Abstract:
An improved method of screening crystal growth conditions is provided wherein molecules are crystallized from solutions containing dyes. These dyes are selectively incorporated or associated with crystals of particular character thereby rendering crystals of particular character colored and improving detection of the dyed crystals. A preferred method involves use of dyes in protein solutions overlayed by oil. Use of oil allows the use of small volumes of solution and facilitates the screening of large numbers of crystallization conditions in arrays using automated devices that dispense appropriate solutions to generate crystallization trials, overlay crystallization trials with an oil, provide appropriate conditions conducive to crystallization and enhance detection of dyed (colored) or undyed (uncolored) crystals that result.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims the benefit of priority of U.S. Provisional Application No. 60/308,698, filed Jul. 30, 2001, to Provisional Application Serial No. 60/328,958, filed Oct. 12, 2001, and to U.S. application Ser. No. 09/543,326, filed Apr. 5, 2000, which claims benefit of U.S. Provisional Application 60/128,018, filed Apr. 6, 1999, each of which are hereby incorporated in their entirety herein by reference. 
     
    
     STATEMENT OF GOVERNMENT SUPPORT  
       [0002] This invention was made with government support under NASA Cooperative Agreement No. NCC8-246. The government may have certain rights in the invention. 
     
    
     
       FIELD OF THE INVENTION  
         [0003]    This invention relates generally to the method of using detectable agents in crystallization of proteins, wherein the detectable agents are preferentially incorporated in the crystals of proteins or preferentially not incorporated in the crystals of proteins such that protein crystals can be readily distinguished from crystals of other substances which can form under the crystallization conditions, thereby allowing rapid and straightforward characterization of crystals and evaluation of crystallization conditions used to obtain the crystals.  
         SUMMARY OF THE INVENTION  
         [0004]    In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention relates to a method of using dyes to facilitate the characterization of protein crystals present in volumes of one microliter or less. Specifically, it relates to the crystallization of proteins from protein solutions containing dyes under conditions wherein the proteins which crystallize and the other components of the protein solutions which crystallize can be distinguished from one another on the basis of whether or not they form dyed crystals.  
           [0005]    In a first aspect, this invention relates to a method for forming a dyed protein crystal including the steps of: (1) providing a protein solution, wherein the protein solution contains at least one dye, which dye is capable of dyeing at least a portion of protein crystals formed from the protein solution; (2) subjecting the protein solution to environmental conditions effective to form protein crystals; and (3) detecting the presence of dyed protein crystals, whereby the presence of the dyed protein crystals indicates the presence of protein crystals.  
           [0006]    In various preferred embodiments of the first aspect of the invention, the dye can be chosen from the group consisting of methylene blue, methylene green, Izit 1  and crystal violet.  
           [0007]    In various preferred embodiments of the first aspect of the invention, the protein solution is partitioned from the atmosphere. When the protein solution is partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solution occurs. The protein solution can be partitioned from the atmosphere by overlaying the protein solution with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL&#39;s oil 1  (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the protein solution can  
           [0008]    In various preferred embodiments of the first aspect of the invention, the protein solution further includes a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof. The component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals. The conditions under which the component crystallizes, can include the step or process of adding a precipitate solution. The component crystals formed can be undyed component crystals.  
           [0009]    In various preferred embodiments of the first aspect of the invention, any crystals formed in the provided protein solution can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy.  
           [0010]    In a second aspect, this invention relates to a method for screening protein crystal growth conditions, including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of protein crystals formed from the protein solution and which does not dye a significant portion of component crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of dyed protein crystals and/or undyed component crystals, wherein the presence of the dyed protein crystals indicates the formation of protein crystals; and the presence of undyed component crystals indicates formation of component crystals.  
           [0011]    In various preferred embodiments of the second aspect of the invention, the set of protein solutions consists of greater than 10, 50, 100, 250, 500, 1000, 1500, 2000 or 5000 protein solutions.  
