457   1.00   22.61   C       ATOM   550   C   PHE   A   51   80.646   −4.603   26.612   1.00   34.01   C       ATOM   551   O   PHE   A   51   80.550   −5.401   27.590   1.00   25.28   O       ATOM   552   CB   PHE   A   51   78.389   −3.898   25.751   1.00   22.63   C       ATOM   553   CG   PHE   A   51   77.158   −3.140   26.170   1.00   27.58   C       ATOM   554   CD1   PHE   A   51   76.426   −3.525   27.280   1.00   21.78   C       ATOM   555   CD2   PHE   A   51   76.663   −2.100   25.380   1.00   19.55   C       ATOM   556   CE1   PHE   A   51   75.267   −2.796   27.662   1.00   28.34   C       ATOM   557   CE2   PHE   A   51   75.492   −1.403   25.734   1.00   14.47   C       ATOM   558   CZ   PHE   A   51   74.797   −1.744   26.878   1.00   14.55   C       ATOM   559   N   ALA   A   52   81.576   −4.706   25.659   1.00   26.43   N       ATOM   560   CA   ALA   A   52   82.587   −5.793   25.714   1.00   29.44   C       ATOM   561   C   ALA   A   52   83.687   −5.560   26.768   1.00   43.76   C       ATOM   562   O   ALA   A   52   84.502   −6.446   27.022   1.00   40.33   O       ATOM   563   CB   ALA   A   52   83.228   −6.049   24.344   1.00   24.25   C       ATOM   564   N   SER   A   53   83.702   −4.382   27.385   1.00   31.96   N       ATOM   565   CA   SER   A   53   84.705   −4.090   28.377   1.00   21.06   C       ATOM   566   C   SER   A   53   84.196   −3.625   29.709   1.00   26.41   C       ATOM   567   O   SER   A   53   84.985   −3.492   30.611   1.00   36.12   O       ATOM   568   CB   SER   A   53   85.709   −3.088   27.843   1.00   14.22   C       ATOM   569   OG   SER   A   53   85.140   −1.807   27.790   1.00   56.90   O       ATOM   570   N   GLU   A   54   82.892   −3.431   29.874   1.00   22.38   N       ATOM   571   CA   GLU   A   54   82.380   −2.893   31.139   1.00   17.27   C       ATOM   572   C   GLU   A   54   81.584   −3.735   32.118   1.00   26.32   C       ATOM   573   O   GLU   A   54   81.229   −3.281   33.191   1.00   37.43   O       ATOM   574   CB   GLU   A   54   81.677   −1.563   30.906   1.00   27.30   C       ATOM   575   CG   GLU   A   54   82.573   −0.543   30.262   1.00   44.77   C       ATOM   576   CD   GLU   A   54   83.669   −0.142   31.194   1.00   86.31   C       ATOM   577   OE1   GLU   A   54   83.392   −0.232   32.428   1.00   50.11   O       ATOM   578   OE2   GLU   A   54   84.785   0.198   30.692   1.00   50.99   O       ATOM   579   N   ARG   A   55   81.268   −4.971   31.804   1.00   29.63   N       ATOM   580   CA   ARG   A   55   80.636   −5.748   32.854   1.00   33.32   C       ATOM   581   C   ARG   A   55   79.347   −5.149   33.378   1.00   38.45   C       ATOM   582   O   ARG   A   55   79.214   −4.897   34.576   1.00   40.18   O       ATOM   583   CB   ARG   A   55   81.621   −5.875   34.045   1.00   57.61   C       ATOM   584   CG   ARG   A   55   82.666   −7.028   33.960   1.00   100.00   C       ATOM   585   CD   ARG   A   55   82.805   −7.805   35.305   1.00   100.00   C       ATOM   586   NE   ARG   A   55   82.838   −9.270   35.146   1.00   100.00   N       ATOM   587   CZ   ARG   A   55   83.206   −10.129   36.102   1.00   100.00   C       ATOM   588   NH1   ARG   A   55   83.583   −9.681   37.301   1.00   100.00   N       ATOM   589   NH2   ARG   A   55   83.208   −11.440   35.855   1.00   100.00   N       ATOM   590   N   ILE   A   56   78.367   −5.029   32.491   1.00   42.25   N       ATOM   591   CA   ILE   A   56   77.064   −4.434   32.794   1.00   25.49   C       ATOM   592   C   ILE   A   56   75.982   −5.474   33.244   1.00   20.18   C       ATOM   593   O   ILE   A   56   75.897   −6.579   32.704   1.00   24.74   O       ATOM   594   CB   ILE   A   56   76.672   −3.512   31.531   1.00   26.89   C       ATOM   595   CG1   ILE   A   56   77.643   −2.301   31.442   1.00   18.30   C       ATOM   596   CG2   ILE   A   56   75.214   −3.016   31.549   1.00   19.84   C       ATOM   597   CD1   ILE   A   56   77.998   −1.936   30.026   1.00   60.42   C       ATOM   598   N   ASP   A   57   75.166   −5.133   34.237   1.00   16.84   N       ATOM   599   CA   ASP   A   57   74.040   −5.999   34.630   1.00   16.33   C       ATOM   600   C   ASP   A   57   72.676   −5.451   34.123   1.00   28.40   C       ATOM   601   O   ASP   A   57   71.836   −6.198   33.657   1.00   25.50   O       ATOM   602   CB   ASP   A   57   74.009   −6.194   36.164   1.00   16.94   C       ATOM   603   CG   ASP   A   57   75.369   −6.720   36.703   1.00   34.27   C       ATOM   604   OD1   ASP   A   57   75.875   −7.729   36.141   1.00   31.76   O       ATOM   605   OD2   ASP   A   57   76.040   −6.007   37.499   1.00   28.36   O       ATOM   606   N   GLN   A   58   72.443   −4.152   34.220   1.00   28.91   N       ATOM   607   CA   GLN   A   58   71.183   −3.590   33.755   1.00   25.68   C       ATOM   608   C   GLN   A   58   71.425   −2.364   32.881   1.00   23.21   C       ATOM   609   O   GLN   A   58   72.403   −1.620   33.067   1.00   18.16   O       ATOM   610   CB   GLN   A   58   70.342   −3.151   34.946   1.00   33.14   C       ATOM   611   CG   GLN   A   58   69.798   −4.241   35.807   1.00   30.00   C       ATOM   612   CD   GLN   A   58   69.226   −3.712   37.105   1.00   27.18   C       ATOM   613   OE1   GLN   A   58   68.722   −2.601   37.161   1.00   31.20   O       ATOM   614   NE2   GLN   A   58   69.455   −4.436   38.186   1.00   16.89   N       ATOM   615   N   VAL   A   59   70.496   −2.138   31.961   1.00   18.35   N       ATOM   616   CA   VAL   A   59   70.562   −0.998   31.045   1.00   15.59   C       ATOM   617   C   VAL   A   59   69.238   −0.240   31.039   1.00   26.28   C       ATOM   618   O   VAL   A   59   68.178   −0.820   30.762   1.00   19.51   O       ATOM   619   CB   VAL   A   59   70.707   ‘1.456   29.601   1.00   15.32   C       ATOM   620   CG1   VAL   A   59   70.477   −0.274   28.649   1.00   11.93   C       ATOM   621   CG2   VAL   A   59   72.080   −2.111   29.364   1.00   15.83   C       ATOM   622   N   TYR   A   60   69.306   1.064   31.293   1.00   21.71   N       ATOM   623   CA   TYR   A   60   68.113   1.927   31.197   1.00   21.40   C       ATOM   624   C   TYR   A   60   68.289   2.756   29.928   1.00   18.69   C       ATOM   625   O   TYR   A   60   69.250   3.532   29.796   1.00   15.51   O       ATOM   626   CB   TYR   A   60   68.021   2.817   32.413   1.00   17.24   C       ATOM   627   CG   TYR   A   60   67.493   2.131   33.658   1.00   19.71   C       ATOM   628   CD1   TYR   A   60   68.345   1.583   34.586   1.00   21.14   C       ATOM   629   CD2   TYR   A   60   66.154   2.223   33.991   1.00   20.16   C       ATOM   630   CE1   TYR   A   60   67.835   1.080   35.794   1.00   19.11   C       ATOM   631   CE2   TYR   A   60   65.648   1.698   35.163   1.00   10.77   C       ATOM   632   CZ   TYR   A   60   66.476   1.094   36.054   1.00   20.07   C       ATOM   633   OH   TYR   A   60   65.921   0.585   37.248   1.00   16.04   O       ATOM   634   N   LEU   A   61   67.491   2.452   28.916   1.00   17.46   N       ATOM   635   CA   LEU   A   61   67.685   3.053   27.585   1.00   20.17   C       ATOM   636   C   LEU   A   61   67.003   4.412   27.409   1.00   23.36   C       ATOM   637   O   LEU   A   61   65.925   4.526   26.799   1.00   14.86   O       ATOM   638   CB   LEU   A   61   67.267   2.060   26.485   1.00   14.78   C       ATOM   639   CG   LEU   A   61   68.117   2.142   25.208   1.00   15.52   C       ATOM   640   CD1   LEU   A   61   67.815   1.010   24.109   1.00   7.75   C       ATOM   641   CD2   LEU   A   61   68.087   3.541   24.580   1.00   15.20   C       ATOM   642   N   ALA   A   62   67.656   5.434   27.956   1.00   20.35   N       ATOM   643   CA   ALA   A   62   67.120   6.784   27.963   1.00   18.55   C       ATOM   644   C   ALA   A   62   67.779   7.739   26.949   1.00   18.57   C       ATOM   645   O   ALA   A   62   67.455   8.924   26.920   1.00   24.31   O       ATOM   646   CB   ALA   A   62   67.071   7.377   29.439   1.00   11.69   C       ATOM   647   N   ALA   A   63   68.681   7.231   26.101   1.00   14.09   N       ATOM   648   CA   ALA   A   63   69.249   8.095   25.052   1.00   12.84   C       ATOM   649   C   ALA   A   63   68.310   8.005   23.877   1.00   27.00   C       ATOM   650   O   ALA   A   63   67.845   6.916   23.511   1.00   24.51   O       ATOM   651   CB   ALA   A   63   70.665   7.660   24.634   1.00   4.89   C       ATOM   652   N   ALA   A   64   68.076   9.148   23.262   1.00   21.05   N       ATOM   653   CA   ALA   A   64   67.202   9.286   22.086   1.00   13.50   C       ATOM   654   C   ALA   A   64   67.435   10.664   21.416   1.00   28.08   C       ATOM   655   O   ALA   A   64   67.987   11.600   22.021   1.00   26.63   O       ATOM   656   CB   ALA   A   64   65.642   9.171   22.518   1.00   7.63   C       ATOM   657   N   LYS   A   65   66.953   10.781   20.182   1.00   23.98   N       ATOM   658   CA   LYS   A   65   66.966   12.012   19.409   1.00   20.47   C       ATOM   659   C   LYS   A   65   65.488   12.443   19.551   1.00   24.37   C       ATOM   660   O   LYS   A   65   64.594   11.807   18.976   1.00   20.29   O       ATOM   661   CE   LYS   A   65   67.317   11.658   17.951   1.00   25.59   C       ATOM   662   CG   LYS   A   65   66.808   12.630   16.923   1.00   27.54   C       ATOM   663   CD   LYS   A   65   67.518   13.926   17.169   1.00   21.08   C       ATOM   664   CE   LYS   A   65   67.316   14.905   16.029   1.00   55.15   C       ATOM   665   NZ   LYS   A   65   67.876   16.263   16.392   1.00   81.63   N       ATOM   666   N   VAL   A   66   65.228   13.362   20.485   1.00   22.47   N       ATOM   667   CA   VAL   A   66   63.873   13.850   20.755   1.00   18.99   C       ATOM   668   C   VAL   A   66   63.711   15.343   20.394   1.00   31.44   C       ATOM   669   O   VAL   A   66   64.665   16.107   20.460   1.00   34.61   O       ATOM   670   CE   VAL   A   66   63.440   13.623   22.204   1.00   16.66   C       ATOM   671   CG1   VAL   A   66   64.269   12.623   22.869   1.00   15.01   C       ATOM   672   CG2   VAL   A   66   63.379   14.904   22.950   1.00   19.21   C       ATOM   673   N   GLY   A   67   62.514   15.755   19.994   1.00   18.03   N       ATOM   674   CA   GLY   A   67   62.298   17.149   19.614   1.00   14.90   C       ATOM   675   C   GLY   A   67   60.792   17.518   19.585   1.00   32.35   C       ATOM   676   O   GLY   A   67   59.922   16.666   19.888   1.00   18.88   O       ATOM   677   N   GLY   A   68   60.503   18.787   19.256   1.00   23.21   N       ATOM   678   CA   GLY   A   68   59.132   19.288   19.183   1.00   23.83   C       ATOM   679   C   GLY   A   68   58.540   19.137   17.771   1.00   19.31   C       ATOM   680   O   GLY   A   68   59.165   18.550   16.870   1.00   30.64   O       ATOM   681   N   ILE   A   69   57.343   19.684   17.588   1.00   15.20   N       ATOM   682   CA   ILE   A   69   56.595   19.632   16.317   1.00   16.80   C       ATOM   683   C   ILE   A   69   57.387   20.153   15.112   1.00   19.33   C       ATOM   684   O   ILE   A   69   57.425   19.519   14.061   1.00   14.66   O       ATOM   685   CE   ILE   A   69   55.257   20.432   16.480   1.00   30.11   C       ATOM   686   CG1   ILE   A   69   54.271   19.683   17.385   1.00   24.27   C       ATOM   687   CG2   ILE   A   69   54.610   20.749   15.181   1.00   47.53   C       ATOM   688   CD1   ILE   A   69   53.259   20.608   18.056   1.00   85.71   C       ATOM   689   N   VAL   A   70   58.010   21.327   15.269   1.00   23.03   N       ATOM   690   CA   VAL   A   70   58.797   21.913   14.183   1.00   19.34   C       ATOM   691   C   VAL   A   70   59.983   21.011   13.840   1.00   24.42   C       ATOM   692   O   VAL   A   70   60.335   20.829   12.662   1.00   24.14   O       ATOM   693   CB   VAL   A   70   59.304   23.404   14.467   1.00   21.37   C       ATOM   694   CG1   VAL   A   70   60.137   23.907   13.281   1.00   17.79   C       ATOM   695   CG2   VAL   A   70   58.136   24.410   14.678   1.00   15.74   C       ATOM   696   N   ALA   A   71   60.621   20.450   14.861   1.00   19.68   N       ATOM   697   CA   ALA   A   71   61.782   19.617   14.572   1.00   16.57   C       ATOM   698   C   ALA   A   71   61.427   18.289   13.910   1.00   23.36   C       ATOM   699   O   ALA   A   71   61.980   17.923   12.849   1.00   21.84   O       ATOM   700   CB   ALA   A   71   62.685   19.439   15.805   1.00   9.36   C       ATOM   701   N   ASN   A   72   60.463   17.598   14.511   1.00   16.80   N       ATOM   702   CA   ASN   A   72   59.998   16.357   13.923   1.00   18.84   C       ATOM   703   C   ASN   A   72   59.608   16.539   12.440   1.00   23.87   C       ATOM   704   O   ASN   A   72   59.919   15.696   11.593   1.00   21.52   O       ATOM   705   CB   ASN   A   72   58.835   15.806   14.738   1.00   8.60   C       ATOM   706   CG   ASN   A   72   59.309   15.013   15.911   1.00   23.75   C       ATOM   707   OD1   ASN   A   72   59.558   13.809   15.810   1.00   23.98   O       ATOM   708   ND2   ASN   A   72   59.572   15.701   16.996   1.00   9.96   N       ATOM   709   N   ASN   A   73   58.931   17.647   12.138   1.00   23.07   N       ATOM   710   CA   ASN   A   73   58.521   17.971   10.761   1.00   26.05   C       ATOM   711   C   ASN   A   73   59.665   18.454   9.817   1.00   26.95   C       ATOM   712   O   ASN   A   73   59.613   18.276   8.569   1.00   22.13   O       ATOM   713   CB   ASN   A   73   57.383   19.001   10.800   1.00   14.86   C       ATOM   714   CG   ASN   A   73   56.015   18.349   10.987   1.00   19.88   C       ATOM   715   OD1   ASN   A   73   55.620   17.468   10.217   1.00   27.02   O       ATOM   716   ND2   ASN   A   73   55.322   18.732   12.051   1.00   20.78   N       ATOM   717   N   THR   A   74   60.710   19.029   10.419   1.00   18.69   N       ATOM   718   CA   THR   A   74   61.845   19.540   9.657   1.00   10.07   C       ATOM   719   C   THR   A   74   62.968   18.548   9.375   1.00   21.00   C       ATOM   720   O   THR   A   74   63.537   18.561   8.289   1.00   11.75   O       ATOM   721   CB   THR   A   74   62.411   20.746   10.306   1.00   29.10   C       ATOM   722   OG1   THR   A   74   61.370   21.714   10.457   1.00   23.24   O       ATOM   723   CG2   THR   A   74   63.541   21.299   9.452   1.00   21.63   C       ATOM   724   N   TYR   A   75   63.230   17.636   10.310   1.00   17.10   N       ATOM   725   CA   TYR   A   75   64.267   16.620   10.112   1.00   9.07   C       ATOM   726   C   TYR   A   75   63.733   15.203   10.318   1.00   6.17   C       ATOM   727   O   TYR   A   75   64.143   14.542   11.267   1.00   15.58   O       ATOM   728   CB   TYR   A   75   65.302   16.825   11.188   1.00   11.89   C       ATOM   729   CG   TYR   A   75   65.779   18.234   11.252   1.00   27.12   C       ATOM   730   CD1   TYR   A   75   66.712   18.696   10.321   1.00   28.46   C       ATOM   731   CD2   TYR   A   75   65.234   19.151   12.173   1.00   24.83   C       ATOM   732   CE1   TYR   A   75   67.117   20.045   10.305   1.00   28.34   C       ATOM   733   CE2   TYR   A   75   65.652   20.523   12.180   1.00   21.00   C       ATOM   734   CZ   TYR   A   75   66.593   20.940   11.234   1.00   45.42   C       ATOM   735   OH   TYR   A   75   67.066   22.230   11.215   1.00   35.37   O       ATOM   736   N   PRO   A   76   62.759   14.775   9.532   1.00   13.30   N       ATOM   737   CA   PRO   A   76   62.185   13.438   9.742   1.00   14.64   C       ATOM   738   C   PRO   A   76   63.209   12.264   9.618   1.00   14.40   C       ATOM   739   O   PRO   A   76   63.157   11.335   10.409   1.00   20.54   O       ATOM   740   CB   PRO   A   76   61.055   13.366   8.709   1.00   7.83   C       ATOM   741   CG   PRO   A   76   61.447   14.388   7.617   1.00   12.61   C       ATOM   742   CD   PRO   A   76   62.068   15.504   8.455   1.00   11.18   C       ATOM   743   N   ALA   A   77   64.163   12.339   8.681   1.00   15.25   N       ATOM   744   CA   ALA   A   77   65.206   11.312   8.538   1.00   6.79   C       ATOM   745   C   ALA   A   77   66.053   11.166   9.820   1.00   17.22   C       ATOM   746   O   ALA   A   77   66.306   10.069   10.292   1.00   18.74   O       ATOM   747   CE   ALA   A   77   66.097   11.601   7.330   1.00   9.04   C       ATOM   748   N   ASP   A   78   66.466   12.267   10.424   1.00   10.92   N       ATOM   749   CA   ASP   A   78   67.256   12.191   11.659   1.00   11.87   C       ATOM   750   C   ASP   A   78   66.572   11.486   12.827   1.00   16.09   C       ATOM   751   O   ASP   A   78   67.212   10.741   13.601   1.00   18.07   O       ATOM   752   CB   ASP   A   78   67.578   13.609   12.088   1.00   19.16   C       ATOM   753   CG   ASP   A   78   68.424   14.325   11.068   1.00   26.82   C       ATOM   754   OD1   ASP   A   78   68.836   13.694   10.044   1.00   33.93   O       ATOM   755   OD2   ASP   A   78   68.673   15.514   11.316   1.00   32.06   O       ATOM   756   N   PHE   A   79   65.279   11.771   12.975   1.00   14.70   N       ATOM   757   CA   PHE   A   79   64.471   11.192   14.044   1.00   20.69   C       ATOM   758   C   PHE   A   79   64.224   9.707   13.876   1.00   20.22   C       ATOM   759   O   PHE   A   79   64.269   8.987   14.862   1.00   22.37   O       ATOM   760   CB   PHE   A   79   63.144   11.933   14.219   1.00   27.38   C       ATOM   761   CG   PHE   A   79   63.264   13.218   14.990   1.00   28.59   C       ATOM   762   CD1   PHE   A   79   63.137   13.230   16.386   1.00   27.49   C       ATOM   763   CD2   PHE   A   79   63.509   14.415   14.325   1.00   28.20   C       ATOM   764   CE1   PHE   A   79   63.281   14.413   17.109   1.00   21.76   C       ATOM   765   CE2   PHE   A   79   63.625   15.593   15.037   1.00   31.48   C       ATOM   766   CZ   PHE   A   79   63.509   15.582   16.439   1.00   26.31   C       ATOM   767   N   ILE   A   80   63.942   9.249   12.650   1.00   10.79   N       ATOM   768   CA   ILE   A   80   63.828   7.795   12.410   1.00   18.12   C       ATOM   769   C   ILE   A   80   65.197   7.052   12.432   1.00   10.97   C       ATOM   770   O   ILE   A   80   65.406   6.090   13.195   1.00   8.92   O       ATOM   771   CB   ILE   A   80   62.944   7.408   11.148   1.00   17.41   C       ATOM   772   CG1   ILE   A   80   62.651   5.886   11.105   1.00   10.16   C       ATOM   773   CG2   ILE   A   80   63.583   7.888   9.901   1.00   17.46   C       ATOM   774   CD1   ILE   A   80   61.722   5.410   9.980   1.00   7.30   C       ATOM   775   N   TYR   A   81   66.151   7.539   11.658   1.00   11.18   N       ATOM   776   CA   TYR   A   81   67.488   6.902   11.630   1.00   15.06   C       ATOM   777   C   TYR   A   81   68.237   6.782   12.959   1.00   16.83   C       ATOM   778   O   TYR   A   81   68.714   5.702   13.383   1.00   16.74   O       ATOM   779   CB   TYR   A   81   68.384   7.599   10.616   1.00   9.43   C       ATOM   780   CG   TYR   A   81   69.749   6.966   10.541   1.00   22.54   C       ATOM   781   CD1   TYR   A   81   69.963   5.824   9.747   1.00   22.37   C       ATOM   782   CD2   TYR   A   81   70.818   7.466   11.299   1.00   18.07   C       ATOM   783   CE1   TYR   A   81   71.202   5.163   9.746   1.00   15.02   C       ATOM   784   CE2   TYR   A   81   72.080   6.893   11.201   1.00   17.37   C       ATOM   785   CZ   TYR   A   81   72.255   5.698   10.472   1.00   24.27   C       ATOM   786   OH   TYR   A   81   73.491   5.063   10.409   1.00   19.57   O       ATOM   787   N   GLN   A   82   68.385   7.918   13.612   1.00   11.39   N       ATOM   788   CA   GLN   A   82   69.193   7.930   14.810   1.00   12.23   C       ATOM   789   CG   GLN   A   82   68.544   7.089   15.834   1.00   14.18   C       ATOM   790   O   GLN   A   82   69.180   6.415   16.631   1.00   11.35   O       ATOM   791   CB   GLN   A   82   69.280   9.354   15.291   1.00   18.73   C       ATOM   792   CG   GLN   A   82   69.986   10.209   14.250   1.00   13.54   C       ATOM   793   CD   GLN   A   82   70.285   11.617   14.736   1.00   26.00   C       ATOM   794   OE1   GLN   A   82   70.410   11.850   15.927   1.00   22.99   O       ATOM   795   NE2   GLN   A   82   70.404   12.561   13.808   1.00   16.59   N       ATOM   796   N   ASN   A   83   67.235   7.181   15.869   1.00   11.35   N       ATOM   797   CA   ASN   A   83   66.549   6.408   16.860   1.00   13.71   C       ATOM   798   C   ASN   A   83   66.623   4.902   16.557   1.00   21.43   C       ATOM   799   O   ASN   A   83   66.831   4.101   17.463   1.00   12.10   O       ATOM   800   CB   ASN   A   83   65.132   6.945   17.074   1.00   13.51   C       ATOM   801   CG   ASN   A   83   65.131   8.245   17.871   1.00   28.91   C       ATOM   802   OD1   ASN   A   83   65.628   8.263   18.990   1.00   22.28   O       ATOM   803   ND2   ASN   A   83   64.756   9.354   17.237   1.00   20.17   N       ATOM   804   N   MET   A   84   66.592   4.517   15.290   1.00   15.63   N       ATOM   805   CA   MET   A   84   66.704   3.101   15.007   1.00   15.66   C       ATOM   806   C   MET   A   84   68.054   2.588   15.348   1.00   14.66   C       ATOM   807   O   MET   A   84   68.148   1.514   15.902   1.00   11.45   O       ATOM   808   CB   MET   A   84   66.418   2.815   13.563   1.00   17.59   C       ATOM   809   CG   MET   A   84   64.911   2.894   13.220   1.00   14.40   C       ATOM   810   SD   MET   A   84   64.638   2.811   11.387   1.00   15.99   S       ATOM   811   CE   MET   A   84   65.164   1.105   10.952   1.00   8.90   C       ATOM   812   N   MET   A   85   69.098   3.338   15.024   1.00   11.20   N       ATOM   813   CA   MET   A   85   70.468   2.879   15.321   1.00   11.67   C       ATOM   814   C   MET   A   85   70.779   2.831   16.774   1.00   13.04   C       ATOM   815   O   MET   A   85   71.359   1.893   17.265   1.00   15.26   O       ATOM   816   CB   MET   A   85   71.525   3.798   14.693   1.00   15.07   C       ATOM   817   CG   MET   A   85   71.530   3.726   13.173   1.00   32.01   C       ATOM   818   SD   MET   A   85   71.918   2.027   12.487   1.00   37.79   S       ATOM   819   CE   MET   A   85   73.379   1.801   13.320   1.00   15.94   C       ATOM   820   N   ILE   A   86   70.471   3.892   17.481   1.00   13.92   N       ATOM   821   CA   ILE   A   86   70.760   3.893   18.912   1.00   12.58   C       ATOM   822   C   ILE   A   86   70.159   2.662   19.591   1.00   21.61   C       ATOM   823   O   ILE   A   86   70.813   1.981   20.362   1.00   18.68   O       ATOM   824   CB   ILE   A   86   70.225   5.189   19.606   1.00   11.84   C       ATOM   825   CG1   ILE   A   86   70.978   6.429   19.119   1.00   19.78   C       ATOM   826   CG2   ILE   A   86   70.435   5.132   21.112   1.00   6.59   C       ATOM   827   CD1   ILE   A   86   70.505   7.694   19.772   1.00   20.37   C       ATOM   828   N   GLU   A   87   68.893   2.383   19.316   1.00   18.78   N       ATOM   829   CA   GLU   A   87   68.263   1.237   19.930   1.00   14.00   C       ATOM   830   C   GLU   A   87   68.797   −0.116   19.454   1.00   15.93   C       ATOM   831   O   GLU   A   87   69.017   −0.991   20.268   1.00   11.04   O       ATOM   832   CB   GLU   A   87   66.734   1.324   19.900   1.00   14.89   C       ATOM   833   CG   GLU   A   87   66.085   1.327   18.538   1.00   28.96   C       ATOM   834   CD   GLU   A   87   64.635   1.922   18.544   1.00   11.12   C       ATOM   835   OE1   GLU   A   87   64.307   2.801   19.376   1.00   25.46   O       ATOM   836   OE2   GLU   A   87   63.845   1.547   17.663   1.00   29.87   O       ATOM   837   N   SER   A   88   69.054   −0.259   18.155   1.00   16.18   N       ATOM   838   CA   SER   A   88   69.650   −1.482   17.569   1.00   19.52   C       ATOM   839   C   SER   A   88   71.029   −1.792   18.160   1.00   22.54   C       ATOM   840   O   SER   A   88   71.313   −2.929   18.592   1.00   13.80   O       ATOM   841   CB   SER   A   88   69.815   −1.326   16.023   1.00   14.61   C       ATOM   842   OG   SER   A   88   68.551   −1.201   15.355   1.00   15.41   O       ATOM   843   N   ASN   A   89   71.884   −0.773   18.143   1.00   22.63   N       ATOM   844   CA   ASN   A   89   73.227   −0.869   18.693   1.00   27.23   C       ATOM   845   C   ASN   A   89   73.195   −1.363   20.134   1.00   21.34   C       ATOM   846   O   ASN   A   89   73.795   −2.384   20.476   1.00   23.68   O       ATOM   847   CB   ASN   A   89   73.980   0.487   18.597   1.00   13.71   C       ATOM   848   CG   ASN   A   89   74.440   0.825   17.168   1.00   20.40   C       ATOM   849   OD1   ASN   A   89   74.305   −0.006   16.255   1.00   14.93   O       ATOM   850   ND2   ASN   A   89   74.937   2.067   16.960   1.00   13.32   N       ATOM   851   N   ILE   A   90   72.488   −0.646   20.979   1.00   16.55   N       ATOM   852   CA   ILE   A   90   72.437   −1.014   22.398   1.00   21.51   C       ATOM   853   C   ILE   A   90   71.876   −2.421   22.729   1.00   26.50   C       ATOM   854   O   ILE   A   90   72.384   −3.159   23.590   1.00   19.71   O       ATOM   855   CB   ILE   A   90   71.670   0.070   23.233   1.00   13.32   C       ATOM   856   CG1   ILE   A   90   72.539   1.299   23.401   1.00   11.05   C       ATOM   857   CG2   ILE   A   90   71.371   −0.445   24.637   1.00   7.54   C       ATOM   858   CD1   ILE   A   90   71.749   2.597   23.668   1.00   20.71   C       ATOM   859   N   ILE   A   91   70.755   −2.733   22.114   1.00   14.98   N       ATOM   860   CA   ILE   A   91   70.047   −3.953   22.442   1.00   21.33   C       ATOM   861   C   ILE   A   91   70.927   −5.098   21.994   1.00   26.27   C       ATOM   862   O   ILE   A   91   71.211   −6.011   22.751   1.00   26.56   O       ATOM   863   CB   ILE   A   91   68.556   −3.930   21.814   1.00   20.39   C       ATOM   864   CG1   ILE   A   91   67.692   −2.886   22.552   1.00   13.51   C       ATOM   865   CG2   ILE   A   91   67.841   −5.316   21.845   1.00   11.31   C       ATOM   866   CD1   ILE   A   91   66.320   −2.648   21.907   1.00   16.23   C       ATOM   867   N   HIS   A   92   71.446   −4.983   20.785   1.00   24.12   N       ATOM   868   CA   HIS   A   92   72.293   −6.015   20.243   1.00   26.71   C       ATOM   869   C   HIS   A   92   73.609   −6.251   21.071   1.00   29.30   C       ATOM   870   O   HIS   A   92   73.983   −7.366   21.443   1.00   18.58   O       ATOM   871   CB   HIS   A   92   72.561   −5.682   18.775   1.00   22.23   C       ATOM   872   CG   HIS   A   92   73.366   −6.720   18.077   1.00   26.32   C       ATOM   873   ND1   HIS   A   92   72.798   −7.711   17.307   1.00   27.19   N       ATOM   874   CD2   HIS   A   92   74.699   −6.978   18.106   1.00   21.95   C       ATOM   875   CE1   HIS   A   92   73.755   −8.487   16.826   1.00   23.66   C       ATOM   876   NE2   HIS   A   92   74.918   −8.062   17.296   1.00   17.36   N       ATOM   877   N   ALA   A   93   74.328   −5.187   21.333   1.00   15.66   N       ATOM   878   CA   ALA   A   93   75.530   −5.301   22.110   1.00   11.88   C       ATOM   879   C   ALA   A   93   75.222   −5.900   23.512   1.00   28.78   C       ATOM   880   O   ALA   A   93   75.912   −6.790   24.037   1.00   25.23   O       ATOM   881   CB   ALA   A   93   76.139   −3.959   22.221   1.00   6.30   C       ATOM   882   N   ALA   A   94   74.142   −5.442   24.113   1.00   18.82   N       ATOM   883   CA   ALA   A   94   73.777   −5.971   25.399   1.00   15.61   C       ATOM   884   C   ALA   A   94   73.593   −7.503   25.301   1.00   28.39   C       ATOM   885   O   ALA   A   94   74.133   −8.263   26.099   1.00   21.67   O       ATOM   886   CB   ALA   A   94   72.449   −5.279   25.911   1.00   18.46   C       ATOM   887   N   HIS   A   95   72.814   −7.966   24.329   1.00   26.35   N       ATOM   888   CA   HIS   A   95   72.551   −9.396   24.271   1.00   24.89   C       ATOM   889   C   HIS   A   95   73.845   −10.176   24.140   1.00   22.81   C       ATOM   890   O   HIS   A   95   74.077   −11.136   24.865   1.00   21.44   O       ATOM   891   CB   HIS   A   95   71.571   −9.778   23.129   1.00   22.39   C       ATOM   892   CG   HIS   A   95   71.554   −11.250   22.831   1.00   28.73   C       ATOM   893   ND1   HIS   A   95   70.979   −12.182   23.682   1.00   22.83   N       ATOM   894   CD2   HIS   A   95   72.159   −11.964   21.845   1.00   25.22   C       ATOM   895   CE1   HIS   A   95   71.171   −13.397   23.196   1.00   22.72   C       ATOM   896   NE2   HIS   A   95   71.911   −13.296   22.101   1.00   24.80   N       ATOM   897   N   GLN   A   96   74.709   −9.658   23.281   1.00   19.97   N       ATOM   898   CA   GLN   A   96   75.960   −10.299   22.917   1.00   22.27   C       ATOM   899   C   GLN   A   96   76.877   −10.353   24.086   1.00   26.58   C       ATOM   900   O   GLN   A   96   77.836   −11.093   24.088   1.00   24.17   O       ATOM   901   CS   GLN   A   96   76.642   −9.492   21.818   1.00   23.38   C       ATOM   902   CG   GLN   A   96   77.043   −10.299   20.596   1.00   61.06   C       ATOM   903   CD   GLN   A   96   78.033   −9.557   19.675   1.00   75.83   C       ATOM   904   OE1   GLN   A   96   78.999   −8.941   20.131   1.00   56.89   O       ATOM   905   NE2   GLN   A   96   77.815   −9.668   18.366   1.00   100.00   N       ATOM   906   N   ASN   A   97   76.652   −9.500   25.060   