           [0012]    In various preferred embodiments of the second aspect of the invention, the dye can be chosen from the group consisting of methylene blue, methylene green, Izit 1  and crystal violet.  
           [0013]    In various preferred embodiments of the second aspect of the invention, the protein solutions are partitioned from the atmosphere. When the protein solutions are partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solutions occurs. The protein solutions can be partitioned from the atmosphere by overlaying the protein solutions with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL&#39;s oil 1  (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere.  
           [0014]    In various preferred embodiments of the second aspect of the invention, the protein solutions further include a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof. The component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals. The conditions under which the component crystallizes, can include the step or process of adding a precipitate solution. The component crystals formed can be undyed component crystals.  
           [0015]    In various preferred embodiments of the second aspect of the invention, any crystals formed in the provided protein solutions can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy.  
           [0016]    In various preferred embodiments of the second aspect of the invention, the detection of dyed protein crystals indicates a combination of environmental conditions to promote crystal growth of a protein in the provide protein solutions. When dyed proteins are detected, the dyed protein crystals can be evaluated in respect to protein crystal quality. Evaluation of protein crystal quality can include consideration of criteria selected from the group consisting of, but not limited to, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape.  
           [0017]    In various preferred embodiments of the second aspect of the invention, the suitability of crystallization conditions can include evaluation of the protein crystal quality of the crystals obtained by a certain set of environmental conditions.  
           [0018]    In a third aspect, this invention relates to a method of forming a dyed component crystal including the steps of: (1) providing a component solution, wherein the component solution contains at least one dye, which dye is capable of dyeing at least a portion of component crystals formed from the component solution; (2) subjecting the component solution to environmental conditions effective to form component crystals; and (3) detecting the presence of dyed component crystals, whereby the presence of the dyed component crystals indicates the presence of component crystals.  
           [0019]    In various preferred embodiments of the third aspect of the invention, the component solution is partitioned from the atmosphere. When the component solution is partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent to or from the component solution occurs. The component solution can be partitioned from the atmosphere by overlaying the component solution with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL&#39;s oil 1  (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent to or from the component solution occurs when the component solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the component solution can be selected so as to optimize the rate of solvent transfer to or from the component solution, in particular, from the component solution to the oil, or vice versa, and from the oil to the atmosphere, or vice versa.  
           [0020]    In various preferred embodiments of the third aspect of the invention, the component solution further includes a protein. The protein selected can crystallize under the controlled environmental conditions to which the component solution is subjected, thereby producing protein crystals. The conditions under which the protein crystallizes, can include the step or process of adding a precipitate solution. The protein crystals formed can be dyed protein crystals.  
           [0021]    In various preferred embodiments of the third aspect of the invention, any crystals formed in the provided component solution can be detected. If crystals are detected, protein crystals can be distinguished from component crystals on the basis that the protein crystals are undyed protein crystals and the component crystals are dyed component crystals. The protein crystals and component crystals can be detected by microscopy.  
           [0022]    In a fourth aspect, this invention relates to a method for screening protein crystal growth conditions including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of component crystals formed from the protein solution and which does not dye a significant portion of protein crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of undyed protein crystals and/or dyed component crystals. The presence of the undyed protein crystals can indicate the formation of protein crystals and the presence of the dyed component crystals can indicate formation of component crystals.  
           [0023]    In various preferred embodiments of the fourth aspect of the invention, the set of protein solutions consists of greater than 10, 50, 100, 250, 500, 1000, 1500, 2000 or 5000 protein solutions.  
           [0024]    In various preferred embodiments of the fourth aspect of the invention, the protein solutions are partitioned from the atmosphere. When the protein solutions are partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solutions occurs. The protein solutions can be partitioned from the atmosphere by overlaying the protein solutions with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL&#39;s oil 1  (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere. The overlaying oil used, or the amount of overlaying oil used, to partition any single protein solution from the atmosphere can be the same or can be different than the overlaying oil used to partition any other protein solution from the atmosphere.  