1.00   22.15   N       ATOM   907   CA   ASN   A   97   77.537   −9.536   26.208   1.00   14.74   C       ATOM   908   C   ASN   A   97   76.732   −10.022   27.387   1.00   29.78   C       ATOM   909   O   ASN   A   97   77.049   −9.762   28.564   1.00   27.09   O       ATOM   910   CB   ASN   A   97   78.241   −8.201   26.462   1.00   12.93   C       ATOM   911   CG   ASN   A   97   79.260   −7.897   25.407   1.00   24.91   C       ATOM   912   OD1   ASN   A   97   80.331   −8.518   25.375   1.00   57.17   O       ATOM   913   ND2   ASN   A   97   78.839   −7.135   24.392   1.00   34.88   N       ATOM   914   N   ASP   A   98   75.666   −10.732   27.055   1.00   27.98   N       ATOM   915   CA   ASP   A   98   74.907   −11.361   28.089   1.00   29.25   C       ATOM   916   C   ASP   A   98   74.400   −10.379   29.164   1.00   37.53   C       ATOM   917   O   ASP   A   98   74.505   −10.634   30.367   1.00   36.42   O       ATOM   918   CB   ASP   A   98   75.791   −12.450   28.700   1.00   36.37   C       ATOM   919   CG   ASP   A   98   75.016   −13.712   29.053   1.00   88.62   C       ATOM   920   OD1   ASP   A   98   73.775   −13.749   28.877   1.00   82.53   O       ATOM   921   OD2   ASP   A   98   75.656   −14.670   29.542   1.00   100.00   O       ATOM   922   N   VAL   A   99   73.879   −9.235   28.730   1.00   27.13   N       ATOM   923   CA   VAL   A   99   73.157   −8.351   29.635   1.00   21.57   C       ATOM   924   C   VAL   A   99   71.706   −8.868   29.530   1.00   16.15   C       ATOM   925   O   VAL   A   99   71.159   −9.088   28.422   1.00   19.47   O       ATOM   926   CB   VAL   A   99   73.264   −6.900   29.206   1.00   24.18   C       ATOM   927   CG1   VAL   A   99   72.517   −6.015   30.198   1.00   14.58   C       ATOM   928   CG2   VAL   A   99   74.720   −6.515   29.225   1.00   30.10   C       ATOM   929   N   ASN   A   100   71.149   −9.262   30.662   1.00   17.39   N       ATOM   930   CA   ASN   A   100   69.852   −9.925   30.613   1.00   25.77   C       ATOM   931   C   ASN   A   100   68.648   −9.034   30.910   1.00   24.95   C       ATOM   932   O   ASN   A   100   67.498   −9.377   30.582   1.00   20.88   O       ATOM   933   CB   ASN   A   100   69.846   −11.157   31.527   1.00   14.98   C       ATOM   934   CG   ASN   A   100   68.724   −12.112   31.180   1.00   20.38   C       ATOM   935   OD1   ASN   A   100   68.737   −12.709   30.100   1.00   29.59   O       ATOM   936   ND2   ASN   A   100   67.716   −12.240   32.076   1.00   16.35   N       ATOM   937   N   LYS   A   101   68.941   −7.923   31.584   1.00   17.91   N       ATOM   938   CA   LYS   A   101   67.970   −6.916   31.994   1.00   25.43   C       ATOM   939   C   LYS   A   101   68.107   −5.510   31.323   1.00   25.29   C       ATOM   940   O   LYS   A   101   69.151   −4.850   31.377   1.00   19.88   O       ATOM   941   CB   LYS   A   101   67.996   −6.807   33.521   1.00   29.28   C       ATOM   942   CG   LYS   A   101   67.464   −8.054   34.205   1.00   9.31   C       ATOM   943   CD   LYS   A   101   67.218   −7.719   35.668   1.00   38.93   C       ATOM   944   CE   LYS   A   101   66.206   −6.569   35.885   1.00   13.38   C       ATOM   945   NZ   LYS   A   101   64.750   −7.006   35.825   1.00   15.26   N       ATOM   946   N   LEU   A   102   67.013   −5.043   30.732   1.00   22.22   N       ATOM   947   CA   LEU   A   102   67.003   −3.744   30.092   1.00   15.40   C       ATOM   948   C   LEU   A   102   65.612   −3.115   30.156   1.00   18.55   C       ATOM   949   O   LEU   A   102   64.590   −3.811   30.102   1.00   18.92   O       ATOM   950   CB   LEU   A   102   67.465   −3.898   28.636   1.00   11.23   C       ATOM   951   CG   LEU   A   102   67.553   −2.711   27.651   1.00   15.51   C       ATOM   952   CD1   LEU   A   102   68.628   −2.985   26.559   1.00   9.65   C       ATOM   953   CD2   LEU   A   102   66.162   −2.407   26.995   1.00   13.10   C       ATOM   954   N   LEU   A   103   65.595   −1.798   30.318   1.00   17.05   N       ATOM   955   CA   LEU   A   103   64.356   −1.036   30.265   1.00   16.23   C       ATOM   956   C   LEU   A   103   64.346   −0.072   29.046   1.00   19.65   C       ATOM   957   O   LEU   A   103   65.215   0.789   28.875   1.00   19.68   O       ATOM   958   CB   LEU   A   103   64.099   −0.289   31.562   1.00   12.28   C       ATOM   959   CG   LEU   A   103   62.686   0.259   31.594   1.00   14.13   C       ATOM   960   CD1   LEU   A   103   61.645   −0.822   31.902   1.00   10.31   C       ATOM   961   CD2   LEU   A   103   62.646   1.360   32.601   1.00   12.30   C       ATOM   962   N   PHE   A   104   63.417   −0.333   28.140   1.00   16.41   N       ATOM   963   CA   PHE   A   104   63.215   0.486   26.956   1.00   18.32   C       ATOM   964   C   PHE   A   104   62.126   1.546   27.249   1.00   21.85   C       ATOM   965   O   PHE   A   104   61.168   1.271   27.992   1.00   18.36   O       ATOM   966   CE   PHE   A   104   62.796   −0.386   25.793   1.00   9.86   C       ATOM   967   CG   PHE   A   104   62.732   0.348   24.508   1.00   16.81   C       ATOM   968   CD1   PHE   A   104   63.894   0.714   23.840   1.00   25.04   C       ATOM   969   CD2   PHE   A   104   61.511   0.795   24.005   1.00   22.59   C       ATOM   970   CE1   PHE   A   104   63.836   1.448   22.619   1.00   31.26   C       ATOM   971   CE2   PHE   A   104   61.449   1.535   22.814   1.00   15.59   C       ATOM   972   CZ   PHE   A   104   62.625   1.895   22.139   1.00   11.67   C       ATOM   973   N   LEU   A   105   62.341   2.762   26.734   1.00   20.33   N       ATOM   974   CA   LEU   A   105   61.416   3.897   26.904   1.00   18.10   C       ATOM   975   C   LEU   A   105   60.711   4.237   25.634   1.00   17.04   C       ATOM   976   O   LEU   A   105   61.315   4.680   24.665   1.00   18.83   O       ATOM   977   CB   LEU   A   105   62.178   5.146   27.214   1.00   17.49   C       ATOM   978   CG   LEU   A   105   62.434   5.544   28.644   1.00   27.17   C       ATOM   979   CD1   LEU   A   105   62.630   4.349   29.574   1.00   19.16   C       ATOM   980   CD2   LEU   A   105   63.688   6.347   28.529   1.00   23.59   C       ATOM   981   N   GLY   A   106   59.407   4.153   25.652   1.00   20.66   N       ATOM   982   CA   GLY   A   106   58.679   4.536   24.455   1.00   21.03   C       ATOM   983   C   GLY   A   106   58.080   5.935   24.597   1.00   17.32   C       ATOM   984   O   GLY   A   106   58.690   6.858   25.113   1.00   26.89   O       ATOM   985   N   SER   A   107   56.831   6.047   24.219   1.00   22.05   N       ATOM   986   CA   SER   A   107   56.177   7.317   24.288   1.00   22.12   C       ATOM   987   C   SER   A   107   54.686   7.212   23.923   1.00   19.06   C       ATOM   988   O   SER   A   107   54.314   6.545   22.963   1.00   27.42   O       ATOM   989   CB   SER   A   107   56.882   8.232   23.300   1.00   20.99   C       ATOM   990   OG   SER   A   107   55.947   9.133   22.776   1.00   42.85   O       ATOM   991   N   SER   A   108   53.826   7.890   24.671   1.00   27.42   N       ATOM   992   CA   SER   A   108   52.382   7.947   24.339   1.00   26.43   C       ATOM   993   C   SER   A   108   52.144   8.259   22.842   1.00   30.97   C       ATOM   994   O   SER   A   108   51.242   7.709   22.217   1.00   33.46   O       ATOM   995   CB   SER   A   108   51.710   9.072   25.144   1.00   19.87   C       ATOM   996   OG   SER   A   108   52.495   10.266   25.071   1.00   70.88   O       ATOM   997   N   CYS   A   109   52.927   9.180   22.278   1.00   24.73   N       ATOM   998   CA   CYS   A   109   52.728   9.549   20.880   1.00   25.61   C       ATOM   999   C   CYS   A   109   52.970   8.482   19.815   1.00   21.29   C       ATOM   1000   O   CYS   A   109   52.967   8.737   18.623   1.00   31.31   O       ATOM   1001   CB   CYS   A   109   53.369   10.899   20.544   1.00   39.55   C       ATOM   1002   SG   CYS   A   109   55.153   11.077   20.847   1.00   49.24   S       ATOM   1003   N   ILE   A   110   53.101   7.264   20.258   1.00   18.31   N       ATOM   1004   CA   ILE   A   110   53.329   6.150   19.379   1.00   28.10   C       ATOM   1005   C   ILE   A   110   51.977   5.489   19.082   1.00   15.38   C       ATOM   1006   O   ILE   A   110   51.895   4.592   18.268   1.00   16.52   O       ATOM   1007   CB   ILE   A   110   54.154   5.153   20.206   1.00   40.45   C       ATOM   1008   CG1   ILE   A   110   55.604   5.510   20.136   1.00   39.02   C       ATOM   1009   CG2   ILE   A   110   53.879   3.715   19.875   1.00   61.33   C       ATOM   1010   CD1   ILE   A   110   56.429   4.338   20.549   1.00   82.74   C       ATOM   1011   N   TYR   A   111   50.951   5.842   19.854   1.00   14.91   N       ATOM   1012   CA   TYR   A   111   49.630   5.227   19.678   1.00   13.96   C       ATOM   1013   C   TYR   A   111   48.956   5.831   18.459   1.00   20.40   C       ATOM   1014   O   TYR   A   111   49.302   6.933   18.056   1.00   11.71   O       ATOM   1015   CB   TYR   A   111   48.763   5.468   20.921   1.00   9.63   C       ATOM   1016   CG   TYR   A   111   49.117   4.550   22.065   1.00   14.94   C       ATOM   1017   CD1   TYR   A   111   48.985   3.159   21.938   1.00   9.73   C       ATOM   1018   CD2   TYR   A   111   49.755   5.038   23.216   1.00   14.96   C       ATOM   1019   CE1   TYR   A   111   49.344   2.273   23.014   1.00   6.53   C       ATOM   1020   CE2   TYR   A   111   50.146   4.155   24.272   1.00   13.66   C       ATOM   1021   CZ   TYR   A   111   49.873   2.787   24.171   1.00   17.86   C       ATOM   1022   OH   TYR   A   111   50.266   1.927   25.157   1.00   11.37   O       ATOM   1023   N   PRO   A   112   47.974   5.145   17.872   1.00   22.56   N       ATOM   1024   CA   PRO   A   112   47.279   5.743   16.721   1.00   23.44   C       ATOM   1025   C   PRO   A   112   46.589   7.111   16.988   1.00   17.82   C       ATOM   1026   O   PRO   A   112   46.197   7.453   18.115   1.00   19.72   O       ATOM   1027   CB   PRO   A   112   46.290   4.644   16.252   1.00   15.69   C       ATOM   1028   CG   PRO   A   112   46.895   3.343   16.769   1.00   22.83   C       ATOM   1029   CD   PRO   A   112   47.593   3.733   18.086   1.00   16.10   C       ATOM   1030   N   LYS   A   113   46.418   7.866   15.915   1.00   19.48   N       ATOM   1031   CA   LYS   A   113   45.793   9.167   15.994   1.00   23.50   C       ATOM   1032   C   LYS   A   113   44.396   9.077   16.655   1.00   34.28   C       ATOM   1033   O   LYS   A   113   44.046   9.887   17.524   1.00   46.14   O       ATOM   1034   CB   LYS   A   113   45.675   9.735   14.593   1.00   30.04   C       ATOM   1035   CG   LYS   A   113   46.219   11.124   14.477   1.00   43.78   C       ATOM   1036   CD   LYS   A   113   45.381   11.941   13.515   1.00   100.00   C       ATOM   1037   CE   LYS   A   113   44.361   12.836   14.250   1.00   100.00   C       ATOM   1038   NZ   LYS   A   113   43.480   13.625   13.304   1.00   100.00   N       ATOM   1039   N   LEU   A   114   43.591   8.103   16.250   1.00   26.33   N       ATOM   1040   CA   LEU   A   114   42.267   7.957   16.833   1.00   20.65   C       ATOM   1041   C   LEU   A   114   42.083   6.792   17.760   1.00   18.44   C       ATOM   1042   O   LEU   A   114   41.002   6.278   17.918   1.00   34.04   O       ATOM   1043   CB   LEU   A   114   41.194   8.002   15.780   1.00   24.37   C       ATOM   1044   CG   LEU   A   114   41.587   9.122   14.830   1.00   40.86   C       ATOM   1045   CD1   LEU   A   114   40.991   8.797   13.504   1.00   49.29   C       ATOM   1046   CD2   LEU   A   114   41.139   10.512   15.300   1.00   26.85   C       ATOM   1047   N   ALA   A   115   43.103   6.473   18.527   1.00   29.00   N       ATOM   1048   CA   ALA   A   115   42.920   5.446   19.528   1.00   25.66   C       ATOM   1049   C   ALA   A   115   41.722   5.727   20.454   1.00   28.76   C       ATOM   1050   O   ALA   A   115   41.364   6.855   20.682   1.00   24.12   O       ATOM   1051   CE   ALA   A   115   44.177   5.272   20.326   1.00   16.86   C       ATOM   1052   N   LYS   A   116   41.137   4.675   20.998   1.00   30.21   N       ATOM   1053   CA   LYS   A   116   40.036   4.792   21.928   1.00   25.85   C       ATOM   1054   C   LYS   A   116   40.668   5.248   23.195   1.00   14.18   C       ATOM   1055   O   LYS   A   116   41.750   4.781   23.535   1.00   23.51   O       ATOM   1056   CE   LYS   A   116   39.369   3.415   22.116   1.00   22.05   C       ATOM   1057   CG   LYS   A   116   39.053   3.032   23.524   1.00   55.38   C       ATOM   1058   CD   LYS   A   116   37.963   1.955   23.549   1.00   100.00   C       ATOM   1059   CE   LYS   A   116   37.120   1.953   24.835   1.00   100.00   C       ATOM   1060   NZ   LYS   A   116   35.767   1.310   24.630   1.00   100.00   N       ATOM   1061   N   GLN   A   117   40.021   6.208   23.856   1.00   18.23   N       ATOM   1062   CA   GLN   A   117   40.456   6.757   25.180   1.00   21.01   C       ATOM   1063   C   GLN   A   117   39.695   6.178   26.383   1.00   30.96   C       ATOM   1064   O   GLN   A   117   38.483   6.009   26.345   1.00   27.66   O       ATOM   1065   CB   GLN   A   117   40.215   8.263   25.179   1.00   11.32   C       ATOM   1066   CG   GLN   A   117   40.849   8.912   23.948   1.00   12.12   C       ATOM   1067   CD   GLN   A   117   42.404   8.823   23.954   1.00   24.10   C       ATOM   1068   OE1   GLN   A   117   43.041   8.628   22.896   1.00   47.88   O       ATOM   1069   NE2   GLN   A   117   43.001   8.953   25.131   1.00   14.24   N       ATOM   1070   N   PRO   A   118   40.374   5.992   27.499   1.00   30.02   N       ATOM   1071   CA   PRO   A   118   41.826   6.194   27.655   1.00   26.44   C       ATOM   1072   C   PRO   A   118   42.450   5.050   26.899   1.00   24.37   C       ATOM   1073   O   PRO   A   118   41.792   4.027   26.726   1.00   25.34   O       ATOM   1074   CB   PRO   A   118   42.055   5.994   29.167   1.00   23.89   C       ATOM   1075   CG   PRO   A   118   40.847   5.240   29.654   1.00   23.20   C       ATOM   1076   CD   PRO   A   118   39.695   5.519   28.709   1.00   15.79   C       ATOM   1077   N   MET   A   119   43.684   5.228   26.432   1.00   16.00   N       ATOM   1078   CA   MET   A   119   44.372   4.215   25.644   1.00   10.80   C       ATOM   1079   C   MET   A   119   45.062   3.083   26.444   1.00   23.61   C       ATOM   1080   O   MET   A   119   46.013   3.281   27.209   1.00   18.02   O       ATOM   1081   CB   MET   A   119   45.384   4.894   24.791   1.00   13.52   C       ATOM   1082   CG   MET   A   119   44.801   6.014   23.989   1.00   18.52   C       ATOM   1083   SD   MET   A   119   46.157   7.054   23.271   1.00   26.27   S       ATOM   1084   CE   MET   A   119   46.264   6.524   21.845   1.00   33.79   C       ATOM   1085   N   ALA   A   120   44.559   1.875   26.271   1.00   26.64   N       ATOM   1086   CA   ALA   A   120   45.177   0.712   26.884   1.00   29.17   C       ATOM   1087   C   ALA   A   120   46.356   0.308   25.984   1.00   23.21   C       ATOM   1088   O   ALA   A   120   46.439   0.759   24.833   1.00   20.19   O       ATOM   1089   CB   ALA   A   120   44.169   −0.419   26.944   1.00   26.02   C       ATOM   1090   N   GLU   A   121   47.238   −0.553   26.507   1.00   12.30   N       ATOM   1091   CA   GLU   A   121   48.427   −1.009   25.788   1.00   9.45   C       ATOM   1092   C   GLU   A   121   48.070   −1.697   24.450   1.00   11.68   C       ATOM   1093   O   GLU   A   121   48.828   −1.670   23.450   1.00   14.84   O       ATOM   1094   CB   GLU   A   121   49.321   −1.883   26.715   1.00   16.74   C       ATOM   1095   CG   GLU   A   121   50.132   −1.122   27.763   1.00   18.14   C       ATOM   1096   CD   GLU   A   121   49.458   −1.000   29.137   1.00   13.00   C       ATOM   1097   OE1   GLU   A   121   48.252   −1.294   29.276   1.00   20.79   O       ATOM   1098   OE2   GLU   A   121   50.123   −0.521   30.080   1.00   17.86   O       ATOM   1099   N   SER   A   122   46.887   −2.273   24.409   1.00   11.79   N       ATOM   1100   CA   SER   A   122   46.427   −2.977   23.218   1.00   12.16   C       ATOM   1101   C   SER   A   122   46.030   −2.058   22.100   1.00   11.70   C       ATOM   1102   O   SER   A   122   45.717   −2.529   21.010   1.00   13.91   O       ATOM   1103   CB   SER   A   122   45.186   −3.781   23.568   1.00   21.50   C       ATOM   1104   OG   SER   A   122   44.143   −2.908   23.976   1.00   28.52   O       ATOM   1105   N   GLU   A   123   46.041   −0.754   22.341   1.00   14.65   N       ATOM   1106   CA   GLU   A   123   45.783   0.202   21.243   1.00   17.15   C       ATOM   1107   C   GLU   A   123   46.959   0.313   20.240   1.00   11.48   C       ATOM   1108   O   GLU   A   123   46.821   0.844   19.141   1.00   11.19   O       ATOM   1109   CB   GLU   A   123   45.481   1.600   21.805   1.00   21.66   C       ATOM   1110   CG   GLU   A   123   44.127   1.694   22.523   1.00   24.68   C       ATOM   1111   CD   GLU   A   123   42.984   1.374   21.585   1.00   35.56   C       ATOM   1112   OE1   GLU   A   123   43.019   1.865   20.426   1.00   41.73   O       ATOM   1113   OE2   GLU   A   123   42.158   0.497   21.940   1.00   100.00   O       ATOM   1114   N   LEU   A   124   48.134   −0.185   20.618   1.00   14.02   N       ATOM   1115   CA   LEU   A   124   49.296   −0.082   19.740   1.00   15.32   C       ATOM   1116   C   LEU   A   124   49.082   −0.754   18.458   1.00   17.76   C       ATOM   1117   O   LEU   A   124   48.752   −1.917   18.445   1.00   18.91   O       ATOM   1118   CB   LEU   A   124   50.564   −0.680   20.362   1.00   18.07   C       ATOM   1119   CG   LEU   A   124   51.922   −0.222   19.803   1.00   21.52   C       ATOM   1120   CD1   LEU   A   124   52.080   1.258   20.117   1.00   20.35   C       ATOM   1121   CD2   LEU   A   124   53.042   −0.919   20.550   1.00   14.07   C       ATOM   1122   N   LEU   A   125   49.514   −0.0711   7.409   1.00   18.44   N       ATOM   1123   CA   LEU   A   125   49.445   −0.564   16.052   1.00   19.92   C       ATOM   1124   C   LEU   A   125   48.034   −0.754   15.509   1.00   25.56   C       ATOM   1125   O   LEU   A   125   47.854   −1.188   14.364   1.00   18.26   O       ATOM   1126   CE   LEU   A   125   50.355   −1.800   15.840   1.00   20.79   C       ATOM   1127   CG   LEU   A   125   51.890   −1.511   15.778   1.00   17.21   C       ATOM   1128   CD1   LEU   A   125   52.744   −2.649   16.316   1.00   19.95   C       ATOM   1129   CD2   LEU   A   125   52.334   −1.219   14.338   1.00   5.81   C       ATOM   1130   N   GLN   A   126   47.027   −0.327   16.276   1.00   21.97   N       ATOM   1131   CA   GLN   A   126   45.652   −0.504   15.790   1.00   19.97   C       ATOM   1132   C   GLN   A   126   45.213   0.447   14.724   1.00   28.31   C       ATOM   1133   O   GLN   A   126   44.076   0.391   14.293   1.00   47.49   O       ATOM   1134   CE   GLN   A   126   44.652   −0.404   16.911   1.00   19.87   C       ATOM   1135   CG   GLN   A   126   44.949   −1.312   18.048   1.00   18.39   C       ATOM   1136   CD   GLN   A   126   44.319   −2.626   17.835   1.00   66.80   C       ATOM   1137   OE1   GLN   A   126   44.064   −3.376   18.792   1.00   40.75   O       ATOM   1138   NE2   GLN   A   126   44.015   −2.952   16.565   1.00   71.74   N       ATOM   1139   N   GLY   A   127   46.080   1.330   14.270   1.00   28.29   N       ATOM   1140   CA   GLY   A   127   45.627   2.260   13.252   1.00   23.31   C       ATOM   1141   C   GLY   A   127   46.662   3.315   12.953   1.00   22.90   C       ATOM   1142   O   GLY   A   127   47.755   3.254   13.474   1.00   25.30   O       ATOM   1143   N   THR   A   128   46.311   4.219   12.046   1.00   19.51   N       ATOM   1144   CA   THR   A   128   47.149   5.314   11.588   1.00   22.12   C       ATOM   1145   C   THR   A   128   47.705   6.219   12.695   1.00   22.60   C       ATOM   1146   O   THR   A   128   47.061   6.461   13.731   1.00   18.58   O       ATOM   1147   CB   THR   A   128   46.392   6.182   10.544   1.00   35.98   C       ATOM   1148   OG1   THR   A   128   46.533   5.594   9.239   1.00   58.05   O       ATOM   1149   CG2   THR   A   128   46.942   7.639   10.542   1.00   43.41   C       ATOM   1150   N   LEU   A   129   48.907   6.715   12.425   1.00   18.32   N       ATOM   1151   CA   LEU   A   129   49.674   7.534   13.356   1.00   16.76   C       ATOM   1152   C   LEU   A   129   49.504   8.959   12.967   1.00   4.89   C       ATOM   1153   O   LEU   A   129   49.232   9.260   11.814   1.00   16.14   O       ATOM   1154   CB   LEU   A   129   51.205   7.191   13.261   1.00   17.91   C       ATOM   1155   CG   LEU   A   129   51.769   5.804   13.752   1.00   18.21   C       ATOM   1156   CD1   LEU   A   129   53.132   5.379   13.193   1.00   12.12   C       ATOM   1157   CD2   LEU   A   129   51.683   5.532   15.251   1.00   3.89   C       ATOM   1158   N   GLU   A   130   49.816   9.827   13.917   1.00   10.23   N       ATOM   1159   CA   GLU   A   130   49.912   11.268   13.691   1.00   13.22   C       ATOM   1160   C   GLU   A   130   51.128   11.544   12.775   1.00   23.44   C       ATOM   1161   O   GLU   A   130   52.249   11.162   13.090   1.00   21.23   O       ATOM   1162   CB   GLU   A   130   50.150   11.979   15.035   1.00   18.48   C       ATOM   1163   CG   GLU   A   130   50.754   13.376   14.886   1.00   77.44   C       ATOM   1164   CD   GLU   A   130   49.833   14.328   14.121   1.00   100.00   C       ATOM   1165   OR1   GLU   A   130   48.588   14.205   14.340   1.00   36.19   O       ATOM   1166   OE2   GLU   A   130   50.347   15.161   13.295   1.00   21.03   O       ATOM   1167   N   PRO   A   131   50.920   12.219   11.648   1.00   21.35   N       ATOM   1168   CA   PRO   A   131   52.023   12.409   10.731   1.00   14.78   C       ATOM   1169   C   PRO   A   131   53.201   13.132   11.265   1.00   14.98   C       ATOM   1170   O   PRO   A   131   54.325   12.847   10.853   1.00   20.99   O       ATOM   1171   CB   PRO   A   131   51.413   13.154   9.552   1.00   14.76   C       ATOM   1172   CG   PRO   A   131   50.071   13.485   9.949   1.00   20.99   C       ATOM   1173   CD   PRO   A   131   49.641   12.626   11.047   1.00   17.25   C       ATOM   1174   N   THR   A   132   52.986   14.095   12.159   1.00   18.77   N       ATOM   1175   CA   THR   A   132   54.131   14.838   12.689   1.00   16.44   C       ATOM   1176   C   THR   A   132   55.102   13.951   13.408   1.00   21.91   C       ATOM   1177   O   THR   A   132   56.317   14.088   13.234   1.00   24.17   O       ATOM   1178   CB   THR   A   132   53.716   15.907   13.606   1.00   23.45   C       ATOM   1179   OG1   THR   A   132   52.976   16.883   12.850   1.00   31.15   O       ATOM   1180   CG2   THR   A   132   54.969   16.519   14.341   1.00   9.28   C       ATOM   1181   N   ASN   A   133   54.551   12.970   14.122   1.00   28.59   N       ATOM   1182   CA   ASN   A   133   55.359   12.007   14.875   1.00   26.38   C       ATOM   1183   C   ASN   A   133   55.666   10.682   14.207   1.00   14.85   C       ATOM   1184   O   ASN   A   133   56.446   9.884   14.755   1.00   18.67   O       ATOM   1185   CE   ASN   A   133   54.661   11.699   16.168   1.00   23.70   C       ATOM   1186   CG   ASN   A   133   54.480   12.894   16.968   1.00   50.55   C       ATOM   1187   OD1   ASN   A   133   53.354   13.272   17.252   1.00   40.07   O       ATOM   1188   ND2   ASN   A   133   55.568   13.638   17.163   1.00   40.36   N       ATOM   1189   N   GLU   A   134   55.100   10.469   13.022   1.00   9.98   N       ATOM   1190   CA   GLU   A   134   55.237   9.210   12.365   1.00   9.66   C       ATOM   1191   C   GLU   A   134   56.648   8.530   12.274   1.00   13.86   C       ATOM   1192   O   GLU   A   134   56.814   7.388   12.706   1.00   22.89   O       ATOM   1193   CB   GLU   A   134   54.448   9.200   11.070   1.00   17.55   C       ATOM   1194   CG   GLU   A   134   54.750   7.930   10.227   1.00   20.89   C       ATOM   1195   CD   GLU   A   134   53.926   7.868   8.970   1.00   13.59   C       ATOM   1196   OE1   GLU   A   134   52.678   7.738   9.085   1.00   35.28   O       ATOM   1197   OE2   GLU   A   134   54.497   8.048   7.869   1.00   13.44   O       ATOM   1198   N   PRO   A   135   57.680   9.222   11.789   1.00   15.72   N       ATOM   1199   CA   PRO   A   135   59.014   8.600   11.699   1.00   18.91   C       ATOM   1200   C   PRO   A   135   59.544   8.174   13.073   1.00   18.68   C       ATOM   1201   O   PRO   A   135   60.072   7.069   13.271   1.00   15.69   O       ATOM   1202   CB   PRO   A   135   59.896   9.755   11.169   1.00   13.84   C       ATOM   1203   CG   PRO   A   135   59.036   10.514   10.350   1.00   9.78   C       ATOM   1204   CD   PRO   A   135   57.594   10.395   10.908   1.00   14.43   C       ATOM   1205   N   TYR   A   136   59.449   9.117   13.994   1.00   8.64   N       ATOM   1206   CA   TYR   A   136   59.873   8.915   15.324   1.00   13.27   C       ATOM   1207   C   TYR   A   136   59.056   7.728   15.907   1.00   16.84   C       ATOM   1208   O   TYR   A   136   59.578   6.903   16.658   1.00   12.90   O       ATOM   1209   CB   TYR   A   136   59.604   10.234   16.100   1.00   15.51   C       ATOM   1210   CG   TYR   A   136   59.912   10.168   17.614   1.00   18.26   C       ATOM   1211   CD1   TYR   A   136   61.200   10.062   18.072   1.00   20.53   C       ATOM   1212   CD2   TYR   A   136   58.904   10.150   18.568   1.00   17.38   C       ATOM   1213   CE1   TYR   A   136   61.484   9.959   19.440   1.00   30.44   C       ATOM   1214   CE2   TYR   A   136   59.184   10.084   19.953   1.00   9.85   C       ATOM   1215   CZ   TYR   A   136   60.476   9.949   20.377   1.00   20.65   C       ATOM   1216   OH   TYR   A   136   60.792   9.873   21.734   1.00   24.41   O       ATOM   1217   N   ALA   A   137   57.760   7.687   15.638   1.00   7.19   N       ATOM   1218   CA   ALA   A   137   56.923   6.633   16.227   1.00   12.68   C       ATOM   1219   C   ALA   A   137   57.345   5.265   15.737   1.00   15.21   C       ATOM   1220   O   ALA   A   137   57.425   4.272   16.488   1.00   14.58   O       ATOM   1221   CB   ALA   A   137   55.517   6.849   15.871   1.00   11.40   C       ATOM   1222   N   ILE   A   138   57.567   5.213   14.447   1.00   8.93   N       ATOM   1223   CA   ILE   A   138   57.954   3.971   13.831   1.00   11.77   C       ATOM   1224   C   ILE   A   138   59.246   3.494   14.492   1.00   16.20   C       ATOM   1225   O   ILE   A   138   59.307   2.377   14.970   1.00   13.79   O       ATOM   1226   CB   ILE   A   138   58.064   4.172   12.316   1.00   17.85   C       ATOM   1227   CG1   ILE   A   138   56.680   4.473   11.757   1.00   28.21   C       ATOM   1228   CG2   ILE   A   138   58.674   2.986   11.602   1.00   9.81   C       ATOM   1229   CD1   ILE   A   138   55.695   3.376   11.970   1.00   18.17   C       ATOM   1230   N   ALA   A   139   60.243   4.361   14.625   1.00   11.54   N       ATOM   1231   CA   ALA   A   139   61.494   3.937   15.288   1.00   13.22   C       ATOM   1232   C   ALA   A   139   61.256   3.364   16.675   1.00   18.73   C       ATOM   1233   O   ALA   A   139   61.791   2.318   17.031   1.00   20.44   O       ATOM   1234   CB   ALA   A   139   62.434   5.073   15.390   1.00   13.62   C       ATOM   1235   N   LYS   A   140   60.397   4.033   17.448   1.00   16.36   N       ATOM   1236   CA   LYS   A   140   60.083   3.600   18.815   1.00   15.14   C       ATOM   1237   C   LYS   A   140   59.392   2.262   18.824   1.00   15.18   C       ATOM   1238   O   LYS   A   140   59.824   1.346   19.475   1.00   21.42   O       ATOM   1239   CB   LYS   A   140   59.193   4.606   19.525   1.00   17.86   C       ATOM   1240   CG   LYS   A   140   59.925   5.806   20.152   1.00   21.11   C       ATOM   1241   CD   LYS   A   140   61.208   5.478   20.958   1.00   16.75   C       ATOM   1242   CE   LYS   A   140   61.664   6.735   21.835   1.00   10.06   C       ATOM   1243   NZ   LYS   A   140   62.688   6.496   22.921   1.00   14.40   N       ATOM   1244   N   ILE   A   141   58.356   2.116   18.027   1.00   11.49   N       ATOM   1245   CA   ILE   A   141   57.703   0.828   17.977   1.00   17.92   C       ATOM   1246   C   ILE   A   141   58.729   −0.282   17.577   1.00   13.46   C       ATOM   1247   O   ILE   A   141   58.730   −1.374   18.148   1.00   13.92   O       ATOM   1248   CB   ILE   A   141   56.497   0.925   17.019   1.00   22.59   C       ATOM   1249   CG1   ILE   A   141   55.466   1.906   17.557   1.00   17.61   C       ATOM   1250   CG2   ILE   A   141   55.863   −0.411   16.700   1.00   10.49   C       ATOM   1251   CD1   ILE   A   141   54.530   2.327   16.449   1.00   13.43   C       ATOM   1252   N   ALA   A   142   59.637   0.028   16.650   1.00   10.29   N       ATOM   1253   CA   ALA   A   142   60.657   −0.931   16.228   1.00   7.15   C       ATOM   1254   C   ALA   A   142   61.456   −1.301   17.456   1.00   16.58   C       ATOM   1255   O   ALA   A   142   61.839   −2.454   17.621   1.00   13.04   O       ATOM   1256   CB   ALA   A   142   61.604   −0.288   15.130   1.00   4.44   C       ATOM   1257   N   GLY   A   143   61.703   −0.307   18.316   1.00   9.56   