           [0025]    In various preferred embodiments of the fourth aspect of the invention, the protein solutions can further include a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof. The component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals. The conditions under which the component crystallizes, can include the step or process of adding a precipitate solution. The component crystals formed can be dyed component crystals.  
           [0026]    In various preferred embodiments of the fourth aspect of the invention, crystals formed in the provided protein solutions can be detected. If crystals are detected, protein crystals can be distinguished from component crystals on the basis that the protein crystals are undyed protein crystals and the component crystals are dyed component crystals. The protein crystals and component crystals can be detected by microscopy.  
           [0027]    In various preferred embodiments of the fourth aspect of the invention, the detection of undyed protein crystals indicates a combination of environmental conditions to promote crystal growth of a protein in the provided protein solutions. When undyed proteins are detected, the undyed protein crystals can be evaluated in respect to protein crystal quality. Evaluation of protein crystal quality can include consideration of criteria selected from the group consisting of, but not limited to, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape.  
           [0028]    In various preferred embodiments of the fourth aspect of the invention, the method of screening the suitability of crystallization conditions can include an evaluation of the characteristics, numbers or types of component crystals formed.  
           [0029]    Additional advantages of the invention will be set forth in part in the description, which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.  
           [0030]    For instance, one advantage provided by one aspect of the invention is that the use of the dye to distinguish between salt and protein crystals allows rapid screening of crystals formed in volumes of less than or equal to approximately one microliter to distinguish between those formed of salt and those formed of protein. The methods known to the art at this time that are used to distinguish between crystals of salt and crystals of protein are not able to achieve this result. The methods used now: (1) the “crush test” where an investigator feels and listens for the sound of salt cracking; (2) bombardment of a crystal by x-rays and subsequent diffraction pattern analysis; and (3) addition of dye after a crystal has been grown and observation of staining; each require a crystal of suitable size (0.3 mm×0.3 mm×0.3 mm). Crystals grown from volumes of solution less than or equal to approximately one microliter are too small for these classical methods of analysis. The salt and protein crystals formed are too small to crush or to manipulate effectively for x-ray bombardment. Also, because the volumes in which the crystals are formed are so small, addition of any feasibly manipulated volume of dye solution has significant effects on the composition of the solution containing the crystals and, therefore, significant effects on the crystals therein. Furthermore, as use of such small volumes requires that the crystals be grown in solutions partitioned from the atmosphere, generally by an overlaying oil, it is extremely difficult to add a dye reagent to protein solutions containing crystals. Inclusion of protein solutions prior to crystal growth provides an extremely efficient and simple method to screen for protein crystals in volumes of less than one microliter. Further, this method reduces the time, cost, and skill labor requirements for screening procedures as well as lending itself to forms of automated screening, including for example, methods of automated screening described in U.S. application Ser. No. 09/543,326 incorporated herein by reference. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0031]    [0031]FIG. 1 depicts micrograph images of crystallization trials. (FIG. 1A) Izit under oil. (FIG. 1B) NaCl under oil. (FIG. 1C) NaCl+dye under oil. (FIG. 1D) Lysozyme under oil. (FIG. 1E) Lysozyme+dye under oil. (FIG. 1F) Thaumatin under oil. (FIG. 1G) Thaumatin+dye under oil. (FIG. 1H) Catalase under oil. (FIG. 1I) Catalase+dye under oil. (FIG. 1J) Trypsin under oil. (FIG. 1K) Trypsin+dye under oil using CS15. (FIG. 1L) Trypsin+dye under oil using CS16. In each trial, the oil was AL&#39;s oil, 50:50 mixture of paraffin oil and silicone oil and the dye was Izit.  
         [0032]    [0032]FIG. 2 depicts a lysozyme crystal. This dyed protein crystal, at the lower left quadrant of the well, was grown in the presence of Izit and has incorporated blue dye.  
         [0033]    [0033]FIG. 3 depicts a pepsin crystal. This dyed protein crystal was grown in the presence of Izit and has incorporated blue dye.  