N       ATOM   1258   CA   GLY   A   143   62.448   −0.525   19.527   1.00   5.15   C       ATOM   1259   C   GLY   A   143   61.770   −1.555   20.430   1.00   16.36   C       ATOM   1260   O   GLY   A   143   62.392   −2.482   20.967   1.00   14.11   O       ATOM   1261   N   ILE   A   144   60.476   −1.418   20.564   1.00   20.33   N       ATOM   1262   CA   ILE   A   144   59.725   −2.314   21.407   1.00   15.35   C       ATOM   1263   C   ILE   A   144   59.706   −3.732   20.859   1.00   19.84   C       ATOM   1264   O   ILE   A   144   59.836   −4.700   21.608   1.00   17.93   O       ATOM   1265   CE   ILE   A   144   58.317   −1.819   21.559   1.00   10.60   C       ATOM   1266   CG1   ILE   A   144   58.311   −0.610   22.516   1.00   9.80   C       ATOM   1267   CG2   ILE   A   144   57.410   −2.928   22.122   1.00   9.60   C       ATOM   1268   CD1   ILE   A   144   57.022   0.076   22.517   1.00   18.32   C       ATOM   1269   N   LYS   A   145   59.520   −3.841   19.556   1.00   7.20   N       ATOM   1270   CA   LYS   A   145   59.459   −5.139   18.926   1.00   7.64   C       ATOM   1271   C   LYS   A   145   60.840   −5.788   18.931   1.00   15.32   C       ATOM   1272   O   LYS   A   145   60.923   −6.989   18.981   1.00   14.76   O       ATOM   1273   CB   LYS   A   145   58.891   −5.001   17.516   1.00   11.25   C       ATOM   1274   CG   LYS   A   145   57.414   −4.581   17.489   1.00   12.13   C       ATOM   1275   CD   LYS   A   145   56.642   −5.434   18.495   1.00   25.23   C       ATOM   1276   CE   LYS   A   145   55.189   −4.995   18.692   1.00   13.64   C       ATOM   1277   NZ   LYS   A   145   54.441   −6.111   19.392   1.00   11.94   N       ATOM   1278   N   LEU   A   146   61.934   −5.011   18.986   1.00   26.98   N       ATOM   1279   CA   LEU   A   146   63.261   −5.642   19.167   1.00   19.72   C       ATOM   1280   C   LEU   A   146   63.262   −6.316   20.542   1.00   18.20   C       ATOM   1281   O   LEU   A   146   63.590   −7.511   20.703   1.00   19.86   O       ATOM   1282   CE   LEU   A   146   64.398   −4.618   19.150   1.00   13.56   C       ATOM   1283   CG   LEU   A   146   64.895   −4.258   17.759   1.00   21.84   C       ATOM   1284   CD1   LEU   A   146   65.672   −2.945   17.817   1.00   17.94   C       ATOM   1285   CD2   LEU   A   146   65.745   −5.397   17.102   1.00   16.10   C       ATOM   1286   N   CYS   A   147   62.931   −5.523   21.548   1.00   7.91   N       ATOM   1287   CA   CYS   A   147   62.875   −6.064   22.893   1.00   9.14   C       ATOM   1288   C   CYS   A   147   62.072   −7.378   22.945   1.00   22.72   C       ATOM   1289   O   CYS   A   147   62.568   −8.401   23.383   1.00   16.90   O       ATOM   1290   CE   CYS   A   147   62.232   −5.058   23.809   1.00   12.63   C       ATOM   1291   SG   CYS   A   147   63.411   −3.823   24.316   1.00   15.02   S       ATOM   1292   N   GLU   A   148   60.823   −7.352   22.508   1.00   20.03   N       ATOM   1293   CA   GLU   A   148   60.016   −8.555   22.567   1.00   16.09   C       ATOM   1294   C   GLU   A   148   60.685   −9.715   21.802   1.00   22.61   C       ATOM   1295   O   GLU   A   148   60.651   −10.888   22.226   1.00   12.05   O       ATOM   1296   CE   GLU   A   148   58.597   −8.268   22.046   1.00   14.66   C       ATOM   1297   CG   GLU   A   148   57.864   −7.189   22.840   1.00   11.45   C       ATOM   1298   CD   GLU   A   148   56.471   −6.821   22.277   1.00   11.75   C       ATOM   1299   OE1   GLU   A   148   56.117   −7.055   21.080   1.00   11.65   O       ATOM   1300   OE2   GLU   A   148   55.728   −6.231   23.081   1.00   22.56   O       ATOM   1301   N   SER   A   149   61.368   −9.377   20.715   1.00   15.57   N       ATOM   1302   CA   SER   A   149   61.938   −10.428   19.887   1.00   10.21   C       ATOM   1303   C   SER   A   149   63.040   −11.245   20.502   1.00   15.83   C       ATOM   1304   O   SER   A   149   63.102   −12.458   20.291   1.00   12.72   O       ATOM   1305   CB   SER   A   149   62.270   −9.936   18.488   1.00   9.44   C       ATOM   1306   OG   SER   A   149   61.053   −9.650   17.782   1.00   15.91   O       ATOM   1307   N   TYR   A   150   63.910   −10.546   21.224   1.00   18.44   N       ATOM   1308   CA   TYR   A   150   65.065   −11.100   21.948   1.00   20.50   C       ATOM   1309   C   TYR   A   150   64.514   −11.848   23.158   1.00   21.87   C       ATOM   1310   O   TYR   A   150   64.939   −12.949   23.486   1.00   31.39   O       ATOM   1311   CB   TYR   A   150   66.005   −9.950   22.425   1.00   13.71   C       ATOM   1312   CG   TYR   A   150   66.994   −9.509   21.365   1.00   14.13   C       ATOM   1313   CD1   TYR   A   150   66.611   −8.673   20.317   1.00   14.64   C       ATOM   1314   CD2   TYR   A   150   68.288   −10.000   21.360   1.00   18.32   C       ATOM   1315   CE1   TYR   A   150   67.487   −8.390   19.278   1.00   11.91   C       ATOM   1316   CE2   TYR   A   150   69.198   −9.682   20.345   1.00   11.10   C       ATOM   1317   CZ   TYR   A   150   68.804   −8.900   19.326   1.00   20.95   C       ATOM   1318   OH   TYR   A   150   69.739   −8.685   18.333   1.00   27.73   O       ATOM   1319   N   ASN   A   151   63.536   −11.249   23.801   1.00   14.83   N       ATOM   1320   CA   ASN   A   151   62.903   −11.889   24.937   1.00   23.62   C       ATOM   1321   C   ASN   A   151   62.417   −13.244   24.410   1.00   28.53   C       ATOM   1322   O   ASN   A   151   62.630   −14.248   25.072   1.00   25.89   O       ATOM   1323   CB   ASN   A   151   61.655   −11.113   25.439   1.00   20.95   C       ATOM   1324   CG   ASN   A   151   61.988   −9.867   26.284   1.00   15.07   C       ATOM   1325   OD1   ASN   A   151   61.126   −9.020   26.466   1.00   26.72   O       ATOM   1326   ND2   ASN   A   151   63.231   −9.709   26.700   1.00   6.31   N       ATOM   1327   N   ARG   A   152   61.731   −13.249   23.259   1.00   19.91   N       ATOM   1328   CA   ARG   A   152   61.129   −14.465   22.687   1.00   17.62   C       ATOM   1329   C   ARG   A   152   62.090   −15.523   22.188   1.00   21.34   C       ATOM   1330   O   ARG   A   152   61.959   −16.687   22.542   1.00   15.44   O       ATOM   1331   CB   ARG   A   152   60.086   −14.148   21.610   1.00   15.30   C       ATOM   1332   CG   ARG   A   152   58.672   −13.754   22.157   1.00   17.22   C       ATOM   1333   CD   ARG   A   152   57.652   −13.297   21.049   1.00   9.11   C       ATOM   1334   NE   ARG   A   152   57.161   −14.419   20.241   1.00   21.05   N       ATOM   1335   CZ   ARG   A   152   57.159   −14.447   18.912   1.00   28.61   C       ATOM   1336   NH1   ARG   A   152   57.590   −13.387   18.221   1.00   21.98   N       ATOM   1337   NH2   ARG   A   152   56.717   −15.528   18.262   1.00   26.11   N       ATOM   1338   N   GLN   A   153   63.098   −15.104   21.434   1.00   16.54   N       ATOM   1339   CA   GLN   A   153   64.044   −16.036   20.842   1.00   9.74   C       ATOM   1340   C   GLN   A   153   65.082   −16.443   21.807   1.00   16.70   C       ATOM   1341   O   GLN   A   153   65.529   −17.545   21.763   1.00   24.35   O       ATOM   1342   CB   GLN   A   153   64.789   −15.372   19.714   1.00   8.99   C       ATOM   1343   CG   GLN   A   153   65.935   −16.225   19.116   1.00   4.63   C       ATOM   1344   CD   GLN   A   153   66.315   −15.637   17.762   1.00   14.17   C       ATOM   1345   OE1   GLN   A   153   65.611   −14.763   17.254   1.00   12.53   O       ATOM   1346   NE2   GLN   A   153   67.466   −16.024   17.228   1.00   13.38   N       ATOM   1347   N   TYR   A   154   65.566   −15.518   22.608   1.00   14.35   N       ATOM   1348   CA   TYR   A   154   66.677   −15.839   23.483   1.00   12.16   C       ATOM   1349   C   TYR   A   154   66.323   −15.930   24.954   1.00   19.06   C       ATOM   1350   O   TYR   A   154   67.185   −16.207   25.777   1.00   25.59   O       ATOM   1351   CB   TYR   A   154   67.829   −14.816   23.326   1.00   16.89   C       ATOM   1352   CG   TYR   A   154   68.418   −14.733   21.943   1.00   17.53   C       ATOM   1353   CD1   TYR   A   154   69.259   −15.726   21.467   1.00   18.91   C       ATOM   1354   CD2   TYR   A   154   68.080   −13.712   21.091   1.00   13.97   C       ATOM   1355   CE1   TYR   A   154   69.782   −15.686   20.190   1.00   10.98   C       ATOM   1356   CE2   TYR   A   154   68.621   −13.639   19.806   1.00   23.81   C       ATOM   1357   CZ   TYR   A   154   69.488   −14.634   19.380   1.00   23.08   C       ATOM   1358   OH   TYR   A   154   70.002   −14.619   18.118   1.00   23.87   O       ATOM   1359   N   GLY   A   155   65.080   −15.686   25.313   1.00   12.08   N       ATOM   1360   CA   GLY   A   155   64.747   −15.702   26.731   1.00   15.80   C       ATOM   1361   C   GLY   A   155   65.323   −14.498   27.580   1.00   33.97   C       ATOM   1362   O   GLY   A   155   65.491   −14.640   28.789   1.00   25.76   O       ATOM   1363   N   ARG   A   156   65.564   −13.318   26.981   1.00   25.91   N       ATOM   1364   CA   ARG   A   156   66.066   −12.146   27.734   1.00   14.13   C       ATOM   1365   C   ARG   A   156   64.971   −11.486   28.581   1.00   16.23   C       ATOM   1366   O   ARG   A   156   63.802   −11.919   28.583   1.00   22.61   O       ATOM   1367   CB   ARG   A   156   66.601   −11.124   26.750   1.00   13.16   C       ATOM   1368   CG   ARG   A   156   67.875   −11.570   26.099   1.00   15.18   C       ATOM   1369   CD   ARG   A   156   68.930   −11.418   27.121   1.00   26.42   C       ATOM   1370   NE   ARG   A   156   70.200   −11.912   26.633   1.00   21.25   N       ATOM   1371   CZ   ARG   A   156   71.092   −12.555   27.386   1.00   42.25   C       ATOM   1372   NH1   ARG   A   156   70.870   −12.795   28.679   1.00   20.02   N       ATOM   1373   NH2   ARG   A   156   72.221   −12.966   26.843   1.00   20.88   N       ATOM   1374   N   ASP   A   157   65.343   −10.446   29.321   1.00   16.00   N       ATOM   1375   CA   ASP   A   157   64.370   −9.749   30.166   1.00   16.20   C       ATOM   1376   C   ASP   A   157   64.444   −8.245   29.841   1.00   19.20   C       ATOM   1377   O   ASP   A   157   64.865   −7.429   30.650   1.00   10.71   O       ATOM   1378   CE   ASP   A   157   64.609   −10.061   31.652   1.00   16.50   C       ATOM   1379   CG   ASP   A   157   63.489   −9.560   32.566 1.00   26.45   C       ATOM   1380   OD1   ASP   A   157   62.433   −9.060   32.108   1.00   26.82   O       ATOM   1381   OD2   ASP   A   157   63.673   −9.653   33.784   1.00   21.88   O       ATOM   1382   N   TYR   A   158   64.038   −7.921   28.620   1.00   19.41   N       ATOM   1383   CA   TYR   A   158   64.099   −6.564   28.083   1.00   18.96   C       ATOM   1384   C   TYR   A   158   62.688   −5.977   28.127   1.00   22.62   C       ATOM   1385   O   TYR   A   158   61.854   −6.296   27.282   1.00   10.12   O       ATOM   1386   CB   TYR   A   158   64.562   −6.661   26.631   1.00   16.34   C       ATOM   1387   CG   TYR   A   158   65.982   −7.166   26.484   1.00   12.04   C       ATOM   1388   CD1   TYR   A   158   66.789   −7.415   27.621   1.00   13.76   C       ATOM   1389   CD2   TYR   A   158   66.544   −7.349   25.218   1.00   16.35   C       ATOM   1390   CE1   TYR   A   158   68.135   −7.786   27.482   1.00   8.18   C       ATOM   1391   CE2   TYR   A   158   67.886   −7.732   25.060   1.00   13.73   C       ATOM   1392   CZ   TYR   A   158   68.676   −7.942   26.186   1.00   24.45   C       ATOM   1393   OH   TYR   A   158   69.993   −8.338   25.997   1.00   14.36   O       ATOM   1394   N   ARG   A   159   62.423   −5.200   29.175   1.00   23.53   N       ATOM   1395   CA   ARG   A   159   61.105   −4.603   29.483   1.00   21.15   C       ATOM   1396   C   ARG   A   159   60.930   −3.172   28.878   1.00   23.55   C       ATOM   1397   O   ARG   A   159   61.911   −2.566   28.424   1.00   18.12   O       ATOM   1398   CB   ARG   A   159   60.891   −4.608   31.034   1.00   21.68   C       ATOM   1399   CG   ARG   A   159   60.986   −6.029   31.722   1.00   16.41   C       ATOM   1400   CD   ARG   A   159   61.135   −6.052   33.233   1.00   18.10   C       ATOM   1401   NE   ARG   A   159   61.305   −7.402   33.772   1.00   19.25   N       ATOM   1402   CZ   ARG   A   159   61.164   −7.720   35.058   1.00   36.67   C       ATOM   1403   NH1   ARG   A   159   60.886   −6.776   35.962   1.00   15.32   N       ATOM   1404   NH2   ARG   A   159   61.309   −8.986   35.448   1.00   11.79   N       ATOM   1405   N   SER   A   160   59.689   −2.661   28.859   1.00   24.44   N       ATOM   1406   CA   SER   A   160   59.312   −1.393   28.200   1.00   21.59   C       ATOM   1407   C   SER   A   160   58.242   −0.577   28.950   1.00   25.07   C       ATOM   1408   O   SER   A   160   57.257   −1.127   29.454   1.00   17.02   O       ATOM   1409   CB   SER   A   160   58.719   −1.747   26.797   1.00   13.05   C       ATOM   1410   OG   SER   A   160   59.782   −1.897   25.885   1.00   37.57   O       ATOM   1411   N   VAL   A   161   58.378   0.742   28.927   1.00   21.01   N       ATOM   1412   CA   VAL   A   161   57.369   1.644   29.509   1.00   9.70   C       ATOM   1413   C   VAL   A   161   57.068   2.747   28.504   1.00   16.77   C       ATOM   1414   O   VAL   A   161   57.955   3.149   27.729   1.00   16.33   O       ATOM   1415   CB   VAL   A   161   57.806   2.248   30.862   1.00   17.94   C       ATOM   1416   CG1   VAL   A   161   57.873   1.185   31.984   1.00   16.16   C       ATOM   1417   CG2   VAL   A   161   59.137   2.992   30.750   1.00   21.10   C       ATOM   1418   N   MET   A   162   55.794   3.147   28.443   1.00   22.46   N       ATOM   1419   CA   MET   A   162   55.296   4.185   27.513   1.00   19.23   C       ATOM   1420   C   MET   A   162   54.880   5.312   28.397   1.00   25.19   C       ATOM   1421   O   MET   A   162   53.788   5.269   28.961   1.00   18.35   O       ATOM   1422   CB   MET   A   162   53.979   3.796   26.850   1.00   15.55   C       ATOM   1423   CG   MET   A   162   54.013   2.630   25.949   1.00   37.79   C       ATOM   1424   SD   MET   A   162   54.354   3.100   24.235   1.00   52.07   S       ATOM   1425   CE   MET   A   162   56.193   3.134   24.410   1.00   36.30   C       ATOM   1426   N   PRO   A   163   55.730   6.313   28.521   1.00   18.43   N       ATOM   1427   CA   PRO   A   163   55.390   7.472   29.337   1.00   17.76   C       ATOM   1428   C   PRO   A   163   54.300   8.384   28.667   1.00   21.23   C       ATOM   1429   O   PRO   A   163   54.208   8.448   27.433   1.00   15.20   O       ATOM   1430   CB   PRO   A   163   56.727   8.196   29.423   1.00   11.43   C       ATOM   1431   CG   PRO   A   163   57.352   7.874   28.031   1.00   13.99   C       ATOM   1432   CD   PRO   A   163   57.086   6.401   27.949   1.00   12.24   C       ATOM   1433   N   THR   A   164   53.478   9.060   29.478   1.00   13.95   N       ATOM   1434   CA   THR   A   164   52.581   10.121   28.963   1.00   25.82   C       ATOM   1435   C   THR   A   164   53.406   11.441   28.781   1.00   19.67   C       ATOM   1436   O   THR   A   164   54.633   11.393   28.868   1.00   13.97   O       ATOM   1437   CB   THR   A   164   51.373   10.391   29.903   1.00   25.51   C       ATOM   1438   OG1   THR   A   164   50.470   11.321   29.267   1.00   14.77   O       ATOM   1439   CG2   THR   A   164   51.818   10.886   31.298   1.00   9.06   C       ATOM   1440   N   ASN   A   165   52.751   12.589   28.556   1.00   14.99   N       ATOM   1441   CA   ASN   A   165   53.448   13.901   28.481   1.00   7.83   C       ATOM   1442   C   ASN   A   165   54.167   14.064   29.824   1.00   11.21   C       ATOM   1443   O   ASN   A   165   53.554   13.929   30.894   1.00   17.66   O       ATOM   1444   CE   ASN   A   165   52.434   15.061   28.416   1.00   14.48   C       ATOM   1445   CG   ASN   A   165   51.492   14.941   27.262   1.00   23.70   C       ATOM   1446   OD1   ASN   A   165   51.939   14.800   26.129   1.00   22.37   O       ATOM   1447   ND2   ASN   A   165   50.173   14.925   27.539   1.00   27.22   N       ATOM   1448   N   LEU   A   166   55.418   14.490   29.777   1.00   8.23   N       ATOM   1449   CA   LEU   A   166   56.187   14.604   30.994   1.00   14.40   C       ATOM   1450   C   LEU   A   166   56.629   16.017   31.120   1.00   25.05   C       ATOM   1451   O   LEU   A   166   56.624   16.718   30.125   1.00   25.09   O       ATOM   1452   CE   LEU   A   166   57.460   13.743   30.870   1.00   17.48   C       ATOM   1453   CG   LEU   A   166   57.423   12.218   30.652   1.00   16.63   C       ATOM   1454   CD1   LEU   A   166   58.837   11.639   31.000   1.00   22.52   C       ATOM   1455   CD2   LEU   A   166   56.336   11.539   31.514   1.00   7.46   C       ATOM   1456   N   TYR   A   167   57.146   16.391   32.300   1.00   19.78   N       ATOM   1457   CA   TYR   A   167   57.678   17.760   32.511   1.00   18.58   C       ATOM   1458   C   TYR   A   167   58.534   17.763   33.767   1.00   15.53   C       ATOM   1459   O   TYR   A   167   58.474   16.852   34.575   1.00   16.71   O       ATOM   1460   CB   TYR   A   167   56.509   18.778   32.665   1.00   18.33   C       ATOM   1461   CG   TYR   A   167   55.671   18.561   33.931   1.00   14.23   C       ATOM   1462   CD1   TYR   A   167   54.624   17.618   33.977   1.00   13.35   C       ATOM   1463   CD2   TYR   A   167   55.984   19.258   35.106   1.00   16.52   C       ATOM   1464   CE1   TYR   A   167   53.889   17.446   35.146   1.00   21.17   C       ATOM   1465   CE2   TYR   A   167   55.302   19.084   36.264   1.00   8.26   C       ATOM   1466   CZ   TYR   A   167   54.228   18.203   36.296   1.00   23.56   C       ATOM   1467   OH   TYR   A   167   53.526   18.078   37.504   1.00   22.81   O       ATOM   1468   N   GLY   A   168   59.334   18.797   33.952   1.00   16.59   N       ATOM   1469   CA   GLY   A   168   60.158   18.817   35.152   1.00   18.21   C       ATOM   1470   C   GLY   A   168   61.534   19.428   34.880   1.00   13.69   C       ATOM   1471   O   GLY   A   168   61.746   20.028   33.837   1.00   16.52   O       ATOM   1472   N   PRO   A   169   62.473   19.263   35.817   1.00   20.33   N       ATOM   1473   CA   PRO   A   169   63.801   19.822   35.656   1.00   16.07   C       ATOM   1474   C   PRO   A   169   64.430   19.353   34.387   1.00   27.18   C       ATOM   1475   O   PRO   A   169   64.305   18.186   33.981   1.00   21.23   O       ATOM   1476   CB   PRO   A   169   64.595   19.206   36.805   1.00   17.28   C       ATOM   1477   CG   PRO   A   169   63.649   18.919   37.830   1.00   19.89   C       ATOM   1478   CD   PRO   A   169   62.263   18.772   37.189   1.00   22.47   C       ATOM   1479   N   HIS   A   170   65.226   20.235   33.829   1.00   19.48   N       ATOM   1480   CA   HIS   A   170   65.952   19.877   32.638   1.00   25.56   C       ATOM   1481   C   HIS   A   170   65.096   19.707   31.428   1.00   29.15   C       ATOM   1482   O   HIS   A   170   65.553   19.091   30.479   1.00   29.71   O       ATOM   1483   CB   HIS   A   170   66.783   18.600   32.845   1.00   28.94   C       ATOM   1484   CG   HIS   A   170   67.703   18.671   34.034   1.00   33.88   C       ATOM   1485   ND1   HIS   A   170   68.975   19.203   33.969   1.00   25.46   N       ATOM   1486   CD2   HIS   A   170   67.518   18.298   35.326   1.00   34.77   C       ATOM   1487   CE1   HIS   A   170   69.531   19.151   35.166   1.00   25.63   C       ATOM   1488   NE2   HIS   A   170   68.673   18.603   36.008   1.00   31.72   N       ATOM   1489   N   ASP   A   171   63.881   20.245   31.440   1.00   21.52   N       ATOM   1490   CA   ASP   A   171   63.041   20.267   30.218   1.00   28.63   C       ATOM   1491   C   ASP   A   171   63.630   21.459   29.359   1.00   41.94   C       ATOM   1492   O   ASP   A   171   64.534   22.171   29.835   1.00   29.69   O       ATOM   1493   CB   ASP   A   171   61.552   20.558   30.602   1.00   26.40   C       ATOM   1494   CG   ASP   A   171   60.552   20.097   29.540   1.00   22.32   C       ATOM   1495   OD1   ASP   A   171   60.890   20.067   28.325   1.00   32.03   O       ATOM   1496   OD2   ASP   A   171   59.427   19.719   29.916   1.00   42.13   O       ATOM   1497   N   ASN   A   172   63.141   21.712   28.137   1.00   42.08   N       ATOM   1498   CA   ASN   A   172   63.616   22.893   27.388   1.00   35.95   C       ATOM   1499   C   ASN   A   172   62.665   24.056   27.674   1.00   33.71   C       ATOM   1500   O   ASN   A   172   61.586   24.102   27.104   1.00   32.69   O       ATOM   1501   CB   ASN   A   172   63.632   22.667   25.869   1.00   41.60   C       ATOM   1502   CG   ASN   A   172   63.807   23.987   25.086   1.00   39.09   C       ATOM   1503   OD1   ASN   A   172   62.973   24.347   24.259   1.00   83.94   O       ATOM   1504   ND2   ASN   A   172   64.855   24.740   25.418   1.00   65.07   N       ATOM   1505   N   PHE   A   173   63.021   24.953   28.583   1.00   31.93   N       ATOM   1506   CA   PHE   A   173   62.082   26.030   28.944   1.00   48.24   C       ATOM   1507   C   PHE   A   173   61.989   27.260   28.045   1.00   69.01   C       ATOM   1508   O   PHE   A   173   62.278   28.395   28.465   1.00   58.79   O       ATOM   1509   CB   PHE   A   173   62.225   26.459   30.390   1.00   43.43   C       ATOM   1510   CG   PHE   A   173   61.867   25.399   31.356   1.00   34.19   C       ATOM   1511   CD1   PHE   A   173   62.810   24.488   31.751   1.00   24.68   C       ATOM   1512   CD2   PHE   A   173   60.621   25.354   31.925   1.00   24.84   C       ATOM   1513   CE1   PHE   A   173   62.524   23.548   32.682   1.00   23.64   C       ATOM   1514   CE2   PHE   A   173   60.305   24.366   32.804   1.00   31.32   C       ATOM   1515   CZ   PHE   A   173   61.263   23.457   33.192   1.00   24.30   C       ATOM   1516   N   HIS   A   174   61.510   27.036   26.831   1.00   68.16   N       ATOM   1517   CA   HIS   A   174   61.401   28.109   25.871   1.00   64.53   C       ATOM   1518   C   HIS   A   174   59.973   28.221   25.400   1.00   71.58   C       ATOM   1519   O   HIS   A   174   59.309   27.186   25.249   1.00   73.20   O       ATOM   1520   CB   HIS   A   174   62.418   27.870   24.736   1.00   71.71   C       ATOM   1521   CG   HIS   A   174   63.835   27.868   25.229   1.00   92.29   C       ATOM   1522   ND1   HIS   A   174   64.921   27.539   24.440   1.00   100.00   N       ATOM   1523   CD2   HIS   A   174   64.338   28.133   26.463   1.00   100.00   C       ATOM   1524   CE1   HIS   A   174   66.032   27.628   25.160   1.00   100.00   C       ATOM   1525   NE2   HIS   A   174   65.705   27.981   26.393   1.00   100.00   N       ATOM   1526   N   PRO   A   175   59.469   29.461   25.262   1.00   65.71   N       ATOM   1527   CA   PRO   A   175   58.109   29.658   24.770   1.00   55.72   C       ATOM   1528   C   PRO   A   175   58.233   29.297   23.267   1.00   75.83   C       ATOM   1529   O   PRO   A   175   57.224   29.226   22.554   1.00   69.59   O       ATOM   1530   CE   PRO   A   175   57.866   31.142   25.026   1.00   49.14   C       ATOM   1531   CG   PRO   A   175   59.258   31.790   24.901   1.00   42.23   C       ATOM   1532   CD   PRO   A   175   60.286   30.695   25.109   1.00   49.59   C       ATOM   1533   N   SER   A   176   59.480   28.954   22.879   1.00   85.09   N       ATOM   1534   CA   SER   A   176   59.954   28.474   21.548   1.00   81.18   C       ATOM   1535   C   SER   A   176   59.660   26.965   21.343   1.00   73.90   C       ATOM   1536   O   SER   A   176   59.617   26.458   20.213   1.00   57.03   O       ATOM   1537   CB   SER   A   176   61.493   28.666   21.447   1.00   71.32   C       ATOM   1538   OG   SER   A   176   62.048   29.349   22.578   1.00   51.93   O       ATOM   1539   N   ASN   A   177   59.520   26.276   22.480   1.00   66.23   N       ATOM   1540   CA   ASN   A   177   59.274   24.847   22.619   1.00   56.41   C       ATOM   1541   C   ASN   A   177   57.810   24.497   22.353   1.00   60.91   C       ATOM   1542   O   ASN   A   177   56.914   25.215   22.811   1.00   55.58   O       ATOM   1543   CB   ASN   A   177   59.619   24.469   24.065   1.00   50.45   C       ATOM   1544   CG   ASN   A   177   59.562   22.970   24.319   1.00   66.57   C       ATOM   1545   OD1   ASN   A   177   59.095   22.216   23.476   1.00   100.00   O       ATOM   1546   ND2   ASN   A   177   60.099   22.546   25.464   1.00   35.61   N       ATOM   1547   N   SER   A   178   57.583   23.387   21.627   1.00   57.10   N       ATOM   1548   CA   SER   A   178   56.234   22.853   21.279   1.00   50.50   C       ATOM   1549   C   SER   A   178   55.557   22.159   22.491   1.00   76.24   C       ATOM   1550   O   SER   A   178   54.575   21.400   22.304   1.00   99.63   O       ATOM   1551   CB   SER   A   178   56.316   21.800   20.118   1.00   10.17   C       ATOM   1552   OG   SER   A   178   57.397   22.112   19.217   1.00   71.69   O       ATOM   1553   N   HIS   A   179   56.134   22.284   23.694   1.00   37.39   N       ATOM   1554   CA   HIS   A   179   55.569   21.587   24.855   1.00   30.96   C       ATOM   1555   C   HIS   A   179   54.961   22.616   25.767   1.00   21.93   C       ATOM   1556   O   HIS   A   179   55.641   23.598   26.138   1.00   25.17   O       ATOM   1557   CB   HIS   A   179   56.634   20.683   25.575   1.00   36.20   C       ATOM   1558   CG   HIS   A   179   56.973   19.419   24.835   1.00   42.90   C       ATOM   1559   ND1   HIS   A   179   56.973   19.335   23.457   1.00   49.52   N       ATOM   1560   CD2   HIS   A   179   57.323   18.190   25.278   1.00   52.42   C       ATOM   1561   CE1   HIS   A   179   57.283   18.109   23.084   1.00   44.78   C       ATOM   1562   NE2   HIS   A   179   57.500   17.393   24.168   1.00   50.49   N       ATOM   1563   N   VAL   A   160   53.661   22.454   26.038   1.00   19.14   N       ATOM   1564   CA   VAL   A   180   52.886   23.449   26.789   1.00   29.03   C       ATOM   1565   C   VAL   A   180   53.373   23.890   28.142   1.00   31.29   C       ATOM   1566   O   VAL   A   180   53.348   25.075   28.447   1.00   19.55   O       ATOM   1567   CB   VAL   A   180   51.403   23.115   26.914   1.00   35.47   C       ATOM   1568   CG1   VAL   A   180   50.630   24.399   27.217   1.00   35.84   C       ATOM   1569   CG2   VAL   A   180   50.923   22.550   25.663   1.00   36.11   C       ATOM   1570   N   ILE   A   181   53.684   22.935   29.005   1.00   26.57   N       ATOM   1571   CA   ILE   A   181   54.138   23.285   30.360   1.00   24.49   C       ATOM   1572   C   ILE   A   181   55.371   24.213   30.361   1.00   16.51   C       ATOM   1573   O   ILE   A   181   55.326   25.315   30.909   1.00   24.42   O       ATOM   1574   CB   ILE   A   181   54.285   22.018   31.264   1.00   20.20   C       ATOM   1575   CG1   ILE   A   181   52.878   21.428   31.528   1.00   18.22   C       ATOM   1576   CG2   ILE   A   181   55.014   22.315   32.581   1.00   13.37   C       ATOM   1577   CD1   ILE   A   181   52.867   20.086   32.286   1.00   8.03   C       ATOM   1578   N   PRO   A   182   56.452   23.779   29.718   1.00   22.21   N       ATOM   1579   CA   PRO   A   182   57.664   24.605   29.640   1.00   22.07   C       ATOM   1580   C   PRO   A   182   57.379   25.852   28.828   1.00   24.18   C       ATOM   1581   O   PRO   A   182   57.811   26.949   29.210   1.00   18.35   O       ATOM   1582   CB   PRO   A   182   58.682   23.725   28.890   1.00   24.97   C       ATOM   1583   CG   PRO   A   182   57.925   22.473   28.471   1.00   25.77   C       ATOM   1584   CD   PRO   A   182   56.727   22.359   29.401   1.00   18.23   C       ATOM   1585   N   ALA   A   183   56.628   25.707   27.729   1.00   21.45   N       ATOM   1586   CA   ALA   A   183   56.261   26.896   26.943   1.00   21.66   C       ATOM   1587   C   ALA   A   183   55.464   27.900   27.811   1.00   26.10   C       ATOM   1588   O   ALA   A   183   55.773   29.091   27.856   1.00   19.50   O       ATOM   1589   CB   ALA   A   183   55.473   26.513   25.703   1.00   13.26   C       ATOM   1590   N   LEU   A   184   54.472   27.389   28.543   1.00   23.34   N       ATOM   1591   CA   LEU   A   184   53.642   28.215   29.401   1.00   19.05   C       ATOM   1592   C   LEU   A   184   54.312   28.693   30.655   1.00   21.91   C       ATOM   1593   O   LEU   A   184   54.017   29.771   31.158   1.00   19.71   O       ATOM   1594   CB   LEU   A   184   52.309   27.553   29.715   1.00   14.41   C       ATOM   1595   CG   LEU   A   184   51.342   27.595   28.525   1.00   23.42   C       ATOM   1596   CD1   LEU   A   184   49.918   27.244   28.928   1.00   31.06   C       ATOM   1597   CD2   LEU   A   184   51.380   28.896   27.690   1.00   21.73   C       ATOM   1598   N   LEU   A   185   55.178   27.879   31.213   1.00   18.39   N       ATOM   1599   CA   LEU   A   185   55.833   28.332   32.417   1.00   16.39   C       ATOM   1600   C   LEU   A   185   56.669   29.528   31.985   1.00   23.67   C       ATOM   1601   O   LEU   A   185   56.681   30.590   32.644   1.00   29.38   O       ATOM   1602   CB   LEU   A   185   56.723   27.233   33.015   1.00   15.05   C       ATOM   1603   CG   LEU   A   185   56.021   26.348   34.041   1.00   15.56   C       ATOM   1604   CD1   LEU   A   185   56.819   25.022   34.301   1.00   21.06   C       ATOM   1605   CD2   LEU   A   185   55.722   27.113   35.321   1.00   11.02   C       ATOM   1606   N   ARG   A   186   57.337   29.397   30.852   1.00   17.09   N       ATOM   1607   CA   ARG   A   186   58.137   30.523   30.429   1.00   18.82   C       ATOM   1608   C   ARG   A   186   57.308   31.752   30.069   1.00   29.00   C       ATOM   1609   O   ARG   A   186   57.629   32.880   30.476   1.00   23.91   O       ATOM   1610   CB   ARG   A   186   59.026   30.146   29.281   1.00   22.06   C       ATOM   1611   CG   ARG   A   186   59.653   31.365   28.652   1.00   38.46   C       ATOM   1612   CD   ARG   A   186   60.825   31.804   29.462   1.00   83.66   C       ATOM   1613   NE   ARG   A   186   62.012   31.861   28.631   1.00   70.77   N       ATOM   1614   CZ   ARG   A   186   63.058   32.622   28.904   1.00   91.68   C       ATOM   1615   NH1   ARG   A   186   63.053   33.386   29.995   1.00   56.56   N       ATOM   1616   NH2   ARG   A   186   64.098   32.639   28.082   1.00   100.00   N       ATOM   1617   N   ARG   A   187   56.234   31.544   29.310   1.00   20.96   N       ATOM   1618   CA   ARG   A   187   55.361   32.662   28.941   1.00   19.32   C       ATOM   1619   C   ARG   A   187   54.765   33.453   30.142   1.00   28.41   C       ATOM   1620   O   ARG   A   187   54.823   34.700   30.193   1.00   17.23   O       ATOM   1621   CB   ARG   A   187   54.270   32.223   27.957   1.00   17.05   C       ATOM   1622   CG   ARG   A   187   54.813   31.546   26.720   1.00   61.42   C       ATOM   1623   CD   ARG   A   187   53.696   31.244   25.757   1.00   44.57   C       ATOM   1624   NE   ARG   A   187   53.033   32.472   25.354   1.00   29.47   N       ATOM   1625   CZ   ARG   A   187   51.831   32.534   24.790   1.00   17.82   C       ATOM   1626   NH1   ARG   A   187   51.136   31.427   24.544   1.00   24.95   N       ATOM   1627   NH2   ARG   A   187   51.341   33.716   24.447   1.00   37.77   N       ATOM   1628   N   PHE   A   188   54.192   32.734   31.101   1.00   23.48   N       ATOM   1629   CA   PHE   A   188   53.604   33.399   32.259   1.00   21.24   C       ATOM   1630   C   PHE   A   188   54.638   34.080   33.095   1.00   21.39   C       ATOM   1631   O   PHE   A   188   54.394   35.126   33.626   1.00   23.90   O       ATOM   1632   CB   PHE   A   188   52.723   32.466   33.077   1.00   19.95   C       ATOM   1633   CG   PHE   A   188   51.389   32.215   32.435   1.00   22.28   C       ATOM   1634   CD1   PHE   A   188   50.440   33.229   32.375   1.00   19.42   C       ATOM   1635   CD2   PHE   A   188   51.144   31.038   31.734   1.00   23.82   C       ATOM   1636   CE1   PHE   A   188   49.191   33.026   31.742   1.00   24.77   C       ATOM   1637   CE2   PHE   A   188   49.936   30.826   31.057   1.00   20.17   C       ATOM   1638   CZ   PHE   A   188   48.945   31.815   31.068   1.00   23.14   C       ATOM   1639   N   HIS   A   189   55.831   33.513   33.118   1.00   24.15   N       ATOM   1640   CA   HIS   A   189   56.933   34.122   33.837   1.00   28.79   C       ATOM   1641   C   HIS   A   189   57.303   35.506   33.315   1.00   28.58   C       ATOM   1642   O   HIS   A   189   57.480   36.463   34.083   1.00   20.07   O       ATOM   1643   CB   HIS   A   189   58.148   33.268   33.641   1.00   31.38   C       ATOM   1644   CG   HIS   A   189   59.364   33.844   34.290   1.00   29.98   C       ATOM   1645   ND1   HIS   A   189   59.548   33.833   35.658   1.00   31.00   N       ATOM   1646   CD2   HIS   A   189   60.449   34.464   33.766   1.00   21.79   C       ATOM   1647   CE1   HIS   A   189   60.722   34.371   35.945   1.00   24.04   C       ATOM   1648   NE2   HIS   A   189   61.257   34.815   34.821   1.00   19.53   N       ATOM   1649   N   GLU   A   190   57.539   35.561   32.006   1.00   28.43   N       ATOM   1650   CA   GLU   A   190   57.876   36.816   31.324   1.00   27.72   C       ATOM   1651   C   GLU   A   190   56.725   37.829   31.437   1.00   32.56   C       ATOM   1652   O   GLU   A   190   56.949   38.995   31.717   1.00   27.06   O       ATOM   1653   CB   GLU   A   190   58.122   36.529   29.849   1.00   28.55   C       ATOM   1654   CG   GLU   A   190   59.150   35.461   29.614   1.00   35.29   C       ATOM   1655   CD   GLU   A   190   60.553   35.941   29.892   1.00   99.81   C       ATOM   1656   OH1   GLU   A   190   60.913   36.037   31.085   1.00   86.56   O       ATOM   1657   OE2   GLU   A   190   61.293   36.167   28.910   1.00   100.00   O       ATOM   1658   N   ALA   A   191   55.493   37.391   31.196   1.00   32.67   N       ATOM   1659   CA   ALA   A   191   54.349   38.286   31.311   1.00   25.30   C       ATOM   1660   C   ALA   A   191   54.287   38.795   32.742   1.00   36.20   C       ATOM   1661   O   ALA   A   191   53.920   39.924   33.014   1.00   27.52   O       ATOM   1662   CB   ALA   A   191   53.055   37.563   31.000   1.00   16.48   C       ATOM   1663   N   THR   A   192   54.549   37.927   33.693   1.00   29.39   N       ATOM   1664   CA   THR   A   192   54.395   38.386   35.041   1.00   19.08   C       ATOM   1665   C   THR   A   192   55.420   39.494   35.298   1.00   44.78   C       ATOM   1666   O   THR   A   192   55.094   40.550   35.839   1.00   40.58   O       ATOM   1667   CB   THR   A   192   54.515   37.235   35.983   1.00   18.99   C       ATOM   1668   OG1   THR   A   192   53.410   36.348   35.755   1.00   34.36   O       ATOM   1669   CG2   THR   A   192   54.461   37.738   37.425   1.00   21.15   C       ATOM   1670   N   ALA   A   193   56.617   39.312   34.757   1.00   48.58   N       ATOM   1671   CA   ALA   A   193   57.705   40.286   34.905   1.00   50.59   C       ATOM   1672   C   ALA   A   193   57.496   41.613   34.145   1.00   54.42   C       ATOM   1673   O   ALA   A   193   57.952   42.698   34.553   1.00   48.28   O       ATOM   1674   CB   ALA   A   193   59.0473   9.640   34.496   1.00   51.78   C       ATOM   1675   N   GLN   A   194   56.810   41.530   33.022   1.00   43.16   N       ATOM   1676   CA   GLN   A   194   56.586   42.722   32.242   1.00   38.03   C       ATOM   1677   C   GLN   A   194   55.264   43.389   32.576   1.00   40.85   C       ATOM   1678   O   GLN   A   194   54.830   44.284   31.845   1.00   51.20   O       ATOM   1679   CB   GLN   A   194   56.599   42.358   30.750   1.00   35.96   C       ATOM   1680   CG   GLN   A   194   57.910   41.692   30.290   1.00   100.00   C       ATOM   1681   CD   GLN   A   194   57.715   40.661   29.158   1.00   100.00   C       ATOM   1682   OE1   GLN   A   194   56.619   40.546   28.579   1.00   100.00   O       ATOM   1683   NE2   GLN   A   194   58.782   39.904   28.848   1.00   100.00   N       ATOM   1684   N   GLY   A   195   54.583   42.949   33.630   1.00   32.29   N       ATOM   1685   CA   GLY   A   195   53.236   43.464   33.864   1.00   36.26   C       ATOM   1686   C   GLY   A   195   52.299   43.332   32.593   1.00   45.33   C       ATOM   1687   O   GLY   A   195   51.515   44.242   32.346   1.00   45.16   O       ATOM   1688   N   GLY   A   196   52.405   42.245   31.788   1.00   36.33   N       ATOM   1689   CA   GLY   A   196   51.515   41.965   30.608   1.00   19.06   C       ATOM   1690   C   GLY   A   196   50.037   41.958   31.117   1.00   22.49   C       ATOM   1691   O   GLY   A   196   49.724   41.479   32.223   1.00   33.09   O       ATOM   1692   N   PRO   A   197   49.144   42.657   30.431   1.00   29.22   N       ATOM   1693   CA   PRO   A   197   47.790   42.732   30.953   1.00   25.29   C       ATOM   1694   C   PRO   A   197   47.091   41.413   30.674   1.00   24.64   C       ATOM   1695   O   PRO   A   197   46.192   40.991   31.411   1.00   24.75   O       ATOM   1696   CB   PRO   A   197   47.162   43.911   30.176   1.00   26.31   C       ATOM   1697   CG   PRO   A   197   48.188   44.407   29.252   1.00   26.56   C       ATOM   1698   CD   PRO   A   197   49.307   43.454   29.203   1.00   30.25   C       ATOM   1699   N   ASP   A   198   47.572   40.723   29.658   1.00   16.88   N       ATOM   1700   CA   ASP   A   198   47.067   39.418   29.405   1.00   21.65   C       ATOM   1701   C   ASP   A   198   48.046   38.522   28.677   1.00   31.28   C       ATOM   1702   O   ASP   A   198   49.062   38.978   28.172   1.00   34.57   O       ATOM   1703   CB   ASP   A   198   45.739   39.507   28.669   1.00   32.80   C       ATOM   1704   CG   ASP   A   198   45.868   40.055   27.256   1.00   46.13   C       ATOM   1705   OD1   ASP   A   198   46.982   40.230   26.725   1.00   57.45   O       ATOM   1706   OD2   ASP   A   198   44.817   40.271   26.640   1.00   67.61   O       ATOM   1707   N   VAL   A   199   47.713   37.234   28.614   1.00   38.67   N       ATOM   1708   CA   VAL   A   199   48.499   36.226   27.901   1.00   27.79   C       ATOM   1709   C   VAL   A   199   47.462   35.469   27.065   1.00   25.88   C       ATOM   1710   O   VAL   A   199   46.460   35.023   27.598   1.00   24.22   O       ATOM   1711   CB   VAL   A   199   49.163   35.229   28.905   1.00   24.37   C       ATOM   1712   CG1   VAL   A   199   49.874   34.047   28.160   1.00   20.28   C       ATOM   1713   CG2   VAL   A   199   50.121   35.942   29.835   1.00   22.25   C       ATOM   1714   N   VAL   A   200   47.661   35.386   25.757   1.00   23.72   N       ATOM   1715   CA   VAL   A   200   46.701   34.694   24.903   1.00   23.99   C       ATOM   1716   C   VAL   A   200   47.167   33.286   24.499   1.00   22.85   C       ATOM   1717   O   VAL   A   200   48.321   33.108   24.188   1.00   29.77   O       ATOM   1718   CB   VAL   A   200   46.358   35.548   23.680   1.00   23.11   C       ATOM   1719   CG1   VAL   A   200   45.561   34.737   22.598   1.00   16.25   C       ATOM   1720   CG2   VAL   A   200   45.652   36.823   24.130   1.00   27.86   C       ATOM   1721   N   VAL   A   201   46.296   32.278   24.632   1.00   27.39   N       ATOM   1722   CA   VAL   A   201   46.588   30.893   24.265   1.00   9.63   C       ATOM   1723   C   VAL   A   201   45.653   30.529   23.165   1.00   19.63   C       ATOM   1724   O   VAL   A   201   44.452   30.755   23.312   1.00   17.61   O       ATOM   1725   CB   VAL   A   201   46.306   29.952   25.426   1.00   19.95   C       ATOM   1726   CG1   VAL   A   201   46.703   28.519   25.054   1.00   20.85   C       ATOM   1727   CG2   VAL   A   201   47.086   30.439   26.661   1.00   16.73   C       ATOM   1728   N   TRP   A   202   46.210   30.080   22.030   1.00   14.36   N       ATOM   1729   CA   TRP   A   202   45.422   29.693   20.865   1.00   18.97   C       ATOM   1730   C   TRP   A   202   44.495   28.572   21.313   1.00   36.22   C       ATOM   1731   O   TRP   A   202   44.934   27.694   22.057   1.00   31.46   O       ATOM   1732   CB   TRP   A   202   46.292   29.055   19.823   1.00   19.14   C       ATOM   1733   CG   TRP   A   202   47.243   29.894   19.066   1.00   33.65   C       ATOM   1734   CD1   TRP   A   202   48.391   29.463   18.429   1.00   35.28   C       ATOM   1735   CD2   TRP   A   202   47.126   31.282   18.772   1.00   39.90   C       ATOM   1736   NE1   TRP   A   202   48.941   30.481   17.693   1.00   37.86   N       ATOM   1737   CE2   TRP   A   202   48.228   31.624   17.922   1.00   38.35   C       ATOM   1738   CE3   TRP   A   202   46.206   32.281   19.138   1.00   39.39   C       ATOM   1739   CZ2   TRP   A   202   48.380   32.884   17.367   1.00   36.15   C       ATOM   1740   CZ3   TRP   A   202   46.356   33.542   18.578   1.00   39.60   C       ATOM   1741   CH2   TRP   A   202   47.428   33.828   17.684   1.00   40.99   C       ATOM   1742   N   GLY   A   203   43.245   28.564   20.842   1.00   25.59   N       ATOM   1743   CA   GLY   A   203   42.332   27.483   21.169   1.00   13.09   C       ATOM   1744   C   GLY   A   203   41.260   27.813   22.193   1.00   21.12   C       ATOM   1745   O   GLY   A   203   41.340   28.815   22.886   1.00   22.86   O       ATOM   1746   N   SER   A   204   40.270   26.919   22.262   1.00   16.88   N       ATOM   1747   CA   SER   A   204   39.163   26.979   23.192   1.00   18.36   C       ATOM   1748   C   SER   A   204   39.561   26.664   24.659   1.00   22.07   C       ATOM   1749   O   SER   A   204   38.888   27.096   25.604   1.00   34.39   O       ATOM   1750   CB   SER   A   204   38.053   25.998   22.740   1.00   9.99   C       ATOM   1751   OG   SER   A   204   38.237   24.695   23.291   1.00   16.37   O       ATOM   1752   N   GLY   A   205   40.562   25.813   24.854   1.00   12.42   N       ATOM   1753   CA   GLY   A   205   40.963   25.411   26.208   1.00   11.64   C       ATOM   1754   C   GLY   A   205   40.208   24.178   26.711   1.00   19.49   C       ATOM   1755   O   GLY   A   205   40.422   23.723   27.838   1.00   13.59   O       ATOM   1756   N   THR   A   206   39.292   23.683   25.881   1.00   15.38   N       ATOM   1757   CA   THR   A   206   38.432   22.594   26.281   1.00   10.80   C       ATOM   1758   C   THR   A   206   39.056   21.221   26.154   1.00   26.39   C       ATOM   1759   O   THR   A   206   38.564   20.267   26.737   1.00   23.28   O       ATOM   1760   CB   THR   A   206   37.124   22.562   25.460   1.00   12.86   C       ATOM   1761   OG1   THR   A   206   37.438   22.395   24.082   1.00   13.12   O       ATOM   1762   CG2   THR   A   206   36.348   23.840   25.620   1.00   10.62   C       ATOM   1763   N   PRO   A   207   40.101   21.083   25.354   1.00   21.10   N       ATOM   1764   CA   PRO   A   207   40.658   19.743   25.175   1.00   18.15   C       ATOM   1765   C   PRO   A   207   41.316   19.181   26.423   1.00   21.75   C       ATOM   1766   O   PRO   A   207   41.951   19.925   27.215   1.00   20.65   O       ATOM   1767   CB   PRO   A   207   41.638   19.909   24.013   1.00   17.51   C       ATOM   1768   CG   PRO   A   207   41.146   21.213   23.307   1.00   21.45   C       ATOM   1769   CD   PRO   A   207   40.698   22.062   24.431   1.00   23.44   C       ATOM   1770   N   MET   A   208   41.112   17.876   26.624   1.00   15.60   N       ATOM   1771   CA   MET   A   208   41.694   17.167   27.775   1.00   22.94   C       ATOM   1772   C   MET   A   208   43.058   16.427   27.579   1.00   21.90   C       ATOM   1773   O   MET   A   208   43.248   15.677   26.633   1.00   23.16   O       ATOM   1774   CB   MET   A   208   40.645   16.273   28.386   1.00   32.86   C       ATOM   1775   CG   MET   A   208   39.630   17.057   29.223   1.00   46.17   C       ATOM   1776   SD   MET   A   208   38.301   15.990   29.826   1.00   57.85   S       ATOM   1777   CE   MET   A   208   37.999   15.028   28.343   1.00   58.23   C       ATOM   1778   N   ARG   A   209   44.022   16.681   28.456   1.00   17.75   N       ATOM   1779   CA   ARG   A   209   45.318   16.042   28.324   1.00   19.88   C       ATOM   1780   C   ARG   A   209   45.871   15.534   29.639   1.00   16.92   C       ATOM   1781   O   ARG   A   209   45.433   15.946   30.697   1.00   16.58   O       ATOM   1782   CB   ARG   A   209   46.340   16.963   27.658   1.00   21.07   C       ATOM   1783   CG   ARG   A   209   45.980   17.478   26.275   1.00   22.57   C       ATOM   1784   CD   ARG   A   209   45.833   16.357   25.282   1.00   28.26   C       ATOM   1785   NE   ARG   A   209   45.586   16.819   23.906   1.00   23.15   N       ATOM   1786   CZ   ARG   A   209   44.420   16.742   23.267   1.00   34.52   C       ATOM   1787   NH1   ARG   A   209   43.336   16.267   23.890   1.00   18.03   N       ATOM   1788   NE2   ARG   A   209   44.339   17.175   22.012   1.00   29.78   N       ATOM   1789   N   GLU   A   210   46.878   14.675   29.547   1.00   20.87   N       ATOM   1790   CA   GLU   A   210   47.530   14.079   30.720   1.00   17.37   C       ATOM   1791   C   GLU   A   210   49.031   14.490   30.851   1.00   20.96   C       ATOM   1792   O   GLU   A   210   49.748   14.622   29.841   1.00   22.44   O       ATOM   1793   CB   GLU   A   210   47.400   12.562   30.571   1.00   16.26   C       ATOM   1794   CG   GLU   A   210   47.807   11.785   31.809   1.00   19.91   C       ATOM   1795   CD   GLU   A   210   48.057   10.304   31.531   1.00   27.81   C       ATOM   1796   OE1   GLU   A   210   48.111   9.919   30.343   1.00   17.29   O       ATOM   1797   OE2   GLU   A   210   48.268   9.540   32.494   1.00   21.63   O       ATOM   1798   N   PHE   A   211   49.504   14.712   32.084   1.00   14.02   N       ATOM   1799   CA   PHE   A   211   50.887   15.159   32.353   1.00   17.48   C       ATOM   1800   C   PHE   A   211   51.458   14.414   33.531   1.00   33.62   C       ATOM   1801   O   PHE   A   211   50.716   14.031   34.443   1.00   27.96   O       ATOM   1802   CB   PHE   A   211   50.933   16.677   32.644   1.00   17.78   C       ATOM   1803   CG   PHE   A   211   50.303   17.490   31.541   1.00   21.49   C       ATOM   1804   CD1   PHE   A   211   51.009   17.676   30.320   1.00   17.36   C       ATOM   1805   CD2   PHE   A   211   48.933   17.844   31.618   1.00   15.09   C       ATOM   1806   CE1   PHE   A   211   50.399   18.334   29.237   1.00   16.37   C       ATOM   1807   CE2   PHE   A   211   48.288   18.491   30.533   1.00   9.61   C       ATOM   1808   CZ   PHE   A   211   49.053   18.756   29.344   1.00   12.71   C       ATOM   1809   N   LEU   A   212   52.761   14.161   33.495   1.00   23.76   N       ATOM   1810   CA   LEU   A   212   53.405   13.448   34.603   1.00   21.24   C       ATOM   1811   C   LEU   A   212   54.772   14.053   34.898   1.00   14.00   C       ATOM   1812   O   LEU   A   212   55.519   14.398   33.985   1.00   13.99   O       ATOM   1813   CB   LEU   A   212   53.548   11.954   34.294   1.00   21.52   C       ATOM   1814   CG   LEU   A   212   54.033   11.039   35.406   1.00   21.09   C       ATOM   1815   CD1   LEU   A   212   52.866   10.634   36.280   1.00   20.84   C       ATOM   1816   CD2   LEU   A   212   54.768   9.829   34.832   1.00   13.18   C       ATOM   1817   N   HIS   A   213   55.023   14.302   36.175   1.00   9.60   N       ATOM   1818   CA   HIS   A   213   56.290   14.864   36.555   1.00   13.66   C       ATOM   1819   C   HIS   A   213   57.380   13.828   36.293   1.00   20.37   C       ATOM   1820   O   HIS   A   213   57.238   12.614   36.542   1.00   16.08   O       ATOM   1821   CB   HIS   A   213   56.280   15.250   38.002   1.00   18.72   C       ATOM   1822   CG   HIS   A   213   57.491   16.017   38.408   1.00   21.22   C       ATOM   1823   ND1   HIS   A   213   58.703   15.406   38.656   1.00   24.29   N       ATOM   1824   CD2   HIS   A   213   57.716   17.353   38.499   1.00   23.67   C       ATOM   1825   CE1   HIS   A   213   59.615   16.331   38.917   1.00   19.13   C       ATOM   1826   NE2   HIS   A   213   59.041   17.523   38.847   1.00   21.99   N       ATOM   1827   N   VAL   A   214   58.459   14.295   35.698   1.00   21.07   N       ATOM   1828   CA   VAL   A   214   59.532   13.383   35.361   1.00   19.23   C       ATOM   1829   C   VAL   A   214   60.067   12.523   36.551   1.00   27.20   C       ATOM   1830   O   VAL   A   214   60.604   11.444   36.359   1.00   22.23   O       ATOM   1831   CE   VAL   A   214   60.625   14.125   34.566   1.00   11.84   C       ATOM   1832   CG1   VAL   A   214   61.390   15.199   35.485   1.00   8.52   C       ATOM   1833   CG2   VAL   A   214   61.560   13.097   33.902   1.00   12.39   C       ATOM   1834   N   ASP   A   215   59.893   12.984   37.790   1.00   25.29   N       ATOM   1835   CA   ASP   A   215   60.406   12.228   38.936   1.00   18.19   C       ATOM   1836   C   ASP   A   215   59.530   11.023   39.230   1.00   13.85   C       ATOM   1837   O   ASP   A   215   59.988   9.981   39.666   1.00   17.44   O       ATOM   1838   CB   ASP   A   215   60.575   13.129   40.155   1.00   16.27   C       ATOM   1839   CG   ASP   A   215   61.859   13.979   40.068   1.00   30.73   C       ATOM   1840   OD1   ASP   A   215   62.782   13.614   39.308   1.00   23.02   O       ATOM   1841   OD2   ASP   A   215   61.957   15.029   40.730   1.00   26.00   O       ATOM   1842   N   ASP   A   216   58.276   11.136   38.863   1.00   20.08   N       ATOM   1843   CA   ASP   A   216   57.378   10.017   39.016   1.00   18.78   C       ATOM   1844   C   ASP   A   216   57.761   9.083   37.894   1.00   23.56   C       ATOM   1845   O   ASP   A   216   57.715   7.880   38.026   1.00   20.79   O       ATOM   1846   CB   ASP   A   216   55.912   10.457   38.821   1.00   17.18   C       ATOM   1847   CG   ASP   A   216   55.193   10.757   40.162   1.00   38.03   C       ATOM   1848   OD1   ASP   A   216   55.503   10.119   41.223   1.00   26.02   O       ATOM   1849   OD2   ASP   A   216   54.249   11.587   40.124   1.00   25.41   O       ATOM   1850   N   MET   A   217   58.092   9.653   36.755   1.00   18.11   N       ATOM   1851   CA   MET   A   217   58.394   8.785   35.636   1.00   22.41   C       ATOM   1852   C   MET   A   217   59.572   7.942   35.992   1.00   27.54   C       ATOM   1853   O   MET   A   217   59.579   6.752   35.710   1.00   20.86   O       ATOM   1854   CB   MET   A   217   58.637   9.592   34.345   1.00   21.24   C       ATOM   1855   CG   MET   A   217   59.478   8.918   33.287   1.00   16.37   C       ATOM   1856   SD   MET   A   217   58.962   7.412   32.473   1.00   30.51   S       ATOM   1857   CE   MET   A   217   57.465   7.608   32.391   1.00   19.57   C       ATOM   1858   N   ALA   A   218   60.561   8.562   36.623   1.00   19.09   N       ATOM   1859   CA   ALA   A   218   61.774   7.841   37.002   1.00   13.65   C       ATOM   1860   C   ALA   A   218   61.436   6.778   38.028   1.00   22.61   C       ATOM   1861   O   ALA   A   218   61.934   5.670   37.967   1.00   19.36   O       ATOM   1862   CB   ALA   A   218   62.809   8.780   37.579   1.00   12.23   C       ATOM   1863   N   ALA   A   219   60.605   7.109   39.000   1.00   19.34   N       ATOM   1864   CA   ALA   A   219   60.310   6.105   40.023   1.00   18.01   C       ATOM   1865   C   ALA   A   219   59.630   4.901   39.413   1.00   23.57   C       ATOM   1866   O   ALA   A   219   59.781   3.777   39.898   1.00   22.71   O       ATOM   1867   CB   ALA   A   219   59.387   6.678   41.083   1.00   10.11   C       ATOM   1868   N   ALA   A   220   58.753   5.174   38.454   1.00   18.99   N       ATOM   1869   CA   ALA   A   220   57.905   4.158   37.855   1.00   14.12   C       ATOM   1870   C   ALA   A   220   58.753   3.213   37.034   1.00   25.33   C       ATOM   1871   O   ALA   A   220   58.584   2.006   37.114   1.00   20.63   O       ATOM   1872   CB   ALA   A   220   56.796   4.798   37.023   1.00   8.53   C       ATOM   1873   N   SER   A   221   59.770   3.772   36.379   1.00   23.92   N       ATOM   1874   CA   SER   A   221   60.702   3.011   35.556   1.00   18.38   C       ATOM   1875   C   SER   A   221   61.537   1.989   36.353   1.00   20.90   C       ATOM   1876   O   SER   A   221   61.683   0.799   35.983   1.00   19.84   O       ATOM   1877   CB   SER   A   221   61.604   3.985   34.804   1.00   10.67   C       ATOM   1878   OG   SER   A   221   60.847   4.744   33.867   1.00   15.61   O       ATOM   1879   N   ILE   A   222   62.083   2.476   37.463   1.00   18.12   N       ATOM   1880   CA   ILE   A   222   62.866   1.644   38.381   1.00   21.56   C       ATOM   1881   C   ILE   A   222   62.020   0.554   39.068   1.00   29.10   C       ATOM   1882   O   ILE   A   222   62.504   −0.566   39.307   1.00   19.03   O       ATOM   1883   CB   ILE   A   222   63.467   2.516   39.432   1.00   24.56   C       ATOM   1884   CG1   ILE   A   222   64.465   3.473   38.765   1.00   32.13   C       ATOM   1885   CG2   ILE   A   222   64.129   1.671   40.500   1.00   28.26   C       ATOM   1886   CD1   ILE   A   222   64.973   4.585   39.649   1.00   15.61   C       ATOM   1887   N   HIS   A   223   60.772   0.907   39.384   1.00   19.34   N       ATOM   1888   CA   HIS   A   223   59.829   −0.031   39.996   1.00   20.46   C       ATOM   1889   C   HIS   A   223   59.599   −1.097   38.964   1.00   24.82   C       ATOM   1890   O   HIS   A   223   59.723   −2.283   39.270   1.00   24.66   O       ATOM   1891   CB   HIS   A   223   58.465   0.637   40.359   1.00   19.53   C       ATOM   1892   CG   HIS   A   223   57.373   −0.333   40.759   1.00   28.64   C       ATOM   1893   ND1   HIS   A   223   57.021   −0.564   42.082   1.00   24.16   N       ATOM   1894   CD2   HIS   A   223   56.497   −1.062   40.004   1.00   30.39   C       ATOM   1895   CE1   HIS   A   223   55.983   −1.399   42.112   1.00   30.39   C       ATOM   1896   NE2   HIS   A   223   55.652   −1.727   40.869   1.00   28.13   N       ATOM   1897   N   VAL   A   224   59.354   −0.684   37.725   1.00   22.06   N       ATOM   1898   CA   VAL   A   224   59.111   −1.657   36.652   1.00   19.15   C       ATOM   1899   C   VAL   A   224   60.350   −2.490   36.333   1.00   25.89   C       ATOM   1900   O   VAL   A   224   60.282   −3.709   36.250   1.00   22.37   O       ATOM   1901   CB   VAL   A   224   58.559   −1.022   35.377   1.00   22.59   C       ATOM   1902   CG1   VAL   A   224   58.512   −2.050   34.231   1.00   22.61   C       ATOM   1903   CG2   VAL   A   224   57.161   −0.491   35.650   1.00   23.44   C       ATOM   1904   N   MET   A   225   61.499   −1.838   36.255   1.00   27.83   N       ATOM   1905   CA   MET   A   225   62.710   −2.577   36.004   1.00   23.69   C       ATOM   1906   C   MET   A   225   62.896   −3.678   37.071   1.00   31.95   C       ATOM   1907   O   MET   A   225   63.290   −4.805   36.785   1.00   24.33   O       ATOM   1908   CB   MET   A   225   63.902   −1.604   36.056   1.00   21.34   C       ATOM   1909   CG   MET   A   225   65.295   −2.296   35.999   1.00   17.83   C       ATOM   1910   SD   MET   A   225   65.750   −2.958   34.306   1.00   23.33   S       ATOM   1911   CE   MET   A   225   67.080   −1.896   33.785   1.00   16.46   C       ATOM   1912   N   GLU   A   226   62.644   −3.319   38.316   1.00   19.54   N       ATOM   1913   CA   GLU   A   226   62.988   −4.161   39.428   1.00   21.58   C       ATOM   1914   C   GLU   A   226   61.999   −5.200   39.918   1.00   30.77   C       ATOM   1915   O   GLU   A   226   62.308   −6.012   40.780   1.00   29.39   O       ATOM   1916   CB   GLU   A   226   63.613   −3.323   40.547   1.00   20.47   C       ATOM   1917   CG   GLU   A   226   64.937   −2.673   40.122   1.00   23.03   C       ATOM   1918   CD   GLU   A   226   65.504   −1.809   41.208   1.00   32.62   C       ATOM   1919   OE1   GLU   A   226   64.721   −1.455   42.122   1.00   26.12   O       ATOM   1920   OE2   GLU   A   226   66.711   −1.479   41.152   1.00   17.67   O       ATOM   1921   N   LEU   A   227   60.837   −5.248   39.295   1.00   34.11   N       ATOM   1922   CA   LEU   A   227   59.883   −6.296   39.642   1.00   35.26   C       ATOM   1923   C   LEU   A   227   60.537   −7.644   39.320   1.00   27.91   C       ATOM   1924   O   LEU   A   227   61.291   −7.766   38.340   1.00   19.89   O       ATOM   1925   CB   LEU   A   227   58.693   −6.236   38.678   1.00   36.48   C       ATOM   1926   CG   LEU   A   227   57.381   −5.569   38.955   1.00   40.30   C       ATOM   1927   CD1   LEU   A   227   57.697   −4.194   39.382   1.00   42.04   C       ATOM   1928   CD2   LEU   A   227   56.610   −5.577   37.647   1.00   46.21   C       ATOM   1929   N   ALA   A   228   60.026   −8.688   39.955   1.00   27.15   N       ATOM   1930   CA   ALA   A   228   60.425   −10.051   39.616   1.00   25.26   C       ATOM   1931   C   ALA   A   228   59.801   −10.435   38.279   1.00   27.93   C       ATOM   1932   O   ALA   A   228   58.624   −10.093   37.934   1.00   31.26   O       ATOM   1933   CB   ALA   A   228   60.003   −11.052   40.703   1.00   22.05   C       ATOM   1934   N   HIS   A   229   60.624   −11.160   37.539   1.00   27.05   N       ATOM   1935   CA   HIS   A   229   60.275   −11.605   36.222   1.00   24.42   C       ATOM   1936   C   HIS   A   229   58.905   −12.260   36.184   1.00   21.74   C       ATOM   1937   O   HIS   A   229   58.015   −11.851   35.398   1.00   22.22   O       ATOM   1938   CB   HIS   A   229   61.351   −12.520   35.698   1.00   17.71   C       ATOM   1939   CG   HIS   A   229   61.284   −12.701   34.220   1.00   27.24   C       ATOM   1940   ND1   HIS   A   229   61.060   −11.650   33.350   1.00   34.38   N       ATOM   1941   CD2   HIS   A   229   61.292   −13.821   33.465   1.00   31.45   C       ATOM   1942   CB1   HIS   A   229   60.992   −12.113   32.115   1.00   30.50   C       ATOM   1943   NE2   HIS   A   229   61.124   −13.427   32.159   1.00   35.23   N       ATOM   1944   N   GLU   A   230   58.681   −13.161   37.140   1.00   20.24   N       ATOM   1945   CA   GLU   A   230   57.425   −13.895   37.209   1.00   29.41   C       ATOM   1946   C   GLU   A   230   56.181   −13.051   37.341   1.00   22.20   C       ATOM   1947   O   GLU   A   230   55.159   −13.359   36.679   1.00   17.78   O       ATOM   1948   CB   GLU   A   230   57.464   −14.997   38.274   1.00   38.51   C       ATOM   1949   CG   GLU   A   230   58.085   −14.582   39.567   1.00   63.09   C       ATOM   1950   CD   GLU   A   230   57.036   −14.473   40.661   1.00   100.00   C       ATOM   1951   OE1   GLU   A   230   55.859   −14.872   40.400   1.00   100.00   O       ATOM   1952   OE2   GLU   