         [0034]    [0034]FIG. 4 depicts a β-lactoglobulin crystal. This dyed protein crystal was grown in the presence of Izit and has incorporated blue dye.  
         [0035]    [0035]FIG. 5 depicts micrograph images of additional crystallization trials. FIGS.  5 A- 5 D depict trials conducted using Izit w/o any added protein under various screening conditions.  5 E- 5 F depict trials with added β-lactoglobulin. FIGS.  5 G- 5 I depict trials with added myristol transferase. FIG. 5J depicts trials with added pepsin. FIGS. 5K and 5L depict trials with protein Renac2. FIGS. 5M and 5N depict trials with protein ROB1. FIG. 5O depicts a trial with protein B10. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0036]    The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.  
         [0037]    Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific methods of using dye to distinguish between crystals of interest and crystals of other character, specific dyes, to specific methods of detecting dyed crystals or to particular uses of the method disclosed herein, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.  
         [0038]    As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a dye” includes mixtures of dyes, reference to “a component” includes mixtures of two or more such components, and the like.  
         [0039]    Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.  
         [0040]    In this specification and in the claims, which follow, reference will be made to a number of terms, which shall be defined to have the following meanings:  
         [0041]    “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example; the phrase “solution optionally containing precipitate” means that the solution may or may not contain precipitate and that the description includes both a solution without precipitate and a solution with a precipitate.  
         [0042]    “Detectable,” as used herein, means that the presence or absence of the molecule or species so described can be determined. Alternatively, “detectable,” as used herein, means that the presence or absence of a certain property or characteristic of a molecule or species so described can be determined. For example, a visually detectable characteristic or property of a species or object, such as a crystal, includes, but is not limited to, the intensity, shade and hue of the species or object.  
         [0043]    “Dye,” as used herein, means an agent which renders another molecule or species detectable by its action on or association with the other molecule or species. Generally, a dye will refer to a visually detectable small molecule, which interacts with, i.e. stains or colors, another molecule. However, dye, as used herein, may also mean an agent that acts on another molecule to render it detectable. Dye, as used herein, may also mean an agent, which absorbs or emits electromagnetic radiation outside of the visible spectrum, e.g., electromagnetic radiation within the ultraviolet spectrum.  
         [0044]    “Environment” or “environmental conditions,” as used herein, means the sum total of all the conditions and elements that make up the surroundings and influence the development of any species subject to the environment. The environmental conditions of a protein in solution can be defined in part by description of a limited number of conditions and elements, including but not limited to the identity and abundance of atoms or molecules in solution, including but not limited to solvents, solutes and precipitates; other conditions such as but not limited to pH, temperature and pressure; and the magnitude and orientation of other forces, including but not limited to gravitational forces, magnetic forces and electrostatic forces.  
         [0045]    It will be recognized by one of skill in the art that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:  
         [0046]    1) Alanine (A), Serine (S), Threonine (T);  
         [0047]    2) Aspartic acid (D), Glutamic acid (E);  
         [0048]    3) Asparagine (N), Glutamine (Q);  
         [0049]    4) Arginine (R), Lysine (K);  
         [0050]    5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).  
         [0051]    Further, it will be recognized by one of skill in the art that two polynucleotides or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman  Adv. Appl. Math.  2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch,  J. Mol. Biol.  48: 443 (1970), by the search for similarity method of Pearson and Lipman,  Proc. Natl. Acad. Sci. U.S.A.  85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.  
         [0052]    The dye(s) used in the practice of the invention may have no effect on the crystallization of a protein contained in the solution. While not being bound by theory, it is contemplated that the nature of the interaction between the selected dyes and protein crystals is such that it has no effect on the structure, stability or growth characteristics of the protein crystals of interest.  
         [0053]    The dye(s) used in the practice of the invention may have a negligible effect on the crystallization of proteins contained in the solution. For example, protein crystals formed in the presence of the dye may be functionally equivalent in respect to their use to determine the structure of the crystallized protein, even though the protein crystals grown in the presence of the dye can be distinguished from the protein crystals grown in the absence of the dye on the basis of criteria that can be used to evaluate protein crystal quality, including the size of crystals, volume of crystals, sharpness of crystal edges and crystal shape.  