A   230   57.409   −14.003   41.768   1.00   81.48   O       ATOM   1953   N   VAL   A   231   56.272   −12.004   38.182   1.00   16.53   N       ATOM   1954   CA   VAL   A   231   55.202   −11.029   38.356   1.00   20.23   C       ATOM   1955   C   VAL   A   231   55.009   −10.164   37.102   1.00   24.45   C       ATOM   1956   O   VAL   A   231   53.864   −9.834   36.705   1.00   2 1.00   O       ATOM   1957   CB   VAL   A   231   55.541   −10.057   39.426   1.00   28.61   C       ATOM   1958   CG1   VAL   A   231   54.362   −9.098   39.610   1.00   29.78   C       ATOM   1959   CG2   VAL   A   231   55.881   −10.757   40.677   1.00   28.96   C       ATOM   1960   N   TRP   A   232   56.133   −9.798   36.486   1.00   17.17   N       ATOM   1961   CA   TRP   A   232   56.052   −9.044   35.262   1.00   21.52   C       ATOM   1962   C   TRP   A   232   55.388   −9.844   34.156   1.00   20.53   C       ATOM   1963   O   TRP   A   232   54.588   −9.306   33.380   1.00   24.31   O       ATOM   1964   CB   TRP   A   232   57.438   −8.644   34.801   1.00   29.88   C       ATOM   1965   CG   TRP   A   232   57.430   −7.843   33.500   1.00   27.65   C       ATOM   1966   CD1   TRP   A   232   57.184   −6.464   33.356   1.00   25.42   C       ATOM   1967   CD2   TRP   A   232   57.714   −8.336   32.169   1.00   27.75   C       ATOM   1968   NE1   TRP   A   232   57.325   −6.095   32.033   1.00   22.53   N       ATOM   1969   CB2   TRP   A   232   57.655   −7.203   31.279   1.00   25.11   C       ATOM   1970   CB3   TRP   A   232   58.037   −9.603   31.640   1.00   22.72   C       ATOM   1971   CZ2   TRP   A   232   57.917   −7.316   29.879   1.00   17.23   C       ATOM   1972   CZ3   TRP   A   232   58.238   −9.720   30.223   1.00   25.97   C       ATOM   1973   CH2   TRP   A   232   58.154   −8.581   29.368   1.00   22.07   C       ATOM   1974   N   LEU   A   233   55.749   −11.121   34.018   1.00   23.80   N       ATOM   1975   CA   LEU   A   233   55.141   −11.949   32.937   1.00   24.78   C       ATOM   1976   C   LEU   A   233   53.652   −12.118   33.122   1.00   24.51   C       ATOM   1977   O   LEU   A   233   52.865   −12.075   32.163   1.00   28.50   O       ATOM   1978   CB   LEU   A   233   55.765   −13.348   32.820   1.00   26.20   C       ATOM   1979   CG   LEU   A   233   57.250   −13.505   32.503   1.00   19.39   C       ATOM   1980   CD1   LEU   A   233   57.745   −14.850   33.023   1.00   19.90   C       ATOM   1981   CD2   LEU   A   233   57.561   −13.287   31.017   1.00   16.01   C       ATOM   1982   N   GLU   A   234   53.298   −12.343   34.372   1.00   25.45   N       ATOM   1983   CA   GLU   A   234   51.929   −12.523   34.822   1.00   30.04   C       ATOM   1984   C   GLU   A   234   51.128   −11.319   34.367   1.00   35.69   C       ATOM   1985   O   GLU   A   234   49.926   −11.390   34.052   1.00   28.25   O       ATOM   1986   CB   GLU   A   234   52.007   −12.468   36.344   1.00   37.30   C       ATOM   1987   CG   GLU   A   234   50.908   −13.133   37.118   1.00   45.39   C       ATOM   1988   CD   GLU   A   234   51.112   −12.881   38.601   1.00   100.00   C       ATOM   1989   OE1   GLU   A   234   52.240   −13.137   39.104   1.00   99.09   O       ATOM   1990   OE2   GLU   A   234   50.211   −12.257   39.211   1.00   100.00   O       ATOM   1991   N   ASN   A   235   51.802   −10.184   34.364   1.00   25.04   N       ATOM   1992   CA   ASN   A   235   51.109   −8.986   33.992   1.00   26.17   C       ATOM   1993   C   ASN   A   235   51.280   −8.494   32.571   1.00   30.46   C       ATOM   1994   O   ASN   A   235   50.824   −7.393   32.259   1.00   22.90   O       ATOM   1995   CB   ASN   A   235   51.427   −7.895   34.981   1.00   29.23   C       ATOM   1996   CG   ASN   A   235   50.878   −8.197   36.342   1.00   39.27   C       ATOM   1997   OD1   ASN   A   235   49.722   −7.882   36.628   1.00   29.06   O       ATOM   1998   ND2   ASN   A   235   51.653   −8.934   37.140   1.00   40.22   N       ATOM   1999   N   THR   A   236   51.935   −9.268   31.708   1.00   20.97   N       ATOM   2000   CA   THR   A   236   52.108   −8.795   30.344   1.00   22.30   C       ATOM   2001   C   THR   A   236   51.867   −9.943   29.419   1.00   29.74   C       ATOM   2002   O   THR   A   236   51.551   −11.033   29.895   1.00   21.23   O       ATOM   2003   CB   THR   A   236   53.545   −8.306   30.161   1.00   22.73   C       ATOM   2004   OG1   THR   A   236   54.422   −9.325   30.636   1.00   21.23   O       ATOM   2005   CG2   THR   A   236   53.801   −7.048   31.041   1.00   19.69   C       ATOM   2006   N   GLN   A   237   52.003   −9.699   28.109   1.00   22.23   N       ATOM   2007   CA   GLN   A   237   52.097   −10.783   27.122   1.00   16.69   C       ATOM   2008   C   GLN   A   237   53.335   −10.507   26.331   1.00   21.02   C       ATOM   2009   O   GLN   A   237   53.729   −9.362   26.204   1.00   22.19   O       ATOM   2010   CB   GLN   A   237   50.913   −10.999   26.189   1.00   8.23   C       ATOM   2011   CG   GLN   A   237   49.639   −11.096   26.904   1.00   21.04   C       ATOM   2012   CD   GLN   A   237   48.907   −9.862   26.606   1.00   62.07   C       ATOM   2013   OE1   GLN   A   237   48.437   ‘9.712   25.460   1.00   59.32   O       ATOM   2014   NE2   GLN   A   237   49.220   −8.847   27.388   1.00   37.82   N       ATOM   2015   N   PRO   A   238   54.002   ‘11.579   25.917   1.00   28.76   N       ATOM   2016   CA   PRO   A   238   55.275   −11.438   25.246   1.00   30.28   C       ATOM   2017   C   PRO   A   238   55.194   −10.643   23.958   1.00   29.08   C       ATOM   2018   O   PRO   A   238   56.181   −10.029   23.600   1.00   15.95   O       ATOM   2019   CB   PRO   A   238   55.733   −12.879   25.011   1.00   22.54   C       ATOM   2020   CG   PRO   A   238   54.898   −13.710   25.886   1.00   18.92   C       ATOM   2021   CD   PRO   A   238   53.626   −12.998   26.068   1.00   11.75   C       ATOM   2022   N   MET   A   239   54.041   −10.635   23.286   1.00   17.26   N       ATOM   2023   CA   MET   A   239   53.924   −9.807   22.104   1.00   17.85   C       ATOM   2024   C   MET   A   239   53.109   −8.509   22.362   1.00   18.63   C       ATOM   2025   O   MET   A   239   52.792   −7.741   21.419   1.00   16.82   O       ATOM   2026   CB   MET   A   239   53.460   −10.588   20.881   1.00   15.22   C       ATOM   2027   CG   MET   A   239   54.536   −11.534   20.261   1.00   12.90   C       ATOM   2028   SD   MET   A   239   53.994   −12.534   18.808   1.00   17.49   S       ATOM   2029   CE   MET   A   239   54.350   −11.357   17.422   1.00   13.12   C       ATOM   2030   N   LEU   A   240   52.847   −8.252   23.646   1.00   18.55   N       ATOM   2031   CA   LEU   A   240   52.159   −7.037   24.131   1.00   16.68   C       ATOM   2032   C   LEU   A   240   52.774   −6.733   25.493   1.00   11.82   C       ATOM   2033   O   LEU   A   240   52.124   −6.803   26.549   1.00   13.84   O       ATOM   2034   CB   LEU   A   240   50.645   −7.249   24.240   1.00   16.91   C       ATOM   2035   CG   LEU   A   240   49.646   −6.120   23.852   1.00   22.29   C       ATOM   2036   CD1   LEU   A   240   48.968   −5.488   25.033   1.00   25.51   C       ATOM   2037   CD2   LEU   A   240   50.070   −5.059   22.815   1.00   28.07   C       ATOM   2038   N   SER   A   241   54.076   −6.467   25.456   1.00   13.09   N       ATOM   2039   CA   SER   A   241   54.842   −6.315   26.682   1.00   24.20   C       ATOM   2040   C   SER   A   241   54.947   −4.938   27.377   1.00   30.52   C       ATOM   2041   O   SER   A   241   55.363   −4.854   28.547   1.00   17.02   O       ATOM   2042   CB   SER   A   241   56.247   −6.900   26.495   1.00   14.04   C       ATOM   2043   OG   SER   A   241   57.062   −6.144   25.598   1.00   13.95   O       ATOM   2044   N   HIS   A   242   54.661   −3.861   26.659   1.00   17.87   N       ATOM   2045   CA   HIS   A   242   54.894   −2.548   27.221   1.00   13.55   C       ATOM   2046   C   HIS   A   242   53.990   −2.254   28.373   1.00   13.70   C       ATOM   2047   O   HIS   A   242   52.974   −2.885   28.539   1.00   13.29   O       ATOM   2048   CB   HIS   A   242   54.826   −1.430   26.130   1.00   16.05   C       ATOM   2049   CG   HIS   A   242   53.595   −1.504   25.272   1.00   18.88   C       ATOM   2050   ND1   HIS   A   242   52.591   −0.553   25.326   1.00   23.24   N       ATOM   2051   CD2   HIS   A   242   53.165   −2.461   24.413   1.00   13.19   C       ATOM   2052   CE1   HIS   A   242   51.629   −0.887   24.483   1.00   17.44   C       ATOM   2053   NE2   HIS   A   242   51.962   −2.031   23.901   1.00   19.54   N       ATOM   2054   N   ILE   A   243   54.310   −1.203   29.095   1.00   15.84   N       ATOM   2055   CA   ILE   A   243   53.492   −0.809   30.192   1.00   19.10   C       ATOM   2056   C   ILE   A   243   53.336   0.714   30.191   1.00   23.23   C       ATOM   2057   O   ILE   A   243   54.312   1.406   30.385   1.00   12.10   O       ATOM   2058   CB   ILE   A   243   54.166   −1.273   31.482   1.00   24.62   C       ATOM   2059   CG1   ILE   A   243   54.014   −2.783   31.576   1.00   25.60   C       ATOM   2060   CG2   ILE   A   243   53.497   −0.665   32.735   1.00   17.37   C       ATOM   2061   CD1   ILE   A   243   54.725   −3.365   32.714   1.00   14.62   C       ATOM   2062   N   ASN   A   244   52.112   1.217   30.013   1.00   16.43   N       ATOM   2063   CA   ASN   A   244   51.824   2.689   30.038   1.00   18.99   C       ATOM   2064   C   ASN   A   244   52.252   3.292   31.348   1.00   18.83   C       ATOM   2065   O   ASN   A   244   51.965   2.727   32.405   1.00   19.58   O       ATOM   2066   CB   ASN   A   244   50.304   2.987   29.910   1.00   15.67   C       ATOM   2067   CG   ASN   A   244   49.768   2.702   28.517   1.00   14.57   C       ATOM   2068   OD1   ASN   A   244   50.546   2.583   27.580   1.00   13.64   O       ATOM   2069   ND2   ASN   A   244   48.443   2.491   28.393   1.00   10.16   N       ATOM   2070   N   VAL   A   245   52.800   4.499   31.326   1.00   13.50   N       ATOM   2071   CA   VAL   A   245   53.159   5.134   32.602   1.00   13.49   C       ATOM   2072   C   VAL   A   245   52.528   6.566   32.644   1.00   16.25   C       ATOM   2073   O   VAL   A   245   52.786   7.405   31.770   1.00   15.20   O       ATOM   2074   CB   VAL   A   245   54.754   5.163   32.810   1.00   21.07   C       ATOM   2075   CG1   VAL   A   245   55.154   6.085   33.937   1.00   15.08   C       ATOM   2076   CG2   VAL   A   245   55.280   3.817   33.143   1.00   15.82   C       ATOM   2077   N   GLY   A   246   51.696   6.843   33.649   1.00   14.03   N       ATOM   2078   CA   GLY   A   246   51.027   8.136   33.707   1.00   16.87   C       ATOM   2079   C   GLY   A   246   50.146   8.203   34.939   1.00   26.95   C       ATOM   2080   O   GLY   A   246   50.323   7.401   35.850   1.00   23.04   O       ATOM   2081   N   THR   A   247   49.207   9.161   34.963   1.00   21.44   N       ATOM   2082   CA   THR   A   247   48.232   9.276   36.063   1.00   21.39   C       ATOM   2083   C   THR   A   247   46.868   8.677   35.673   1.00   24.08   C       ATOM   2084   O   THR   A   247   46.069   8.306   36.508   1.00   21.03   O       ATOM   2085   CB   THR   A   247   47.988   10.730   36.404   1.00   22.24   C       ATOM   2086   OG1   THR   A   247   47.409   11.389   35.265   1.00   18.62   O       ATOM   2087   CG2   THR   A   247   49.275   11.378   36.724   1.00   18.99   C       ATOM   2088   N   GLY   A   248   46.583   8.651   34.384   1.00   24.95   N       ATOM   2089   CA   GLY   A   248   45.319   8.143   33.924   1.00   22.61   C       ATOM   2090   C   GLY   A   248   44.223   9.160   34.226   1.00   21.42   C       ATOM   2091   O   GLY   A   248   43.059   8.866   34.137   1.00   25.70   O       ATOM   2092   N   VAL   A   249   44.615   10.386   34.521   1.00   30.72   N       ATOM   2093   CA   VAL   A   249   43.673   11.464   34.827   1.00   26.09   C       ATOM   2094   C   VAL   A   249   43.747   12.596   33.786   1.00   32.70   C       ATOM   2095   O   VAL   A   249   44.853   13.006   33.387   1.00   26.92   O       ATOM   2096   CB   VAL   A   249   44.020   12.085   36.214   1.00   38.59   C       ATOM   2097   CG1   VAL   A   249   43.225   13.324   36.470   1.00   36.11   C       ATOM   2098   CG2   VAL   A   249   43.782   11.083   37.306   1.00   41.30   C       ATOM   2099   N   ASP   A   250   42.581   13.125   33.397   1.00   27.95   N       ATOM   2100   CA   ASP   A   250   42.488   14.232   32.439   1.00   20.64   C       ATOM   2101   C   ASP   A   250   42.611   15.581   33.155   1.00   27.63   C       ATOM   2102   O   ASP   A   250   42.188   15.783   34.308   1.00   26.23   O       ATOM   2103   CB   ASP   A   250   41.075   14.302   31.827   1.00   23.89   C       ATOM   2104   CG   ASP   A   250   40.768   13.180   30.850   1.00   39.52   C       ATOM   2105   OD1   ASP   A   250   41.283   13.184   29.688   1.00   39.96   O       ATOM   2106   OD2   ASP   A   250   39.767   12.501   31.153   1.00   45.34   O       ATOM   2107   N   CYS   A   251   43.0291   6.566   32.388   1.00   20.12   N       ATOM   2108   CA   CYS   A   251   42.962   17.906   32.851   1.00   27.20   C       ATOM   2109   C   CYS   A   251   42.918   18.779   31.577   1.00   26.47   C       ATOM   2110   O   CYS   A   251   43.699   18.560   30.633   1.00   19.45   O       ATOM   2111   CB   CYS   A   251   44.148   18.157   33.778   1.00   34.86   C       ATOM   2112   SG   CYS   A   251   45.129   19.619   33.453   1.00   29.47   S       ATOM   2113   N   THR   A   252   41.932   19.673   31.494   1.00   14.85   N       ATOM   2114   CA   THR   A   252   41.834   20.588   30.335   1.00   21.21   C       ATOM   2115   C   THR   A   252   42.999   21.592   30.236   1.00   20.53   C       ATOM   2116   O   THR   A   252   43.657   21.926   31.249   1.00   15.24   O       ATOM   2117   CB   THR   A   252   40.506   21.407   30.329   1.00   32.08   C       ATOM   2118   OG1   THR   A   252   40.460   22.304   31.447   1.00   19.26   O       ATOM   2119   CG2   THR   A   252   39.309   20.495   30.372   1.00   13.91   C       ATOM   2120   N   ILE   A   253   43.228   22.095   29.024   1.00   14.81   N       ATOM   2121   CA   ILE   A   253   44.264   23.118   28.812   1.00   16.90   C       ATOM   2122   C   ILE   A   253   43.934   24.383   29.627   1.00   23.41   C       ATOM   2123   O   ILE   A   253   44.834   25.012   30.247   1.00   15.27   O       ATOM   2124   CB   ILE   A   253   44.404   23.452   27.302   1.00   24.05   C       ATOM   2125   CG1   ILE   A   253   44.862   22.200   26.561   1.00   27.33   C       ATOM   2126   CG2   ILE   A   253   45.473   24.479   27.077   1.00   9.22   C       ATOM   2127   CD1   ILE   A   253   45.662   21.276   27.452   1.00   49.56   C       ATOM   2128   N   ARG   A   254   42.637   24.709   29.707   1.00   19.56   N       ATOM   2129   CA   ARG   A   254   42.228   25.865   30.522   1.00   19.41   C       ATOM   2130   C   ARG   A   254   42.712   25.713   31.970   1.00   18.10   C       ATOM   2131   O   ARG   A   254   43.311   26.616   32.515   1.00   13.89   O       ATOM   2132   CB   ARG   A   254   40.704   26.101   30.480   1.00   15.98   C       ATOM   2133   CG   ARG   A   254   40.282   27.378   31.255   1.00   9.96   C       ATOM   2134   CD   ARG   A   254   38.809   27.702   31.218   1.00   24.79   C       ATOM   2135   NE   ARG   A   254   38.498   28.414   29.997   1.00   29.42   N       ATOM   2136   CZ   ARG   A   254   38.693   29.723   29.794   1.00   59.85   C       ATOM   2137   NH1   ARG   A   254   39.194   30.527   30.732   1.00   42.58   N       ATOM   2138   NH2   ARG   A   254   38.377   30.245   28.620   1.00   18.44   N       ATOM   2139   N   ASP   A   255   42.406   24.564   32.586   1.00   20.22   N       ATOM   2140   CA   ASP   A   255   42.795   24.205   33.974   1.00   16.48   C       ATOM   2141   C   ASP   A   255   44.321   24.372   34.069   1.00   22.43   C       ATOM   2142   O   ASP   A   255   44.868   24.897   35.060   1.00   18.53   O       ATOM   2143   CB   ASP   A   255   42.478   22.686   34.157   1.00   19.17   C       ATOM   2144   CG   ASP   A   255   42.144   22.246   35.610   1.00   47.08   C       ATOM   2145   OD1   ASP   A   255   41.780   23.090   36.429   1.00   49.66   O       ATOM   2146   OD2   ASP   A   255   42.020   21.016   35.880   1.00   48.12   O       ATOM   2147   N   LEU   A   256   45.014   23.809   33.078   1.00   15.98   N       ATOM   2148   CA   LEU   A   256   46.465   23.844   33.069   1.00   21.76   C       ATOM   2149   C   LEU   A   256   47.020   25.275   33.076   1.00   16.79   C       ATOM   2150   O   LEU   A   256   47.825   25.697   33.946   1.00   15.24   O       ATOM   2151   CB   LEU   A   256   46.967   23.056   31.859   1.00   23.33   C       ATOM   2152   CG   LEU   A   256   48.491   23.100   31.765   1.00   26.80   C       ATOM   2153   CD1   LEU   A   256   49.171   22.334   32.984   1.00   17.13   C       ATOM   2154   CD2   LEU   A   256   49.040   22.724   30.346   1.00   15.42   C       ATOM   2155   N   ALA   A   257   46.520   26.048   32.140   1.00   13.77   N       ATOM   2156   CA   ALA   A   257   46.938   27.436   32.025   1.00   12.70   C       ATOM   2157   C   ALA   A   257   46.656   28.237   33.267   1.00   10.73   C       ATOM   2158   O   ALA   A   257   47.451   29.073   33.672   1.00   20.33   O       ATOM   2159   CB   ALA   A   257   46.208   28.073   30.834   1.00   13.34   C       ATOM   2160   N   GLN   A   258   45.470   28.080   33.835   1.00   12.40   N       ATOM   2161   CA   GLN   A   258   45.102   28.911   34.981   1.00   8.39   C       ATOM   2162   C   GLN   A   258   45.879   28.480   36.166   1.00   13.48   C       ATOM   2163   O   GLN   A   258   46.178   29.281   37.029   1.00   22.96   O       ATOM   2164   CB   GLN   A   258   43.614   28.761   35.305   1.00   16.12   C       ATOM   2165   CG   GLN   A   258   42.674   29.096   34.130   1.00   30.19   C       ATOM   2166   CD   GLN   A   258   42.574   30.585   33.781   1.00   37.29   C       ATOM   2167   OE1   GLN   A   258   42.911   31.471   34.610   1.00   21.24   O       ATOM   2168   NE2   GLN   A   258   42.021   30.876   32.572   1.00   15.94   N       ATOM   2169   N   THR   A   259   46.179   27.182   36.232   1.00   16.21   N       ATOM   2170   CA   THR   A   259   46.982   26.678   37.336   1.00   16.85   C       ATOM   2171   C   THR   A   259   48.410   27.186   37.233   1.00   20.56   C       ATOM   2172   O   THR   A   259   49.002   27.621   38.214   1.00   21.44   O       ATOM   2173   CB   THR   A   259   47.066   25.192   37.361   1.00   27.56   C       ATOM   2174   OG1   THR   A   259   45.752   24.620   37.509   1.00   20.92   O       ATOM   2175   CG2   THR   A   259   47.936   24.796   38.545   1.00   12.85   C       ATOM   2176   N   ILE   A   260   48.952   27.170   36.028   1.00   19.96   N       ATOM   2177   CA   ILE   A   260   50.292   27.704   35.839   1.00   23.01   C       ATOM   2178   C   ILE   A   260   50.313   29.180   36.225   1.00   31.73   C       ATOM   2179   O   ILE   A   260   51.211   29.627   36.993   1.00   25.90   O       ATOM   2180   CB   ILE   A   260   50.835   27.456   34.390   1.00   22.46   C       ATOM   2181   CG1   ILE   A   260   51.153   25.940   34.232   1.00   24.12   C       ATOM   2182   CG2   ILE   A   260   52.099   28.361   34.106   1.00   13.47   C       ATOM   2183   CD1   ILE   A   260   51.501   25.443   32.810   1.00   12.58   C       ATOM   2184   N   ALA   A   261   49.28   029.910   35.764   1.00   15.35   N       ATOM   2185   CA   ALA   A   261   49.177   31.355   36.048   1.00   16.00   C       ATOM   2186   C   ALA   A   261   49.316   31.604   37.550   1.00   20.58   C       ATOM   2187   O   ALA   A   261   50.104   32.443   37.987   1.00   16.09   O       ATOM   2188   CB   ALA   A   261   47.832   31.958   35.487   1.00   13.65   C       ATOM   2189   N   LYS   A   262   48.551   30.843   38.323   1.00   11.50   N       ATOM   2190   CA   LYS   A   262   48.578   30.905   39.770   1.00   10.13   C       ATOM   2191   C   LYS   A   262   49.968   30.460   40.296   1.00   28.08   C       ATOM   2192   O   LYS   A   262   50.503   31.084   41.205   1.00   29.37   O       ATOM   2193   CB   LYS   A   262   47.453   30.032   40.335   1.00   12.50   C       ATOM   2194   CG   LYS   A   262   47.332   29.962   41.888   1.00   16.51   C       ATOM   2195   CD   LYS   A   262   46.092   29.092   42.371   1.00   46.61   C       ATOM   2196   CE   LYS   A   262   46.344   27.555   42.661   1.00   99.70   C       ATOM   2197   NZ   LYS   A   262   45.157   26.703   43.200   1.00   36.59   N       ATOM   2198   N   VAL   A   263   50.589   29.443   39.705   1.00   17.44   N       ATOM   2199   CA   VAL   A   263   51.915   29.039   40.171   1.00   16.72   C       ATOM   2200   C   VAL   A   263   52.997   30.170   39.997   1.00   32.12   C       ATOM   2201   O   VAL   A   263   53.871   30.412   40.834   1.00   21.18   O       ATOM   2202   CS   VAL   A   263   52.389   27.709   39.476   1.00   16.35   C       ATOM   2203   CG1   VAL   A   263   53.920   27.518   39.647   1.00   11.83   C       ATOM   2204   CG2   VAL   A   263   51.646   26.522   40.093   1.00   14.99   C       ATOM   2205   N   VAL   A   264   52.913   30.899   38.909   1.00   21.75   N       ATOM   2206   CA   VAL   A   264   53.917   31.877   38.653   1.00   19.81   C       ATOM   2207   C   VAL   A   264   53.719   33.208   39.377   1.00   35.79   C       ATOM   2208   O   VAL   A   264   54.632   34.032   39.482   1.00   28.99   O       ATOM   2209   CS   VAL   A   264   54.059   32.014   37.175   1.00   24.27   C       ATOM   2210   CG1   VAL   A   264   54.728   33.269   36.822   1.00   33.58   C       ATOM   2211   CG2   VAL   A   264   54.840   30.808   36.674   1.00   23.01   C       ATOM   2212   N   GLY   A   265   52.550   33.378   39.969   1.00   25.30   N       ATOM   2213   CA   GLY   A   265   52.241   34.620   40.636   1.00   24.14   C       ATOM   2214   C   GLY   A   265   51.730   35.694   39.632   1.00   35.03   C       ATOM   2215   O   GLY   A   265   51.773   36.911   39.962   1.00   33.71   O       ATOM   2216   N   TYR   A   266   51.294   35.257   38.428   1.00   26.25   N       ATOM   2217   CA   TYR   A   266   50.698   36.151   37.373   1.00   26.55   C       ATOM   2218   C   TYR   A   266   49.364   36.745   37.818   1.00   31.01   C       ATOM   2219   O   TYR   A   266   48.532   36.067   38.456   1.00   27.99   O       ATOM   2220   CB   TYR   A   266   50.501   35.463   36.008   1.00   24.31   C       ATOM   2221   CG   TYR   A   266   49.994   36.381   34.884   1.00   28.64   C       ATOM   2222   CD1   TYR   A   266   50.670   37.582   34.542   1.00   35.05   C       ATOM   2223   CD2   TYR   A   266   48.860   36.038   34.118   1.00   22.60   C       ATOM   2224   CE1   TYR   A   266   50.212   38.434   33.472   1.00   20.73   C       ATOM   2225   CE2   TYR   A   266   48.428   36.859   33.012   1.00   20.91   C       ATOM   2226   CZ   TYR   A   266   49.088   38.062   32.735   1.00   23.85   C       ATOM   2227   OH   TYR   A   266   48.622   38.851   31.710   1.00   33.40   O       ATOM   2228   N   LYS   A   267   49.217   38.043   37.604   1.00   25.72   N       ATOM   2229   CA   LYS   A   267   47.988   38.697   38.009   1.00   30.77   C       ATOM   2230   C   LYS   A   267   47.217   39.280   36.798   1.00   28.85   C       ATOM   2231   O   LYS   A   267   46.179   39.894   36.949   1.00   31.17   O       ATOM   2232   CB   LYS   A   267   48.279   39.741   39.092   1.00   27.13   C       ATOM   2233   CG   LYS   A   267   48.728   39.128   40.403   1.00   23.18   C       ATOM   2234   CD   LYS   A   267   48.420   40.096   41.562   1.00   30.98   C       ATOM   2235   CE   LYS   A   267   47.933   39.358   42.820   1.00   48.52   C       ATOM   2236   NZ   LYS   A   267   47.005   38.208   42.505   1.00   100.00   N       ATOM   2237   N   GLY   A   268   47.716   39.054   35.594   1.00   22.67   N       ATOM   2238   CA   GLY   A   268   47.019   39.518   34.394   1.00   21.38   C       ATOM   2239   C   GLY   A   268   45.856   38.568   34.085   1.00   31.03   C       ATOM   2240   O   GLY   A   268   45.455   37.728   34.911   1.00   19.71   O       ATOM   2241   N   ARG   A   269   45.387   38.645   32.849   1.00   30.40   N       ATOM   2242   CA   ARG   A   269   44.263   37.846   32.399   1.00   26.47   C       ATOM   2243   C   ARG   A   269   44.680   36.705   31.489   1.00   22.35   C       ATOM   2244   O   ARG   A   269   45.378   36.926   30.524   1.00   22.75   O       ATOM   2245   CB   ARG   A   269   43.297   38.753   31.626   1.00   22.65   C       ATOM   2246   CG   ARG   A   269   42.201   39.390   32.463   1.00   24.21   C       ATOM   2247   CD   ARG   A   269   40.936   39.465   31.568   1.00   83.45   C       ATOM   2248   NE   ARG   A   269   40.113   40.676   31.762   1.00   100.00   N       ATOM   2249   CZ   ARG   A   269   38.808   40.751   31.431   1.00   100.00   C       ATOM   2250   NH1   ARG   A   269   38.201   39.691   30.921   1.00   99.93   N       ATOM   2251   NH2   ARG   A   269   38.094   41.865   31.663   1.00   100.00   N       ATOM   2252   N   VAL   A   270   44.195   35.494   31.758   1.00   19.87   N       ATOM   2253   CA   VAL   A   270   44.468   34.389   30.856   1.00   24.82   C       ATOM   2254   C   VAL   A   270   43.319   34.456   29.824   1.00   22.51   C       ATOM   2255   O   VAL   A   270   42.145   34.501   30.181   1.00   25.79   O       ATOM   2256   CB   VAL   A   270   44.436   32.979   31.571   1.00   24.03   C       ATOM   2257   CG1   VAL   A   270   44.576   31.861   30.533   1.00   20.72   C       ATOM   2258   CG2   VAL   A   270   45.506   32.849   32.639   1.00   11.27   C       ATOM   2259   N   VAL   A   271   43.660   34.409   28.554   1.00   25.18   N       ATOM   2260   CA   VAL   A   271   42.666   34.492   27.487   1.00   28.32   C       ATOM   2261   C   VAL   A   271   42.819   33.370   26.442   1.00   24.89   C       ATOM   2262   O   VAL   A   271   43.923   33.115   25.980   1.00   21.98   O       ATOM   2263   CB   VAL   A   271   42.901   35.813   26.736   1.00   29.25   C       ATOM   2264   CG1   VAL   A   271   42.256   35.773   25.370   1.00   31.91   C       ATOM   2265   CG2   VAL   A   271   42.421   36.989   27.565   1.00   18.72   C       ATOM   2266   N   PHE   A   272   41.716   32.758   26.019   1.00   26.14   N       ATOM   2267   CA   PHE   A   272   41.752   31.747   24.963   1.00   24.34   C       ATOM   2268   C   PHE   A   272   41.236   32.266   23.623   1.00   28.95   C       ATOM   2269   O   PHE   A   272   40.155   32.826   23.582   1.00   22.01   O       ATOM   2270   CB   PHE   A   272   40.960   30.506   25.391   1.00   20.97   C       ATOM   2271   CG   PHE   A   272   41.764   29.570   26.243   1.00   21.77   C       ATOM   2272   CD1   PHE   A   272   41.940   29.842   27.610   1.00   14.60   C       ATOM   2273   CD2   PHE   A   272   42.504   28.550   25.656   1.00   22.19   C       ATOM   2274   CE1   PHE   A   272   42.763   29.041   28.434   1.00   17.89   C       ATOM   2275   CE2   PHE   A   272   43.336   27.726   26.454   1.00   27.64   C       ATOM   2276   CZ   PHE   A   272   43.478   27.979   27.851   1.00   25.14   C       ATOM   2277   N   ASP   A   273   42.012   32.114   22.542   1.00   29.45   N       ATOM   2278   CA   ASP   A   273   41.557   32.536   21.214   1.00   22.33   C       ATOM   2279   C   ASP   A   273   40.896   31.365   20.493   1.00   25.67   C       ATOM   2280   O   ASP   A   273   41.539   30.570   19.793   1.00   17.81   O       ATOM   2281   CB   ASP   A   273   42.672   33.114   20.343   1.00   21.45   C       ATOM   2282   CG   ASP   A   273   42.131   33.626   18.990   1.00   26.89   C       ATOM   2283   OD1   ASP   A   273   40.975   33.249   18.598   1.00   27.76   O       ATOM   2284   OD2   ASP   A   273   42.838   34.421   18.327   1.00   30.06   O       ATOM   2285   N   ALA   A   274   39.589   31.284   20.649   1.00   15.59   N       ATOM   2286   CA   ALA   A   274   38.932   30.128   20.128   1.00   23.75   C       ATOM   2287   C   ALA   A   274   38.853   30.168   18.653   1.00   32.30   C       ATOM   2288   O   ALA   A   274   38.284   29.256   18.029   1.00   29.37   O       ATOM   2289   CB   ALA   A   274   37.567   29.905   20.777   1.00   18.87   C       ATOM   2290   N   SER   A   275   39.372   31.243   18.081   1.00   21.10   N       ATOM   2291   CA   SER   A   275   39.343   31.288   16.631   1.00   26.90   C       ATOM   2292   C   SER   A   275   40.390   30.300   16.116   1.00   43.37   C       ATOM   2293   O   SER   A   275   40.421   29.949   14.927   1.00   46.32   O       ATOM   2294   CB   SER   A   275   39.547   32.683   16.074   1.00   15.19   C       ATOM   2295   OG   SER   A   275   40.904   33.070   16.078   1.00   28.71   O       ATOM   2296   N   LYS   A   276   41.192   29.780   17.037   1.00   22.98   N       ATOM   2297   CA   LYS   A   276   42.178   28.791   16.638   1.00   23.28   C       ATOM   2298   C   LYS   A   276   41.645   27.405   16.976   1.00   29.73   C       ATOM   2299   O   LYS   A   276   40.992   27.206   18.010   1.00   25.10   O       ATOM   2300   CS   LYS   A   276   43.544   29.051   17.275   1.00   19.19   C       ATOM   2301   CG   LYS   A   276   43.957   30.496   17.218   1.00   32.11   C       ATOM   2302   CD   LYS   A   276   44.062   30.852   15.798   1.00   22.43   C       ATOM   2303   CE   LYS   A   276   44.930   32.067   15.570   1.00   23.18   C       ATOM   2304   NZ   LYS   A   276   45.454   32.117   14.152   1.00   29.42   N       ATOM   2305   N   PRO   A   277   41.892   26.476   16.055   1.00   36.04   N       ATOM   2306   CA   PRO   A   277   41.446   25.087   16.170   1.00   35.93   C       ATOM   2307   C   PRO   A   277   42.022   24.332   17.363   1.00   29.30   C       ATOM   2308   O   PRO   A   277   43.103   24.650   17.885   1.00   30.54   O       ATOM   2309   CB   PRO   A   277   41.975   24.453   14.878   1.00   39.65   C       ATOM   2310   CG   PRO   A   277   43.249   25.261   14.566   1.00   42.90   C       ATOM   2311   CD   PRO   A   277   42.787   26.670   14.892   1.00   37.84   C       ATOM   2312   N   ASP   A   278   41.273   23.339   17.809   1.00   22.35   N       ATOM   2313   CA   ASP   A   278   41.745   22.501   18.903   1.00   22.16   C       ATOM   2314   C   ASP   A   278   42.184   21.189   18.272   1.00   19.66   C       ATOM   2315   O   ASP   A   278   41.905   20.917   17.117   1.00   23.49   O       ATOM   2316   CB   ASP   A   278   40.636   22.241   19.971   1.00   15.09   C       ATOM   2317   CG   ASP   A   278   40.216   23.503   20.702   1.00   22.86   C       ATOM   2318   OD1   ASP   A   278   41.113   24.254   21.096   1.00   25.18   O       ATOM   2319   OD2   ASP   A   278   38.999   23.787   20.812   1.00   39.55   O       ATOM   2320   N   GLY   A   279   42.846   20.355   19.044   1.00   30.65   N       ATOM   2321   CA   GLY   A   279   43.229   19.034   18.546   1.00   33.78   C       ATOM   2322   C   GLY   A   279   42.115   18.099   18.944   1.00   38.10   C       ATOM   2323   O   GLY   A   279   40.963   18.517   19.068   1.00   47.52   O       ATOM   2324   N   THR   A   280   42.419   16.839   19.177   1.00   29.44   N       ATOM   2325   CA   THR   A   280   41.328   15.990   19.587   1.00   26.68   C       ATOM   2326   C   THR   A   280   40.889   16.439   20.972   1.00   23.52   C       ATOM   2327   O   THR   A   280   41.670   17.067   21.713   1.00   23.62   O       ATOM   2328   CB   THR   A   280   41.695   14.492   19.540   1.00   40.78   C       ATOM   2329   OG1   THR   A   280   42.889   14.272   20.296   1.00   25.56   O       ATOM   2330   CG2   THR   A   280   41.893   14.054   18.095   1.00   37.71   C       ATOM   2331   N   PRO   A   281   39.672   16.063   21.346   1.00   25.54   N       ATOM   2332   CA   PRO   A   281   39.129   16.454   22.628   1.00   25.72   C       ATOM   2333   C   PRO   A   281   39.776   15.778   23.800   1.00   26.02   C       ATOM   2334   O   PRO   A   281   39.752   16.314   24.915   1.00   22.68   O       ATOM   2335   CB   PRO   A   281   37.650   15.990   22.559   1.00   28.89   C       ATOM   2336   CG   PRO   A   281   37.417   15.540   21.201   1.00   29.39   C       ATOM   2337   CD   PRO   A   281   38.761   15.138   20.646   1.00   26.82   C       ATOM   2338   N   ARG   A   282   40.281   14.567   23.587   1.00   27.88   N       ATOM   2339   CA   ARG   A   282   40.806   13.817   24.720   1.00   34.08   C       ATOM   2340   C   ARG   A   282   41.977   12.918   24.384   1.00   27.62   C       ATOM   2341   O   ARG   A   282   41.913   12.182   23.425   1.00   23.83   O       ATOM   2342   CB   ARG   A   282   39.676   13.017   25.405   1.00   20.89   C       ATOM   2343   CG   ARG   A   282   40.035   12.467   26.775   1.00   22.81   C       ATOM   2344   CD   ARG   A   282   38.762   11.925   27.442   1.00   26.77   C       ATOM   2345   NE   ARG   A   282   38.963   11.345   28.781   1.00   36.48   N       ATOM   2346   CZ   ARG   A   282   38.518   10.139   29.164   1.00   37.74   C       ATOM   2347   NH1   ARG   A   282   37.813   9.360   28.346   1.00   28.45   N       ATOM   2348   NH2   ARG   A   282   38.754   9.700   30.384   1.00   27.25   N       ATOM   2349   N   LYS   A   283   43.016   12.963   25.223   1.00   28.91   N       ATOM   2350   CA   LYS   A   283   44.217   12.171   25.051   1.00   24.32   C       ATOM   2351   C   LYS   A   283   44.796   11.766   26.404   1.00   29.57   C       ATOM   2352   O   LYS   A   283   45.262   12.626   27.138   1.00   33.16   O       ATOM   2353   CB   LYS   A   283   45.226   13.008   24.287   1.00   21.93   C       ATOM   2354   CG   LYS   A   283   46.111   12.251   23.316   1.00   32.38   C       ATOM   2355   CD   LYS   A   283   46.526   13.171   22.143   1.00   95.77   C       ATOM   2356   CE   LYS   A   283   45.710   12.937   20.836   1.00   100.00   C       ATOM   2357   NZ   LYS   A   283   46.418   13.332   19.535   1.00   100.00   N       ATOM   2358   N   LEU   A   284   44.747   10.467   26.734   1.00   23.37   N       ATOM   2359   CA   LEU   A   284   45.327   9.905   27.997   1.00   16.08   C       ATOM   2360   C   LEU   A   284   45.463   8.386   28.047   1.00   20.46   C       ATOM   2361   O   LEU   A   284   44.679   7.655   27.446   1.00   25.45   O       ATOM   2362   CB   LEU   A   284   44.641   10.387   29.284   1.00   16.30   C       ATOM   2363   CG   LEU   A   284   43.334   9.700   29.714   1.00   25.97   C       ATOM   2364   CD1   LEU   A   284   42.881   10.089   31.152   1.00   22.11   C       ATOM   2365   CD2   LEU   A   284   42.203   9.953   28.693   1.00   23.92   C       ATOM   2366   N   LEU   A   285   46.453   7.939   28.820   1.00   18.51   N       ATOM   2367   CA   LEU   A   285   46.792   6.527   29.003   1.00   16.77   C       ATOM   2368   C   LEU   A   285   45.880   5.865   30.006   1.00   30.75   C       ATOM   2369   O   LEU   A   285   45.576   6.439   31.058   1.00   22.02   O       ATOM   2370   CB   LEU   A   285   48.229   6.389   29.585   1.00   15.85   C       ATOM   2371   CG   LEU   A   285   49.307   6.970   28.672   1.00   21.51   C       ATOM   2372   CD1   LEU   A   285   50.703   6.705   29.122   1.00   15.15   C       ATOM   2373   CD2   LEU   A   285   49.051   6.368   27.330   1.00   16.94   C       ATOM   2374   N   ASP   A   286   45.565   4.599   29.734   1.00   26.62   N       ATOM   2375   CA   ASP   A   286   44.945   3.726   30.698   1.00   10.90   C       ATOM   2376   C   ASP   A   286   46.128   3.055   31.498   1.00   20.54   C       ATOM   2377   O   ASP   A   286   46.991   2.372   30.938   1.00   23.38   O       ATOM   2378   CB   ASP   A   286   44.073   2.702   29.970   1.00   14.65   C       ATOM   2379   CG   ASP   A   286   43.409   1.699   30.943   1.00   24.60   C       ATOM   2380   OD1   ASP   A   286   43.932   1.437   32.083   1.00   24.60   O       ATOM   2381   OD2   ASP   A   286   42.316   1.231   30.583   1.00   26.03   O       ATOM   2382   N   VAL   A   287   46.230   3.317   32.791   1.00   15.44   N       ATOM   2383   CA   VAL   A   287   47.354   2.816   33.556   1.00   15.58   C       ATOM   2384   C   VAL   A   287   46.973   1.695   34.521   1.00   16.48   C       ATOM   2385   O   VAL   A   287   47.613   1.473   35.572   1.00   16.63   O       ATOM   2386   CB   VAL   A   287   48.101   4.006   34.260   1.00   29.84   C       ATOM   2387   CG1   VAL   A   287   48.534   5.085   33.224   1.00   18.39   C       ATOM   2388   CG2   VAL   A   287   47.173   4.670   35.258   1.00   37.79   C       ATOM   2389   N   THR   A   288   45.904   0.992   34.152   1.00   22.27   N       ATOM   2390   CA   THR   A   288   45.428   −0.152   34.956   1.00   19.34   C       ATOM   2391   C   THR   A   288   46.561   −1.177   35.227   1.00   27.47   C       ATOM   2392   O   THR   A   288   46.778   −1.586   36.365   1.00   24.87   O       ATOM   2393   CB   THR   A   288   44.288   −0.909   34.244   1.00   22.86   C       ATOM   2394   OG1   THR   A   288   43.120   −0.096   34.106   1.00   24.84   O       ATOM   2395   CG2   THR   A   288   43.916   −2.113   35.024   1.00   25.08   C       ATOM   2396   N   ARG   A   289   47.290   −1.585   34.179   1.00   26.08   N       ATOM   2397   CA   ARG   A   289   48.428   −2.506   34.319   1.00   16.92   C       ATOM   2398   C   ARG   A   289   49.405   −2.037   35.408   1.00   22.96   C       ATOM   2399   O   ARG   A   289   49.847   −2.790   36.275   1.00   23.03   O       ATOM   2400   CB   ARG   A   289   49.208   −2.607   32.976   1.00   12.43   C       ATOM   2401   CG   ARG   A   289   48.934   −3.804   32.103   1.00   29.39   C       ATOM   2402   CD   ARG   A   289   50.016   −4.102   31.037   1.00   25.88   C       ATOM   2403   NE   ARG   A   289   49.441   −4.996   30.020   1.00   17.26   N       ATOM   2404   CZ   ARG   A   289   50.053   −5.459   28.930   1.00   38.82   C       ATOM   2405   NH1   ARG   A   289   51.306   −5.153   28.660   1.00   13.51   N       ATOM   2406   NH2   ARG   A   289   49.400   −6.262   28.096   1.00   37.68   N       ATOM   2407   N   LEU   A   290   49.815   −0.786   35.306   1.00   26.60   N       ATOM   2408   CA   LEU   A   290   50.809   −0.254   36.219   1.00   25.42   C       ATOM   2409   C   LEU   A   290   50.324   −0.376   37.656   1.00   24.17   C       ATOM   2410   O   LEU   A   290   51.072   −0.759   38.574   1.00   19.94   O       ATOM   2411   CB   LEU   A   290   51.000   1.219   35.876   1.00   24.66   C       ATOM   2412   CG   LEU   A   290   52.281   2.019   36.066   1.00   24.67   C       ATOM   2413   CD1   LEU   A   290   51.992   3.479   36.504   1.00   29.25   C       ATOM   2414   CD2   LEU   A   290   53.450   1.335   36.788   1.00   15.82   C       ATOM   2415   N   HIS   A   291   49.093   0.075   37.868   1.00   30.10   N       ATOM   2416   CA   HIS   A   291   48.513   0.074   39.212   1.00   34.17   C       ATOM   2417   C   HIS   A   291   48.411   −1.367   39.730   1.00   43.41   C       ATOM   2418   O   HIS   A   291   48.621   −1.654   40.929   1.00   38.81   O       ATOM   2419   CB   HIS   A   291   47.113   0.674   39.143   1.00   28.01   C       ATOM   2420   CG   HIS   A   291   47.097   2.153   38.984   1.00   29.68   C       ATOM   2421   ND1   HIS   A   291   48.242   2.921   39.015   1.00   35.63   N       ATOM   2422   CD2   HIS   A   291   46.068   3.024   38.855   1.00   31.18   C       ATOM   2423   CE1   HIS   A   291   47.926   4.197   38.845   1.00   24.20   C       ATOM   2424   NE2   HIS   A   291   46.612   4.289   38.747   1.00   21.92   N       ATOM   2425   N   GLN   A   292   48.048   −2.260   38.821   1.00   30.71   N       ATOM   2426   CA   GLN   A   292   47.950   −3.654   39.181   1.00   34.82   C       ATOM   2427   C   GLN   A   292   49.287   −4.197   39.622   1.00   36.93   C       ATOM   2428   O   GLN   A   292   49.323   −5.040   40.510   1.00   27.56   O       ATOM   2429   CB   GLN   A   292   47.322   −4.487   38.069   1.00   28.23   C       ATOM   2430   CG   GLN   A   292   45.798   −4.405   38.171   1.00   81.15   C       ATOM   2431   CD   GLN   A   292   45.023   ‘4.954   36.963   1.00   100.00   C       ATOM   2432   OE1   GLN   A   292   45.597   −5.410   35.951   1.00   99.65   O       ATOM   2433   NE2   GLN   A   292   43.687   −4.895   37.073   1.00   40.86   N       ATOM   2434   N   LEU   A   293   50.375   −3.658   39.058   1.00   31.75   N       ATOM   2435   CA   LEU   A   293   51.750   −4.072   39.383   1.00   22.67   C       ATOM   2436   C   LEU   A   293   52.238   −3.323   40.613   1.00   28.64   C       ATOM   2437   O   LEU   A   293   53.420   −3.377   41.017   1.00   22.27   O       ATOM   2438   CB   LEU   A   293   52.665   −3.769   38.205   1.00   25.57   C       ATOM   2439   CG   LEU   A   293   52.497   −4.703   37.016   1.00   35.11   C       ATOM   2440   CD1   LEU   A   293   53.306   −4.170   35.836   1.00   28.25   C       ATOM   2441   CD2   LEU   A   293   52.965   −6.110   37.439   1.00   47.81   C       ATOM   2442   N   GLY   A   294   51.316   −2.510   41.111   1.00   33.08   N       ATOM   2443   CA   GLY   A   294   51.488   −1.793   42.347   1.00   24.90   C       ATOM   2444   C   GLY   A   294   52.272   −0.512   42.326   1.00   29.31   C       ATOM   2445   O   GLY   A   294   53.070   −0.249   43.223   1.00   25.25   O       ATOM   2446   N   TRP   A   295   52.000   0.347   41.368   1.00   27.83   N       ATOM   2447   CA   TRP   A   295   52.687   1.623   41.385   1.00   19.45   C       ATOM   2448   C   TRP   A   295   51.684   2.731   41.081   1.00   25.79   C       ATOM   2449   O   TRP   A   295   50.765   2.527   40.297   1.00   20.43   O       ATOM   2450   CB   TRP   A   295   53.961   1.614   40.524   1.00   12.85   C       ATOM   2451   CG   TRP   A   295   54.750   2.911   40.618   1.00   23.04   C       ATOM   2452   CD1   TRP   A   295   55.897   3.161   41.368   1.00   23.68   C       ATOM   2453   CD2   TRP   A   295   54.415   4.159   39.979   1.00   20.72   C       ATOM   2454   NE1   TRP   A   295   56.258   4.493   41.244   1.00   18.67   N       ATOM   2455   CE2   TRP   A   295   55.389   5.113   40.373   1.00   20.95   C       ATOM   2456   CE3   TRP   A   295   53.406   4.550   39.102   1.00   21.47   C       ATOM   2457   CZ2   TRP   A   295   55.338   6.439   39.958   1.00   17.58   C       ATOM   2456   CZ3   TRP   A   295   53.403   5.873   38.632   1.00   21.57   C       ATOM   2459   CH2   TRP   A   295   54.368   6.787   39.058   1.00   19.45   C       ATOM   2460   N   TYR   A   296   51.709   3.797   41.884   1.00   25.17   N       ATOM   2461   CA   TYR   A   296   50.720   4.883   41.731   1.00   24.90   C       ATOM   2462   C   TYR   A   296   51.517   6.178   41.857   1.00   30.85   C       ATOM   2463   O   TYR   A   296   52.363   6.272   42.745   1.00   21.27   O       ATOM   2464   CB   TYR   A   296   49.654   4.813   42.840   1.00   25.16   C       ATOM   2465   CG   TYR   A   296   48.685   3.651   42.744   1.00   23.04   C       ATOM   2466   CD1   TYR   A   296   49.078   2.343   43.088   1.00   31.62   C       ATOM   2467   CD2   TYR   A   296   47.380   3.853   42.289   1.00   26.02   C       ATOM   2468   CE1   TYR   A   296   48.203   1.268   42.935   1.00   24.42   C       ATOM   2469   CE2   TYR   A   296   46.493   2.770   42.127   1.00   24.81   C       ATOM   2470   CZ   TYR   A   296   46.902   1.483   42.464   1.00   39.41   C       ATOM   2471   OH   TYR   A   296   45.984   0.434   42.337   1.00   66.19   O       ATOM   2472   N   HIS   A   297   51.324   7.123   40.924   1.00   20.95   N       ATOM   2473   CA   HIS   A   297   52.130   8.343   40.938   1.00   26.86   C       ATOM   2474   C   HIS   A   297   51.947   9.175   42.210   1.00   35.01   C       ATOM   2475   O   HIS   A   297   50.885   9.132   42.874   1.00   26.92   O       ATOM   2476   CB   HIS   A   297   51.819   9.192   39.733   1.00   25.77   C       ATOM   2477   CG   HIS   A   297   50.489   9.842   39.803   1.00   31.16   C       ATOM   2478   ND1   HIS   A   297   49.314   9.145   39.633   1.00   34.21   N       ATOM   2479   CD2   HIS   A   297   50.135   11.094   40.167   1.00   25.83   C       ATOM   2480   CE1   HIS   A   297   48.290   9.972   39.776   1.00   24.14   C       ATOM   2481   NE2   HIS   A   297   48.761   11.164   40.087   1.00   23.35   N       ATOM   2482   N   GLU   A   298   52.983   9.926   42.554   1.00   24.98   N       ATOM   2483   CA   GLU   A   298   52.957   10.683   43.798   1.00   27.65   C       ATOM   2484   C   GLU   A   298   52.831   12.187   43.741   1.00   36.86   C       ATOM   2485   O   GLU   A   298   52.433   12.792   44.718   1.00   43.61   O       ATOM   2486   CB   GLU   A   298   54.153   10.319   44.686   1.00   22.02   C       ATOM   2487   CG   GLU   A   298   54.004   8.943   45.285   1.00   36.42   C       ATOM   2488   CD   GLU   A   298   54.999   8.664   46.406   1.00   100.00   C       ATOM   2489   OE1   GLU   A   298   56.223   8.561   46.152   1.00   44.79   O       ATOM   2490   OE2   GLU   A   298   54.526   8.470   47.547   1.00   100.00   O       ATOM   2491   N   ILE   A   299   53.232   12.800   42.639   1.00   23.49   N       ATOM   2492   CA   ILE   A   299   53.268   14.244   42.562   1.00   13.25   C       ATOM   2493   C   ILE   A   299   52.016   14.848   41.906   1.00   27.05   C       ATOM   2494   O   ILE   A   299   51.681   14.530   40.757   1.00   26.73   O       ATOM   2495   CB   ILE   A   299   54.586   14.711   41.862   1.00   15.93   C       ATOM   2496   CG1   ILE   A   299   55.836   14.183   42.606   1.00   23.83   C       ATOM   2497   CG2   ILE   A   299   54.596   16.213   41.541   1.00   17.37   C       ATOM   2498   CD1   ILE   A   299   57.232   14.221   41.787   1.00   21.32   C       ATOM   2499   N   SER   A   300   51.323   15.716   42.648   1.00   18.55   N       ATOM   2500   CA   SER   A   300   50.177   16.449   42.091   1.00   19.58   C       ATOM   2501   C   SER   A   300   50.714   17.415   41.042   1.00   17.29   C       ATOM   2502   O   SER   A   300   51.824   17.941   41.178   1.00   21.06   O       ATOM   2503   CB   SER   A   300   49.542   17.307   43.181   1.00   16.78   C       ATOM   2504   OG   SER   A   300   50.548   17.969   43.923   1.00   75.80   O       ATOM   2505   N   LEU   A   301   49.870   17.755   40.075   1.00   16.13   N       ATOM   2506   CA   LEU   A   301   50.246   18.675   39.014   1.00   17.70   C       ATOM   2507   C   LEU   A   301   50.689   19.964   39.646   1.00   20.11   C       ATOM   2508   O   LEU   A   301   51.714   20.568   39.303   1.00   20.46   O       ATOM   2509   CB   LEU   A   301   48.990   18.981   38.197   1.00   17.92   C       ATOM   2510   CG   LEU   A   301   49.182   20.030   37.112   1.00   25.15   C       ATOM   2511   CD1   LEU   A   301   50.233   19.552   36.086   1.00   18.82   C       ATOM   2512   CD2   LEU   A   301   47.854   20.177   36.436   1.00   25.88   C       ATOM   2513   N   GLU   A   302   49.845   20.398   40.554   1.00   27.01   N       ATOM   2514   CA   GLU   A   302   50.053   21.636   41.280   1.00   37.72   C       ATOM   2515   C   GLU   A   302   51.410   21.618   41.996   1.00   29.99   C       ATOM   2516   O   GLU   A   302   52.245   22.514   41.798   1.00   27.15   O       ATOM   2517   CB   GLU   A   302   48.899   21.841   42.275   1.00   43.10   C       ATOM   2518   CG   GLU   A   302   49.061   23.061   43.174   1.00   90.85   C       ATOM   2519   CD   GLU   A   302   48.451   24.324   42.580   1.00   100.00   C       ATOM   2520   OE1   GLU   A   302   47.566   24.209   41.706   1.00   100.00   O       ATOM   2521   OE2   GLU   A   302   48.808   25.432   43.036   1.00   64.50   O       ATOM   2522   N   ALA   A   303   51.646   20.591   42.801   1.00   8.72   N       ATOM   2523   CA   ALA   A   303   52.937   20.455   43.459   1.00   15.03   C       ATOM   2524   C   ALA   A   303   54.102   20.355   42.450   1.00   19.85   C       ATOM   2525   O   ALA   A   303   55.104   21.090   42.553   1.00   22.24   O       ATOM   2526   CB   ALA   A   303   52.938   19.258   44.410   1.00   18.97   C       ATOM   2527   N   GLY   A   304   53.953   19.472   41.467   1.00   13.05   N       ATOM   2528   CA   GLY   A   304   54.970   19.321   40.448   1.00   8.94   C       ATOM   2529   C   GLY   A   304   55.239   20.621   39.695   1.00   20.31   C       ATOM   2530   O   GLY   A   304   56.394   20.900   39.322   1.00   14.30   O       ATOM   2531   N   LEU   A   305   54.191   21.383   39.361   1.00   10.76   N       ATOM   2532   CA   LEU   A   305   54.483   22.622   38.611   1.00   20.29   C       ATOM   2533   C   LEU   A   305   55.281   23.669   39.456   1.00   28.92   C       ATOM   2534   O   LEU   A   305   56.194   24.385   38.974   1.00   17.69   O       ATOM   2535   CB   LEU   A   305   53.202   23.245   38.033   1.00   24.03   C       ATOM   2536   CG   LEU   A   305   52.357   22.647   36.880   1.00   27.66   C       ATOM   2537   CD1   LEU   A   305   50.975   23.384   36.789   1.00   13.44   C       ATOM   2538   CD2   LEU   A   305   53.079   22.724   35.543   1.00   18.39   C       ATOM   2539   N   ALA   A   306   54.904   23.757   40.724   1.00   19.94   N       ATOM   2540   CA   ALA   A   306   55.544   24.660   41.655   1.00   24.79   C       ATOM   2541   C   ALA   A   306   57.035   24.380   41.743   1.00   27.51   C       ATOM   2542   O   ALA   A   306   57.852   25.280   41.662   1.00   29.68   O       ATOM   2543   CB   ALA   A   306   54.937   24.471   43.002   1.00   17.87   C       ATOM   2544   N   SER   A   307   57.378   23.137   42.011   1.00   18.46   N       ATOM   2545   CA   SER   A   307   58.793   22.756   42.162   1.00   16.31   C       ATOM   2546   C   SER   A   307   59.547   22.885   40.832   1.00   22.66   C       ATOM   2547   O   SER   A   307   60.742   23.212   40.786   1.00   28.47   O       ATOM   2548   CB   SER   A   307   58.851   21.304   42.622   1.00   20.47   C       ATOM   2549   OG   SER   A   307   58.517   20.454   41.526   1.00   29.03   O       ATOM   2550   N   THR   A   308   58.849   22.631   39.735   1.00   27.31   N       ATOM   2551   CA   THR   A   308   59.458   22.738   38.413   1.00   22.89   C       ATOM   2552   C   THR   A   308   59.757   24.216   38.107   1.00   26.06   C       ATOM   2553   O   THR   A   308   60.819   24.546   37.591   1.00   29.89   O       ATOM   2554   CB   THR   A   308   58.536   22.115   37.318   1.00   18.72   C       ATOM   2555   OG1   THR   A   308   58.356   20.714   37.545   1.00   20.17   O       ATOM   2556   CG2   THR   A   308   59.094   22.330   35.923   1.00   12.37   C       ATOM   2557   N   TYR   A   309   58.846   25.118   38.453   1.00   28.20   N       ATOM   2558   CA   TYR   A   309   59.110   26.549   38.241   1.00   31.09   C       ATOM   2559   C   TYR   A   309   60.383   27.059   39.045   1.00   16.31   C       ATOM   2560   O   TYR   A   309   61.179   27.858   38.577   1.00   16.91   O       ATOM   2561   CB   TYR   A   309   57.819   27.373   38.533   1.00   31.19   C       ATOM   2562   CG   TYR   A   309   57.944   28.895   38.392   1.00   14.57   C       ATOM   2563   CD1   TYR   A   309   58.397   29.457   37.224   1.00   17.51   C       ATOM   2564   CD2   TYR   A   309   57.575   29.757   39.442   1.00   24.99   C       ATOM   2565   CE1   TYR   A   309   58.527   30.801   37.100   1.00   18.41   C       ATOM   2566   CE2   TYR   A   309   57.744   31.129   39.351   1.00   19.04   C       ATOM   2567   CZ   TYR   A   309   58.212   31.641   38.164   1.00   29.13   C       ATOM   2568   OH   TYR   A   309   58.300   33.004   37.966   1.00   28.22   O       ATOM   2569   N   GLN   A   310   60.560   26.579   40.260   1.00   15.41   N       ATOM   2570   CA   GLN   A   310   61.705   26.964   41.087   1.00   22.35   C       ATOM   2571   C   GLN   A   310   63.001   26.492   40.446   1.00   31.46   C       ATOM   2572   O   GLN   A   310   64.009   27.191   40.442   1.00   33.42   O       ATOM   2573   CB   GLN   A   310   61.587   26.335   42.482   1.00   17.67   C       ATOM   2574   CG   GLN   A   310   62.579   26.921   43.461   1.00   57.58   C       ATOM   2575   CD   GLN   A   310   62.287   28.370   43.782   1.00   65.14   C       ATOM   2576   OE1   GLN   A   310   61.134   28.754   44.000   1.00   41.94   O       ATOM   2577   NE2   GLN   A   310   63.330   29.194   43.801   1.00   99.09   N       ATOM   2578   N   TRP   A   311   62.957   25.321   39.830   1.00   28.76   N       ATOM   2579   CA   TRP   A   311   64.146   24.822   39.163   1.00   26.29   C       ATOM   2580   C   TRP   A   311   64.474   25.769   38.040   1.00   17.91   C       ATOM   2581   O   TRP   A   311   65.599   26.193   37.880   1.00   22.89   O       ATOM   2582   CB   TRP   A   311   63.938   23.383   38.643   1.00   27.53   C       ATOM   2583   CG   TRP   A   311   65.176   22.784   38.119   1.00   17.82   C       ATOM   2584   CD1   TRP   A   311   66.132   22.090   38.826   1.00   20.21   C       ATOM   2585   CD2   TRP   A   311   65.652   22.881   36.784   1.00   17.99   C       ATOM   2586   NE1   TRP   A   311   67.197   21.776   37.992   1.00   20.39   N       ATOM   2587   CE2   TRP   A   311   66.933   22.284   36.746   1.00   19.57   C       ATOM   2588   CE3   TRP   A   311   65.141   23.461   35.621   1.00   20.26   C       ATOM   2589   CZ2   TRP   A   311   67.686   22.236   35.599   1.00   14.25   C       ATOM   2590   CZ3   TRP   A   311   65.901   23.446   34.501   1.00   18.59   C       ATOM   2591   CH2   TRP   A   311   67.169   22.831   34.494   1.00   16.86   C       ATOM   2592   N   PHE   A   312   63.469   26.109   37.256   1.00   17.47   N       ATOM   2593   CA   PHE   A   312   63.665   27.064   36.179   1.00   20.14   C       ATOM   2594   C   PHE   A   312   64.224   28.371   36.733   1.00   18.33   C       ATOM   2595   O   PHE   A   312   65.080   29.024   36.104   1.00   24.76   O       ATOM   2596   CB   PHE   A   312   62.328   27.318   35.458   1.00   29.51   C       ATOM   2597   CG   PHE   A   312   62.328   28.544   34.603   1.00   28.52   C       ATOM   2598   CD1   PHE   A   312   62.883   28.508   33.338   1.00   30.53   C       ATOM   2599   CD2   PHE   A   312   61.825   29.758   35.104   1.00   29.31   C       ATOM   2600   CE1   PHE   A   312   62.936   29.660   32.554   1.00   34.73   C       ATOM   2601   CE2   PHE   A   312   61.900   30.904   34.362   1.00   38.40   C       ATOM   2602   CZ   PHE   A   312   62.432   30.860   33.063   1.00   40.73   C       ATOM   2603   N   LEU   A   313   63.697   28.787   37.876   1.00   22.46   N       ATOM   2604   CA   LEU   A   313   64.170   30.025   38.516   1.00   28.47   C       ATOM   2605   C   LEU   A   313   65.627   29.827   38.898   1.00   37.53   C       ATOM   2606   O   LEU   A   313   66.452   30.693   38.629   1.00   34.20   O       ATOM   2607   CB   LEU   A   313   63.375   30.410   39.783   1.00   20.44   C       ATOM   2608   CG   LEU   A   313   61.955   30.897   39.555   1.00   16.29   C       ATOM   2609   CD1   LEU   A   313   61.499   31.399   40.871   1.00   15.94   C       ATOM   2610   CD2   LEU   A   313   61.959   31.961   38.524   1.00   14.44   C       ATOM   2611   N   GLU   A   314   65.953   28.685   39.508   1.00   30.70   N       ATOM   2612   CA   GLU   A   314   67.353   28.432   39.875   1.00   24.15   C       ATOM   2613   C   GLU   A   314   68.291   28.149   38.703   1.00   36.34   C       ATOM   2614   O   GLU   A   314   69.485   28.047   38.890   1.00   43.10   O       ATOM   2615   CB   GLU   A   314   67.459   27.366   40.947   1.00   19.90   C       ATOM   2616   CG   GLU   A   314   66.634   27.754   42.141   1.00   27.37   C       ATOM   2617   CD   GLU   A   314   66.450   26.666   43.182   1.00   31.09   C       ATOM   2618   OE1   GLU   A   314   67.157   25.648   43.085   1.00   59.60   O       ATOM   2619   OE2   GLU   A   314   65.634   26.872   44.125   1.00   46.20   O       ATOM   2620   N   ASN   A   315   67.778   28.114   37.479   1.00   40.17   N       ATOM   2621   CA   ASN   A   315   68.637   27.802   36.343   1.00   37.76   C       ATOM   2622   C   ASN   A   315   68.383   28.578   35.112   1.00   43.75   C       ATOM   2623   O   ASN   A   315   68.591   28.001   34.047   1.00   39.15   O       ATOM   2624   CB   ASN   A   315   68.425   26.360   35.884   1.00   33.74   C       ATOM   2625   CG   ASN   A   315   69.028   25.383   36.801   1.00   53.18   C       ATOM   2626   OD1   ASN   A   315   68.456   25.087   37.835   1.00   49.13   O       ATOM   2627   ND2   ASN   A   315   70.239   24.926   36.479   1.00   97.72   N       ATOM   2628   N   GLN   A   316   67.852   29.803   35.197   1.00   49.87   N       ATOM   2629   CA   GLN   A   316   67.627   30.550   33.957   1.00   77.90   C       ATOM   2630   C   GLN   A   316   68.797   31.448   33.525   1.00   100.00   C       ATOM   2631   O   GLN   A   316   69.272   31.387   32.375   1.00   51.33   O       ATOM   2632   CB   GLN   A   316   66.280   31.276   33.902   1.00   75.89   C       ATOM   2633   CG   GLN   A   316   65.683   31.589   35.231   1.00   80.97   C       ATOM   2634   CD   GLN   A   316   65.233   33.036   35.350   1.00   54.58   C       ATOM   2635   OE1   GLN   A   316   64.881   33.699   34.367   1.00   46.46   O       ATOM   2636   NE2   GLN   A   316   65.257   33.538   36.566   1.00   33.46   N       TER   2637   GLN   A   316       CONECT   110   111       CONECT   111   110   112       CONECT   112   111   113   114       CONECT   113   112   118       CONECT   114   112   115   116       CONECT   115   114       CONECT   116   114   117   118       CONECT   117   116   129       CONECT   118   113   116       CONECT   120   121       CONECT   121   120   122       CONECT   122   121   123   124       CONECT   123   122   128       CONECT   124   122   125   126       CONECT   125   124       CONECT   126   124   127   128       CONECT   127   126       CONECT   128   123   126       CONECT   129   117   130   131   132       CONECT   130   129       CONECT   131   129       CONECT   132   129                                                                 MASTER   208   O   1   13   10   O   3   6   2636   1   22   25       END                  
 