         [0054]    The dye(s) used in the practice of the invention may have a significant effect on the crystallization of proteins contained in the solution. For example, the protein crystals&#39; growth may be significantly slower when the protein solution contains the dye or the protein crystals&#39; growth may be significantly faster when the protein solution contains the dye. Significant differences in the growth rate of protein crystals are those greater than 5, 10, 15, 25, 50, 75, 100, 500 or 1000 fold different. Also, the quality of the crystals may be significantly different, as measured by the criteria for determining a protein crystal&#39;s quality, for example, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape.  
         [0055]    It will be recognized that under some conditions, for example, when using Izit dye under high salt conditions and at low pH, colored dye crystals can be formed in the absence of protein. It will also be recognized that under some conditions, certain buffer components or salts can crystal with the dyes used to form colored crystals. Formation of such colored dye-only or colored component crystals can be detected by screening for the formation of colored crystals in the absence of any added protein or biomolecule of interest. Detection of the presence or absence of such colored crystals under such conditions can be used by one of skill in the art to evaluate the authenticity of colored crystals formed under such conditions when protein or the biomolecule of interest is present.  
         [0056]    Screening protein crystal growth conditions, as is described herein, refers broadly to the process of providing at least two protein solutions and subjecting them to different conditions and then determining whether or not the conditions allow for the crystallization of proteins included in the protein solution. The protein solutions, unless otherwise indicated, contain the same protein species, which is the protein of interest. When the protein solutions contain the same protein of interest, the environmental conditions that a given protein solution is subjected to can differ. The protein solutions can also contain different protein species. When the protein species contained in the different protein solutions differ, the protein species that differ may be either the protein of interest and/or other proteins. The protein of interest, as will be recognized by those of skill in the art, is the protein for which the evaluation of protein crystallization conditions is intended. In other words, if an investigator is seeking to establish suitable conditions for the crystallization of protein X, then protein X is the protein of interest.  
         [0057]    The volume of a protein solution used in the method of forming dyed or undyed protein crystals or in screening protein crystallization conditions, as is described herein, can be greater than 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800 or 900 pico-, nano-, or micro-liters. The volume of a protein solution used in the method of forming dyed or undyed protein crystals or in screening protein crystallization conditions, as is described herein, can be less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 milli-, micro-, nano- or pico-liters.  
         [0058]    “Partitioned,” as used herein, refers to the segregation of the protein solution from the atmosphere such that the transfer of at least one species present in the solution from the solution to the atmosphere occurs at a lower rate than when the protein solution is not partitioned from the atmosphere. For example, segregation of the protein solution from the atmosphere by the interposition of a semipermeable membrane, which allows the passage of certain solvent molecules, but not others, would partition the protein solution from the atmosphere. When the protein solution is partitioned from the atmosphere, the partitioning may be done so as to lower the rate at which transfer of solvent from the protein solution occurs. The protein solution may be partitioned from the atmosphere by overlaying the protein solution with an oil. The overlaying oil may be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL&#39;s oil 1  (a 1:1 mixture of paraffin and silicone oil). The particular overlaying oil selected may be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil. Paraffin oil is more restrictive to evaporation of water from the overlayed protein solution than either silicone oil or mixtures containing paraffin oil and silicone oil. Mixtures of the two oils can be used where the rate of evaporation to be allowed falls between the rate allowed when 100% paraffin oil is used and when 100% silicone oil is used. Mixtures of the two oils having higher paraffin oil/silicone oil ratios are more restrictive to transfer of water molecules than those mixtures of oil having lower paraffin oil/silicone oil ratios. In mixtures of the two oils, the %(v/v) of either of the two oils, either the paraffin oil or the silicone oil, can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.  