     [0227] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. However, it is to be expressly understood that such modifications and adaptations are within the spirit and scope of the present invention, as set forth in the following claims.  
    
     
       
         1 
         
           
             15  
           
           
             1  
             1583  
             DNA  
             Arabidopsis thaliana  
             
               CDS  
               (49)..(990)  
             
           
            1 

tagtctttaa tttcgcagcg tttttataat tgtgcagagg tttcgtcc atg tct gac      57 
                                                     Met Ser Asp 
                                                       1 

aaa tct gcc aaa atc ttc gtc gcg ggt cat cgt ggt ttg gtt gga tct      105 
Lys Ser Ala Lys Ile Phe Val Ala Gly His Arg Gly Leu Val Gly Ser 
      5                  10                  15 

gcc att gtc cgc aag ctt cag gaa caa ggt ttc acc aat ctc gtt ctt      153 
Ala Ile Val Arg Lys Leu Gln Glu Gln Gly Phe Thr Asn Leu Val Leu 
 20                  25                  30                  35 

aaa aca cac gcc gag ctt gat ctc act cgt caa gcc gat gtt gaa tcc      201 
Lys Thr His Ala Glu Leu Asp Leu Thr Arg Gln Ala Asp Val Glu Ser 
                 40                  45                  50 

ttc ttt tct caa gag aag cca gtt tat gta atc cta gca gca gct aaa      249 
Phe Phe Ser Gln Glu Lys Pro Val Tyr Val Ile Leu Ala Ala Ala Lys 
             55                  60                  65 

gtt ggt ggt att cac gct aac aac acc tat cct gct gat ttc att ggt      297 
Val Gly Gly Ile His Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile Gly 
         70                  75                  80 

gtc aat ctc cag att cag acc aat gtg atc cac tct gca tat gag cac      345 
Val Asn Leu Gln Ile Gln Thr Asn Val Ile His Ser Ala Tyr Glu His 
     85                  90                  95 

ggt gtg aag aag ctt ctc ttc ctt gga tca tcc tgc att tac cct aaa      393 
Gly Val Lys Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys 
100                 105                 110                 115 

ttt gct cct cag cca att cct gag tct gct ttg tta aca gca tcg ctt      441 
Phe Ala Pro Gln Pro Ile Pro Glu Ser Ala Leu Leu Thr Ala Ser Leu 
                120                 125                 130 

gaa cca act aat gag tgg tat gct att gct aag atc gct ggg att aag      489 
Glu Pro Thr Asn Glu Trp Tyr Ala Ile Ala Lys Ile Ala Gly Ile Lys 
            135                 140                 145 

act tgt cag gct tat agg att cag cac gga tgg gat gca atc tct ggc      537 
Thr Cys Gln Ala Tyr Arg Ile Gln His Gly Trp Asp Ala Ile Ser Gly 
        150                 155                 160 

atg cct act aat ctc tat ggt cct aat gac aat ttc cac ccg gag tct      585 
Met Pro Thr Asn Leu Tyr Gly Pro Asn Asp Asn Phe His Pro Glu Ser 
    165                 170                 175 

cat gtg ctt cct gct ctt atg agg agg ttc cac gag gcg aaa gtg aat      633 
His Val Leu Pro Ala Leu Met Arg Arg Phe His Glu Ala Lys Val Asn 
180                 185                 190                 195 

tgg agc gga gga agt tgt ggt gtg ggg tac aag gta gtc ccg ttg gaa      681 
Trp Ser Gly Gly Ser Cys Gly Val Gly Tyr Lys Val Val Pro Leu Glu 
                200                 205                 210 

ggg aag ttc ttg cat gtt gat gat ttg gct gat gct tgt gtt ttc ttg      729 
Gly Lys Phe Leu His Val Asp Asp Leu Ala Asp Ala Cys Val Phe Leu 
            215                 220                 225 

ctg gat cgg ata cag cgg ggg ttg gag cat gtt aac att gga agt ggt      777 
Leu Asp Arg Ile Gln Arg Gly Leu Glu His Val Asn Ile Gly Ser Gly 
        230                 235                 240 

caa gaa gtg act att aga gag ttg gct gag ttg gtg aaa gag gtt gtt      825 
Gln Glu Val Thr Ile Arg Glu Leu Ala Glu Leu Val Lys Glu Val Val 
    245                 250                 255 

ggt ttt gaa ggg aag ctt gga tgg gat tgc act aag cca gat ggc aca      873 
Gly Phe Glu Gly Lys Leu Gly Trp Asp Cys Thr Lys Pro Asp Gly Thr 
260                 265                 270                 275 

ccg agg aaa ctt atg gac agc tca aag ctc gcg tct ttg ggt tgg aca      921 
Pro Arg Lys Leu Met Asp Ser Ser Lys Leu Ala Ser Leu Gly Trp Thr 
                280                 285                 290 

cct aag gtt tct ctt aga gat ggt ctg agc caa act tat gat tgg tat      969 
Pro Lys Val Ser Leu Arg Asp Gly Leu Ser Gln Thr Tyr Asp Trp Tyr 
            295                 300                 305 

ttg aag aat gtt tgc aac cga taagttaatg gtttctcttc tcatatatac        1020 
Leu Lys Asn Val Cys Asn Arg 
        310 

acaactattg agtctcaggt aaatcagctt atcaccacat tgtgatttaa acctttcttt   1080 

gagattcgag aattgctttt ttttttatca aaattgattc atttagagat aagacttgct   1140 

tctttataca acattgtctg aggaatttta attttggatc tccgagtatg gtctattatt   1200 

agctctcttc tatacaaatt atcaaaacag ttgtaagaag tttcaagaaa aacatttgat   1260 

atctcactaa tttggctatc cttgcaagtt gcaacgctaa aatgacaaat aatgaattct   1320 

cggcccaatg ggcttacaca agccttgtta aagatagcgt gaacaaaacg cggctcacta   1380 

gccctaacct gtctctcttt cgcttacctt cttcttcgtc ttcgttggct cagtcacttg   1440 

acttcacggc ccgctcaagc tctgacacga aactcatttc aaattaattt aataaaacct   1500 

taatcacaaa aggggcaaaa gcaatcgccg gcgattatgc cttctcctcc ggtgccggag   1560 

acggttgtga gccaacccgt tcg                                           1583 

 
           
             2  
             314  
             PRT  
             Arabidopsis thaliana  
           
            2 

Met Ser Asp Lys Ser Ala Lys Ile Phe Val Ala Gly His Arg Gly Leu 
  1               5                  10                  15 

Val Gly Ser Ala Ile Val Arg Lys Leu Gln Glu Gln Gly Phe Thr Asn 
             20                  25                  30 

Leu Val Leu Lys Thr His Ala Glu Leu Asp Leu Thr Arg Gln Ala Asp 
         35                  40                  45 

Val Glu Ser Phe Phe Ser Gln Glu Lys Pro Val Tyr Val Ile Leu Ala 
     50                  55                  60 

Ala Ala Lys Val Gly Gly Ile His Ala Asn Asn Thr Tyr Pro Ala Asp 
 65                  70                  75                  80 

Phe Ile Gly Val Asn Leu Gln Ile Gln Thr Asn Val Ile His Ser Ala 
                 85                  90                  95 

Tyr Glu His Gly Val Lys Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile 
            100                 105                 110 

Tyr Pro Lys Phe Ala Pro Gln Pro Ile Pro Glu Ser Ala Leu Leu Thr 
        115                 120                 125 

Ala Ser Leu Glu Pro Thr Asn Glu Trp Tyr Ala Ile Ala Lys Ile Ala 
    130                 135                 140 

Gly Ile Lys Thr Cys Gln Ala Tyr Arg Ile Gln His Gly Trp Asp Ala 
145                 150                 155                 160 

Ile Ser Gly Met Pro Thr Asn Leu Tyr Gly Pro Asn Asp Asn Phe His 
                165                 170                 175 

Pro Glu Ser His Val Leu Pro Ala Leu Met Arg Arg Phe His Glu Ala 
            180                 185                 190 

Lys Val Asn Trp Ser Gly Gly Ser Cys Gly Val Gly Tyr Lys Val Val 
        195                 200                 205 

Pro Leu Glu Gly Lys Phe Leu His Val Asp Asp Leu Ala Asp Ala Cys 
    210                 215                 220 

Val Phe Leu Leu Asp Arg Ile Gln Arg Gly Leu Glu His Val Asn Ile 
225                 230                 235                 240 

Gly Ser Gly Gln Glu Val Thr Ile Arg Glu Leu Ala Glu Leu Val Lys 
                245                 250                 255 

Glu Val Val Gly Phe Glu Gly Lys Leu Gly Trp Asp Cys Thr Lys Pro 
            260                 265                 270 

Asp Gly Thr Pro Arg Lys Leu Met Asp Ser Ser Lys Leu Ala Ser Leu 
        275                 280                 285 

Gly Trp Thr Pro Lys Val Ser Leu Arg Asp Gly Leu Ser Gln Thr Tyr 
    290                 295                 300 

Asp Trp Tyr Leu Lys Asn Val Cys Asn Arg 
305                 310 

 
           
             3  
             966  
             DNA  
             Escherichia coli  
             
               CDS  
               (1)..(966)  
             
           
            3 

atg agt aaa caa cga gtt ttt att gct ggt cat cgc ggg atg gtc ggt       48 
Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly 
  1               5                  10                  15 

tcc gcc atc agg cgg cag ctc gaa cag cgc ggt gat gtg gaa ctg gta       96 
Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val 
             20                  25                  30 

tta cgc acc cgc gac gag ctg aac ctg ctg gac agc cgc gcc gtg cat      144 
Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His 
         35                  40                  45 

gat ttc ttt gcc agc gaa cgt att gac cag gtc tat ctg gcg gcg gcg      192 
Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala 
     50                  55                  60 

aaa gtg ggc ggc att gtt gcc aac aac acc tat ccg gcg gat ttc atc      240 
Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile 
 65                  70                  75                  80 

tac cag aac atg atg att gag agc aac atc att cac gcc gcg cat cag      288 
Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln 
                 85                  90                  95 

aac gac gtg aac aaa ctg ctg ttt ctc gga tcg tcc tgc atc tac ccg      336 
Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro 
            100                 105                 110 

aaa ctg gca aaa cag ccg atg gca gaa agc gag ttg ttg cag ggc acg      384 
Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr 
        115                 120                 125 

ctg gag ccg act aac gag cct tat gct att gcc aaa atc gcc ggg atc      432 
Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile 
    130                 135                 140 

aaa ctg tgc gaa tca tac aac cgc cag tac gga cgc gat tac cgc tca      480 
Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser 
145                 150                 155                 160 

gtc atg ccg acc aac ctg tac ggg cca cac gac aac ttc cac ccg agt      528 
Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser 
                165                 170                 175 

aat tcg cat gtg atc cca gca ttg ctg cgt cgc ttc cac gag gcg acg      576 
Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr 
            180                 185                 190 

gca cag aat gcg ccg gac gtg gtg gta tgg ggc agc ggt aca ccg atg      624 
Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met 
        195                 200                 205 

cgc gaa ttt ctg cac gtc gat gat atg gcg gcg gcg agc att cat gtc      672 
Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val 
    210                 215                 220 

atg gag ctg gcg cat gaa gtc tgg ctg gag aac acc cag ccg atg ttg      720 
Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu 
225                 230                 235                 240 

tcg cac att aac gtc ggc acg ggc gtt gac tgc act atc cgc gac gtg      768 
Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Asp Val 
                245                 250                 255 

gcg caa acc atc gcc aaa gtg gtg ggt tac aaa ggc cgg gtg gtt ttt      816 
Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe 
            260                 265                 270 

gat gcc agc aaa ccg gat ggc acg ccg cgc aaa ctg ctg gat gtg acg      864 
Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr 
        275                 280                 285 

cgc ctg cat cag ctt ggc tgg tat cac gaa atc tca ctg gaa gcg ggg      912 
Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly 
    290                 295                 300 

ctt gcc agc act tac cag tgg ttc ctt gag aat caa gac cgc ttt cgg      960 
Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg 
305                 310                 315                 320 

ggg taa                                                              966 
Gly 

 
           
             4  
             321  
             PRT  
             Escherichia coli  
           
            4 

Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly 
  1               5                  10                  15 

Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val 
             20                  25                  30 

Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His 
         35                  40                  45 

Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala 
     50                  55                  60 

Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile 
 65                  70                  75                  80 

Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln 
                 85                  90                  95 

Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro 
            100                 105                 110 

Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr 
        115                 120                 125 

Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile 
    130                 135                 140 

Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser 
145                 150                 155                 160 

Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser 
                165                 170                 175 

Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr 
            180                 185                 190 

Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met 
        195                 200                 205 

Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val 
    210                 215                 220 

Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu 
225                 230                 235                 240 

Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Asp Val 
                245                 250                 255 

Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe 
            260                 265                 270 

Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr 
        275                 280                 285 

Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly 
    290                 295                 300 

Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg 
305                 310                 315                 320 

Gly 

 
           
             5  
             1340  
             DNA  
             Homo sapiens  
             
               CDS  
               (75)..(1040)  
             
           
            5 

ctagaattca gcggccgctg aattctagct agaattcagc ggccgctgaa ttctagaacc     60 

caggtgcaac tgac atg ggt gaa ccc cag gga tcc atg cgg att cta gtg      110 
                Met Gly Glu Pro Gln Gly Ser Met Arg Ile Leu Val 
                  1               5                  10 

aca ggg ggc tct ggg ctg gta ggc aaa gcc atc cag aag gtg gta gca      158 
Thr Gly Gly Ser Gly Leu Val Gly Lys Ala Ile Gln Lys Val Val Ala 
         15                  20                  25 

gat gga gct gga ctt cct gga gag gac tgg gtg ttt gtc tcc tct aaa      206 
Asp Gly Ala Gly Leu Pro Gly Glu Asp Trp Val Phe Val Ser Ser Lys 
     30                  35                  40 

gac gcc gat ctc acg gat aca gca cag acc cgc gcc ctg ttt gag aag      254 
Asp Ala Asp Leu Thr Asp Thr Ala Gln Thr Arg Ala Leu Phe Glu Lys 
 45                  50                  55                  60 

gtc caa ccc aca cac gtc atc cat ctt gct gca atg gtg ggg ggc ctg      302 
Val Gln Pro Thr His Val Ile His Leu Ala Ala Met Val Gly Gly Leu 
                 65                  70                  75 

ttc cgg aat atc aaa tac aat ttg gac ttc tgg agg aaa aac gtg cac      350 
Phe Arg Asn Ile Lys Tyr Asn Leu Asp Phe Trp Arg Lys Asn Val His 
             80                  85                  90 

atg aac gac aac gtc ctg cac tcg gcc ttt gag gtg ggg gcc cgc aag      398 
Met Asn Asp Asn Val Leu His Ser Ala Phe Glu Val Gly Ala Arg Lys 
         95                 100                 105 

gtg gtg tcc tgc ctg tcc acc tgt atc ttc cct gac aag acg acc tac      446 
Val Val Ser Cys Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr Thr Tyr 
    110                 115                 120 

ccg ata gat gag acc atg atc cac aat ggg cct ccc cac aac agc aat      494 
Pro Ile Asp Glu Thr Met Ile His Asn Gly Pro Pro His Asn Ser Asn 
125                 130                 135                 140 

ttt ggg tac tcg tat gcc aag agg atg atc gac gtg cag aac agg gcc      542 
Phe Gly Tyr Ser Tyr Ala Lys Arg Met Ile Asp Val Gln Asn Arg Ala 
                145                 150                 155 

tac ttc cag cag tac ggc tgc acc ttc acc gct gtc atc ccc acc aac      590 
Tyr Phe Gln Gln Tyr Gly Cys Thr Phe Thr Ala Val Ile Pro Thr Asn 
            160                 165                 170 

gtt ttc ggg ccc cac gac aac ttc aac atc gag gat ggc cac gtg ctg      638 
Val Phe Gly Pro His Asp Asn Phe Asn Ile Glu Asp Gly His Val Leu 
        175                 180                 185 

cct ggc ctc atc cac aag gtg cac ctg gcc aag agc agc ggc tcg gcc      686 
Pro Gly Leu Ile His Lys Val His Leu Ala Lys Ser Ser Gly Ser Ala 
    190                 195                 200 

ctg acg gtg tgg ggt aca ggg aat ccg cgg agg cag ttc ata tac tcg      734 
Leu Thr Val Trp Gly Thr Gly Asn Pro Arg Arg Gln Phe Ile Tyr Ser 
205                 210                 215                 220 

ctg gac ctg gcc cag ctc ttt atc tgg gtc ctg cgg gag tac aat gaa      782 
Leu Asp Leu Ala Gln Leu Phe Ile Trp Val Leu Arg Glu Tyr Asn Glu 
                225                 230                 235 

gtg gag ccc atc atc ctc tcc gtg ggc gag gaa gat gag gtc tcc atc      830 
Val Glu Pro Ile Ile Leu Ser Val Gly Glu Glu Asp Glu Val Ser Ile 
            240                 245                 250 

aag gag gca gcc gag gcg gtg gtg gag gcc atg gac ttc cat ggg gaa      878 
Lys Glu Ala Ala Glu Ala Val Val Glu Ala Met Asp Phe His Gly Glu 
        255                 260                 265 

gtc acc ttt gat aca acc aag tcg gat ggg cag ttt aag aag aca gcc      926 
Val Thr Phe Asp Thr Thr Lys Ser Asp Gly Gln Phe Lys Lys Thr Ala 
    270                 275                 280 

agt aac agc aag ctg agg acc tac ctg ccc gac ttc cgg ttc aca ccc      974 
Ser Asn Ser Lys Leu Arg Thr Tyr Leu Pro Asp Phe Arg Phe Thr Pro 
285                 290                 295                 300 

ttc aag cag gcg gtg aag gag acc tgt gct tgg ttc act gac aac tac     1022 
Phe Lys Gln Ala Val Lys Glu Thr Cys Ala Trp Phe Thr Asp Asn Tyr 
                305                 310                 315 

gag cag gcc cgg aag tga agctggaaga caggatcagg tgccagcgga            1070 
Glu Gln Ala Arg Lys 
            320 

ccatcggctg gcagagccca gcggccacca cccgtcaacc ctgccaggag ctgagggcac   1130 

cacccagcaa cctgggcctg cattccatcc gctctgcagc cccaagcatc tttccagtgg   1190 

ggcccccatt cacgttggtc ctcagggaaa ccagggtccg gggcaggccc ggcgctttgc   1250 

tccccacacc agccccctgc gcgtgtccac tctgatcctg catcccactc cctgggagcc   1310 

aataaagtgc attttcacag aaaaaaaaaa                                    1340 

 
           
             6  
             321  
             PRT  
             Homo sapiens  
           
            6 

Met Gly Glu Pro Gln Gly Ser Met Arg Ile Leu Val Thr Gly Gly Ser 
  1               5                  10                  15 

Gly Leu Val Gly Lys Ala Ile Gln Lys Val Val Ala Asp Gly Ala Gly 
             20                  25                  30 

Leu Pro Gly Glu Asp Trp Val Phe Val Ser Ser Lys Asp Ala Asp Leu 
         35                  40                  45 

Thr Asp Thr Ala Gln Thr Arg Ala Leu Phe Glu Lys Val Gln Pro Thr 
     50                  55                  60 

His Val Ile His Leu Ala Ala Met Val Gly Gly Leu Phe Arg Asn Ile 
 65                  70                  75                  80 

Lys Tyr Asn Leu Asp Phe Trp Arg Lys Asn Val His Met Asn Asp Asn 
                 85                  90                  95 

Val Leu His Ser Ala Phe Glu Val Gly Ala Arg Lys Val Val Ser Cys 
            100                 105                 110 

Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr Thr Tyr Pro Ile Asp Glu 
        115                 120                 125 

Thr Met Ile His Asn Gly Pro Pro His Asn Ser Asn Phe Gly Tyr Ser 
    130                 135                 140 

Tyr Ala Lys Arg Met Ile Asp Val Gln Asn Arg Ala Tyr Phe Gln Gln 
145                 150                 155                 160 

Tyr Gly Cys Thr Phe Thr Ala Val Ile Pro Thr Asn Val Phe Gly Pro 
                165                 170                 175 

His Asp Asn Phe Asn Ile Glu Asp Gly His Val Leu Pro Gly Leu Ile 
            180                 185                 190 

His Lys Val His Leu Ala Lys Ser Ser Gly Ser Ala Leu Thr Val Trp 
        195                 200                 205 

Gly Thr Gly Asn Pro Arg Arg Gln Phe Ile Tyr Ser Leu Asp Leu Ala 
    210                 215                 220 

Gln Leu Phe Ile Trp Val Leu Arg Glu Tyr Asn Glu Val Glu Pro Ile 
225                 230                 235                 240 

Ile Leu Ser Val Gly Glu Glu Asp Glu Val Ser Ile Lys Glu Ala Ala 
                245                 250                 255 

Glu Ala Val Val Glu Ala Met Asp Phe His Gly Glu Val Thr Phe Asp 
            260                 265                 270 

Thr Thr Lys Ser Asp Gly Gln Phe Lys Lys Thr Ala Ser Asn Ser Lys 
        275                 280                 285 

Leu Arg Thr Tyr Leu Pro Asp Phe Arg Phe Thr Pro Phe Lys Gln Ala 
    290                 295                 300 

Val Lys Glu Thr Cys Ala Trp Phe Thr Asp Asn Tyr Glu Gln Ala Arg 
305                 310                 315                 320 

Lys 

 
           
             7  
             1017  
             DNA  
             Escherichia coli  
             
               CDS  
               (1)..(1017)  
             
           
            7 

atg aga gtt ctg gtt acc ggt ggt agc ggt tac att gga agt cat acc       48 
Met Arg Val Leu Val Thr Gly Gly Ser Gly Tyr Ile Gly Ser His Thr 
  1               5                  10                  15 

tgt gtg caa tta ctg caa aac ggt cat gat gtc atc att ctt gat aac       96 
Cys Val Gln Leu Leu Gln Asn Gly His Asp Val Ile Ile Leu Asp Asn 
             20                  25                  30 

ctc tgt aac agt aag cgc agc gta ctg cct gtt atc gag cgt tta ggc      144 
Leu Cys Asn Ser Lys Arg Ser Val Leu Pro Val Ile Glu Arg Leu Gly 
         35                  40                  45 

ggc aaa cat cca acg ttt gtt gaa ggc gat att cgt aac gaa gcg ttg      192 
Gly Lys His Pro Thr Phe Val Glu Gly Asp Ile Arg Asn Glu Ala Leu 
     50                  55                  60 

atg acc gag atc ctg cac gat cac gct atc gac acc gtg atc cac ttc      240 
Met Thr Glu Ile Leu His Asp His Ala Ile Asp Thr Val Ile His Phe 
 65                  70                  75                  80 

gcc ggg ctg aaa gcc gtg ggc gaa tcg gta caa aaa ccg ctg gaa tat      288 
Ala Gly Leu Lys Ala Val Gly Glu Ser Val Gln Lys Pro Leu Glu Tyr 
                 85                  90                  95 

tac gac aac aat gtc aac ggc act ctg cgc ctg att agc gcc atg cgc      336 
Tyr Asp Asn Asn Val Asn Gly Thr Leu Arg Leu Ile Ser Ala Met Arg 
            100                 105                 110 

gcc gct aac gtc aaa aac ttt att ttt agc tcc tcc gcc acc gtt tat      384 
Ala Ala Asn Val Lys Asn Phe Ile Phe Ser Ser Ser Ala Thr Val Tyr 
        115                 120                 125 

ggc gat cag ccc aaa att cca tac gtt gaa agc ttc ccg acc ggc aca      432 
Gly Asp Gln Pro Lys Ile Pro Tyr Val Glu Ser Phe Pro Thr Gly Thr 
    130                 135                 140 

ccg caa agc cct tac ggc aaa agc aag ctg atg gtg gaa cag atc ctc      480 
Pro Gln Ser Pro Tyr Gly Lys Ser Lys Leu Met Val Glu Gln Ile Leu 
145                 150                 155                 160 

acc gat ctg caa aaa gcc cag ccg gac tgg agc att gcc ctg ctg cgc      528 
Thr Asp Leu Gln Lys Ala Gln Pro Asp Trp Ser Ile Ala Leu Leu Arg 
                165                 170                 175 

tac ttc aac ccg gtt ggc gcg cat ccg tcg ggc gat atg ggc gaa gat      576 
Tyr Phe Asn Pro Val Gly Ala His Pro Ser Gly Asp Met Gly Glu Asp 
            180                 185                 190 

ccg caa ggc att ccg aat aac ctg atg cca tac atc gcc cag gtt gct      624 
Pro Gln Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Ala Gln Val Ala 
        195                 200                 205 

gta ggc cgt cgc gac tcg ctg gcg att ttt ggt aac gat tat ccg acc      672 
Val Gly Arg Arg Asp Ser Leu Ala Ile Phe Gly Asn Asp Tyr Pro Thr 
    210                 215                 220 

gaa gat ggt act ggc gta cgc gat tac atc cac gta atg gat ctg gcg      720 
Glu Asp Gly Thr Gly Val Arg Asp Tyr Ile His Val Met Asp Leu Ala 
225                 230                 235                 240 

gac ggt cac gtc gtg gcg atg gaa aaa ctg gcg aac aag cca ggc gta      768 
Asp Gly His Val Val Ala Met Glu Lys Leu Ala Asn Lys Pro Gly Val 
                245                 250                 255 

cac atc tac aac ctc ggc gct ggc gta ggc aac agc gtg ctg gac gtg      816 
His Ile Tyr Asn Leu Gly Ala Gly Val Gly Asn Ser Val Leu Asp Val 
            260                 265                 270 

gtt aat gcc ttc agc aaa gcc tgc ggc aaa ccg gtt aat tat cat ttt      864 
Val Asn Ala Phe Ser Lys Ala Cys Gly Lys Pro Val Asn Tyr His Phe 
        275                 280                 285 

gca ccg cgt cgc gag ggc gac ctt ccg gcc tac tgg gcg gac gcc agc      912 
Ala Pro Arg Arg Glu Gly Asp Leu Pro Ala Tyr Trp Ala Asp Ala Ser 
    290                 295                 300 

aaa gcc gac cgt gaa ctg aac tgg cgc gta acg cgc aca ctc gat gaa      960 
Lys Ala Asp Arg Glu Leu Asn Trp Arg Val Thr Arg Thr Leu Asp Glu 
305                 310                 315                 320 

atg gcg cag gac acc tgg cac tgg cag tca cgc cat cca cag gga tat     1008 
Met Ala Gln Asp Thr Trp His Trp Gln Ser Arg His Pro Gln Gly Tyr 
                325                 330                 335 

ccc gat taa                                                         1017 
Pro Asp 

 
           
             8  
             338  
             PRT  
             Escherichia coli  
           
            8 

Met Arg Val Leu Val Thr Gly Gly Ser Gly Tyr Ile Gly Ser His Thr 
  1               5                  10                  15 

Cys Val Gln Leu Leu Gln Asn Gly His Asp Val Ile Ile Leu Asp Asn 
             20                  25                  30 

Leu Cys Asn Ser Lys Arg Ser Val Leu Pro Val Ile Glu Arg Leu Gly 
         35                  40                  45 

Gly Lys His Pro Thr Phe Val Glu Gly Asp Ile Arg Asn Glu Ala Leu 
     50                  55                  60 

Met Thr Glu Ile Leu His Asp His Ala Ile Asp Thr Val Ile His Phe 
 65                  70                  75                  80 

Ala Gly Leu Lys Ala Val Gly Glu Ser Val Gln Lys Pro Leu Glu Tyr 
                 85                  90                  95 

Tyr Asp Asn Asn Val Asn Gly Thr Leu Arg Leu Ile Ser Ala Met Arg 
            100                 105                 110 

Ala Ala Asn Val Lys Asn Phe Ile Phe Ser Ser Ser Ala Thr Val Tyr 
        115                 120                 125 

Gly Asp Gln Pro Lys Ile Pro Tyr Val Glu Ser Phe Pro Thr Gly Thr 
    130                 135                 140 

Pro Gln Ser Pro Tyr Gly Lys Ser Lys Leu Met Val Glu Gln Ile Leu 
145                 150                 155                 160 

Thr Asp Leu Gln Lys Ala Gln Pro Asp Trp Ser Ile Ala Leu Leu Arg 
                165                 170                 175 

Tyr Phe Asn Pro Val Gly Ala His Pro Ser Gly Asp Met Gly Glu Asp 
            180                 185                 190 

Pro Gln Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Ala Gln Val Ala 
        195                 200                 205 

Val Gly Arg Arg Asp Ser Leu Ala Ile Phe Gly Asn Asp Tyr Pro Thr 
    210                 215                 220 

Glu Asp Gly Thr Gly Val Arg Asp Tyr Ile His Val Met Asp Leu Ala 
225                 230                 235                 240 

Asp Gly His Val Val Ala Met Glu Lys Leu Ala Asn Lys Pro Gly Val 
                245                 250                 255 

His Ile Tyr Asn Leu Gly Ala Gly Val Gly Asn Ser Val Leu Asp Val 
            260                 265                 270 

Val Asn Ala Phe Ser Lys Ala Cys Gly Lys Pro Val Asn Tyr His Phe 
        275                 280                 285 

Ala Pro Arg Arg Glu Gly Asp Leu Pro Ala Tyr Trp Ala Asp Ala Ser 
    290                 295                 300 

Lys Ala Asp Arg Glu Leu Asn Trp Arg Val Thr Arg Thr Leu Asp Glu 
305                 310                 315                 320 

Met Ala Gln Asp Thr Trp His Trp Gln Ser Arg His Pro Gln Gly Tyr 
                325                 330                 335 

Pro Asp 

 
           
             9  
             1047  
             DNA  
             Homo sapiens  
             
               CDS  
               (1)..(1047)  
             
           
            9 

atg gca gag aag gtg ctg gta aca ggt ggg gct ggc tac att ggc agc       48 
Met Ala Glu Lys Val Leu Val Thr Gly Gly Ala Gly Tyr Ile Gly Ser 
  1               5                  10                  15 

cac acg gtg ctg gag ctg ctg gag gct ggc tac ttg cct gtg gtc atc       96 
His Thr Val Leu Glu Leu Leu Glu Ala Gly Tyr Leu Pro Val Val Ile 
             20                  25                  30 

gat aac ttc cat aat gcc ttc cgt gga ggg ggc tcc ctg cct gag agc      144 
Asp Asn Phe His Asn Ala Phe Arg Gly Gly Gly Ser Leu Pro Glu Ser 
         35                  40                  45 

ctg cgg cgg gtc cag gag ctg aca ggc cgc tct gtg gag ttt gag gag      192 
Leu Arg Arg Val Gln Glu Leu Thr Gly Arg Ser Val Glu Phe Glu Glu 
     50                  55                  60 

atg gac att ttg gac cag gga gcc cta cag cgt ctc ttc aaa aag tac      240 
Met Asp Ile Leu Asp Gln Gly Ala Leu Gln Arg Leu Phe Lys Lys Tyr 
 65                  70                  75                  80 

agc ttt atg gcg gtc atc cac ttt gcg ggg ctc aag gcc gtg ggc gag      288 
Ser Phe Met Ala Val Ile His Phe Ala Gly Leu Lys Ala Val Gly Glu 
                 85                  90                  95 

tcg gtg cag aag cct ctg gat tat tac aga gtt aac ctg acc ggg acc      336 
Ser Val Gln Lys Pro Leu Asp Tyr Tyr Arg Val Asn Leu Thr Gly Thr 
            100                 105                 110 

atc cag ctt ctg gag atc atg aag gcc cac ggg gtg aag aac ctg gtg      384 
Ile Gln Leu Leu Glu Ile Met Lys Ala His Gly Val Lys Asn Leu Val 
        115                 120                 125 

ttc agc agc tca gcc act gtg tac ggg aac ccc cag tac ctg ccc ctt      432 
Phe Ser Ser Ser Ala Thr Val Tyr Gly Asn Pro Gln Tyr Leu Pro Leu 
    130                 135                 140 

gat gag gcc cac ccc acg ggt ggt tgt acc aac cct tac ggc aag tcc      480 
Asp Glu Ala His Pro Thr Gly Gly Cys Thr Asn Pro Tyr Gly Lys Ser 
145                 150                 155                 160 

aag ttc ttc atc gag gaa atg atc cgg gac ctg tgc cag gca gac aag      528 
Lys Phe Phe Ile Glu Glu Met Ile Arg Asp Leu Cys Gln Ala Asp Lys 
                165                 170                 175 

act tgg aac gta gtg ctg ctg cgc tat ttc aac ccc aca ggt gcc cat      576 
Thr Trp Asn Val Val Leu Leu Arg Tyr Phe Asn Pro Thr Gly Ala His 
            180                 185                 190 

gcc tct ggc tgc att ggt gag gat ccc cag ggc ata ccc aac aac ctc      624 
Ala Ser Gly Cys Ile Gly Glu Asp Pro Gln Gly Ile Pro Asn Asn Leu 
        195                 200                 205 

atg cct tat gtc tcc cag gtg gcg atc ggg cga cgg gag gcc ctg aat      672 
Met Pro Tyr Val Ser Gln Val Ala Ile Gly Arg Arg Glu Ala Leu Asn 
    210                 215                 220 

gtc ttt ggc aat gac tat gac aca gag gat ggc aca ggt gtc cgg gat      720 
Val Phe Gly Asn Asp Tyr Asp Thr Glu Asp Gly Thr Gly Val Arg Asp 
225                 230                 235                 240 

tac atc cat gtc gtg gat ctg gcc aag ggc cac att gca gcc tta agg      768 
Tyr Ile His Val Val Asp Leu Ala Lys Gly His Ile Ala Ala Leu Arg 
                245                 250                 255 

aag ctg aaa gaa cag tgt ggc tgc cgg atc tac aac ctg ggc acg ggc      816 
Lys Leu Lys Glu Gln Cys Gly Cys Arg Ile Tyr Asn Leu Gly Thr Gly 
            260                 265                 270 

aca ggc tat tca gtg ctg cag atg gtc cag gct atg gag aag gcc tct      864 
Thr Gly Tyr Ser Val Leu Gln Met Val Gln Ala Met Glu Lys Ala Ser 
        275                 280                 285 

ggg aag aag atc ccg tac aag gtg gtg gca cgg cgg gaa ggt gat gtg      912 
Gly Lys Lys Ile Pro Tyr Lys Val Val Ala Arg Arg Glu Gly Asp Val 
    290                 295                 300 

gca gcc tgt tac gcc aac ccc agc ctg gcc caa gag gag ctg ggg tgg      960 
Ala Ala Cys Tyr Ala Asn Pro Ser Leu Ala Gln Glu Glu Leu Gly Trp 
305                 310                 315                 320 

aca gca gcc tta ggg ctg gac agg atg tgt gag gat ctc tgg cgc tgg     1008 
Thr Ala Ala Leu Gly Leu Asp Arg Met Cys Glu Asp Leu Trp Arg Trp 
                325                 330                 335 

cag aag cag aat cct tca ggc ttt ggc acg caa gcc tga                 1047 
Gln Lys Gln Asn Pro Ser Gly Phe Gly Thr Gln Ala 
            340                 345 

 
           
             10  
             348  
             PRT  
             Homo sapiens  
           
            10 

Met Ala Glu Lys Val Leu Val Thr Gly Gly Ala Gly Tyr Ile Gly Ser 
  1               5                  10                  15 

His Thr Val Leu Glu Leu Leu Glu Ala Gly Tyr Leu Pro Val Val Ile 
             20                  25                  30 

Asp Asn Phe His Asn Ala Phe Arg Gly Gly Gly Ser Leu Pro Glu Ser 
         35                  40                  45 

Leu Arg Arg Val Gln Glu Leu Thr Gly Arg Ser Val Glu Phe Glu Glu 
     50                  55                  60 

Met Asp Ile Leu Asp Gln Gly Ala Leu Gln Arg Leu Phe Lys Lys Tyr 
 65                  70                  75                  80 

Ser Phe Met Ala Val Ile His Phe Ala Gly Leu Lys Ala Val Gly Glu 
                 85                  90                  95 

Ser Val Gln Lys Pro Leu Asp Tyr Tyr Arg Val Asn Leu Thr Gly Thr 
            100                 105                 110 

Ile Gln Leu Leu Glu Ile Met Lys Ala His Gly Val Lys Asn Leu Val 
        115                 120                 125 

Phe Ser Ser Ser Ala Thr Val Tyr Gly Asn Pro Gln Tyr Leu Pro Leu 
    130                 135                 140 

Asp Glu Ala His Pro Thr Gly Gly Cys Thr Asn Pro Tyr Gly Lys Ser 
145                 150                 155                 160 

Lys Phe Phe Ile Glu Glu Met Ile Arg Asp Leu Cys Gln Ala Asp Lys 
                165                 170                 175 

Thr Trp Asn Val Val Leu Leu Arg Tyr Phe Asn Pro Thr Gly Ala His 
            180                 185                 190 

Ala Ser Gly Cys Ile Gly Glu Asp Pro Gln Gly Ile Pro Asn Asn Leu 
        195                 200                 205 

Met Pro Tyr Val Ser Gln Val Ala Ile Gly Arg Arg Glu Ala Leu Asn 
    210                 215                 220 

Val Phe Gly Asn Asp Tyr Asp Thr Glu Asp Gly Thr Gly Val Arg Asp 
225                 230                 235                 240 

Tyr Ile His Val Val Asp Leu Ala Lys Gly His Ile Ala Ala Leu Arg 
                245                 250                 255 

Lys Leu Lys Glu Gln Cys Gly Cys Arg Ile Tyr Asn Leu Gly Thr Gly 
            260                 265                 270 

Thr Gly Tyr Ser Val Leu Gln Met Val Gln Ala Met Glu Lys Ala Ser 
        275                 280                 285 

Gly Lys Lys Ile Pro Tyr Lys Val Val Ala Arg Arg Glu Gly Asp Val 
    290                 295                 300 

Ala Ala Cys Tyr Ala Asn Pro Ser Leu Ala Gln Glu Glu Leu Gly Trp 
305                 310                 315                 320 

Thr Ala Ala Leu Gly Leu Asp Arg Met Cys Glu Asp Leu Trp Arg Trp 
                325                 330                 335 

Gln Lys Gln Asn Pro Ser Gly Phe Gly Thr Gln Ala 
            340                 345 

 
           
             11  
             317  
             PRT  
             Artificial Sequence  
             
               Description of Artificial SequenceCONSENSUS  
             
           
            11 

Xaa Xaa Arg Xaa Xaa Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 
  1               5                  10                  15 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
             20                  25                  30 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
         35                  40                  45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Xaa Ala Xaa Xaa Xaa 
     50                  55                  60 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
 65                  70                  75                  80 

Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
                 85                  90                  95 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
            100                 105                 110 

Xaa Xaa Xaa Pro Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa 
        115                 120                 125 

Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Xaa 
    130                 135                 140 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
145                 150                 155                 160 

Xaa Xaa Asn Xaa Xaa Gly Xaa His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
                165                 170                 175 

Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
            180                 185                 190 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Gly Xaa Xaa Xaa Arg Xaa 
        195                 200                 205 

Xaa Xaa Xaa Xaa Xaa Asp Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
    210                 215                 220 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
225                 230                 235                 240 

Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
                245                 250                 255 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa 
            260                 265                 270 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
        275                 280                 285 

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
    290                 295                 300 

Xaa Thr Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
305                 310                 315 

 
           
             12  
             34  
             DNA  
             Escherichia coli  
           
            12 

tagaattcag taaacaacga gtttttattg ctgg                                 34 

 
           
             13  
             32  
             DNA  
             Escherichia coli  
           
            13 

aactcgagtt acccccaaag cggtcttgat tc                                   32 

 
           
             14  
             30  
             DNA  
             Escherichia coli  
           
            14 

ctggagtcga attcatgagt aaacaacgag                                      30 

 
           
             15  
             33  
             DNA  
             Escherichia coli  
           
            15 

aactgcagtt acccccgaaa gcggtcttga ttc                                  33