         [0059]    The practice of protein crystallization to provide crystals suitable for x-ray diffraction and/or other methods of analysis is often hampered by the difficulty in obtaining crystals of sufficient size and quality for analysis. Thus, the most powerful technique currently available for studying the structure of large molecules can often not be applied to molecules for which there is a need for greater understanding to solve pressing questions relating to the health and welfare of humankind. While the barrier of insufficient crystal quality can often be breached with the application of sufficient resources, the elucidation of the conditions required to produce suitable crystals for x-ray analysis is a significant undertaking. The process for determining suitable conditions generally requires large amounts of both highly skilled labor and highly purified protein.  
         [0060]    The application and use of the invention greatly decreases both the quantity of labor and protein required to screen potential crystallization conditions to obtain effective crystallization conditions to provide the suitable high quality crystals required for x-ray crystallography of biomolecules for the use in structure based drug design and the attendant benefits thereof.  
         [0061]    The significance of the improvement provided by the invention can be appreciated better in light of what is provided by other state-of-the-art practices relating to x-ray crystallographic analysis. In particular, the current automated processes, the availability of intense x-radiation from synchrotron sources and improvements in calculating phases, either from molecular replacement or multiple anomalous dispersion (MAD) strategies, all appear able to handle large numbers of crystallized proteins for structure determination. However, none of these aforementioned processes provide the information necessary, or the means, for identifying the proper environmental conditions to form crystals of molecules for growth, diffraction and/or analysis.  
         [0062]    The method of the present invention produces a significant aid in determining the conditions suitable for crystallizing proteins, thereby greatly facilitating the production of suitable protein crystals for structural determination without the excessive expenditure of limited resources.  
         [0063]    In particular, the presently disclosed method can facilitate a great reduction in the quantity of protein needed for the screening of large numbers of crystallization conditions and the time required to analyze the outcome of each crystallization condition trial. It does so by allowing rapid discrimination between crystals of protein and crystals of other components present in a crystallization trial in very small volumes. Furthermore, the ease in discrimination between protein crystals of interest and crystals of other materials (e.g., salt, buffer, precipitants) is of particular usefulness when the presently disclosed use of dye to distinguish between protein and salt crystals is applied to pico, nano or meso scale crystallization trials performed in a microarray or other micro-device.  
         [0064]    In accomplishing the presently disclosed method, the crystallization trials, or necessary parts thereof, can be automated. As the method used to characterize the nature of crystals present does not require physical contact between the crystal of interest and a probe or other such element, as is required for testing of crystals using conventional means, the method is more amenable to automation than conventional methods now used to determine that a crystal is a protein crystal. Embodiments of the method that are automated are contemplated and provide for the preparation and/or rapid analysis of many samples in high throughput applications.  
         [0065]    Automated systems to form solutions for preparing crystallization trials, to conduct trials and to monitor the resulting crystallizations, can include any of those features and aspects as described in U.S. provisional application 60/128,018 and U.S. utility application Ser. No. 09/543,326 both of which are incorporated herein by reference in their entirety. However, the automated systems or components thereof need not include all features described in the previous applications. Minimally, a fully automated system need only include an automated dispensing system to deliver crystallization trial volumes to receptacles, a capability to provide an environment in which crystals can form under at least some conditions, and an ability to detect the presence or absence of formed crystals. The present invention includes the use of dye to facilitate the detection of crystals. Thus, devices of the present invention adapted for automated screening do provide solutions containing the appropriate selected dye and do provide an environment that allows detection of dyed crystals. In one aspect, provision of that environment is effected by use of wells having clear sides or a clear path for optical detection. As will be recognized by one of skill in the art, such an environment need not provide such a clear optical path provided other means for detecting the presence of the dyed crystals is provided. Examples of such other means include, but are not limited to, detectors present within the wells, devices for removal of crystals from wells, and detectors as are known in the art that do not rely upon the presence of a clear optical path, but instead rely on other physical or chemical properties.  
         [0066]    In particularly useful embodiments contemplated, the method can include use of an automated system that dispenses the appropriate solutions to form a crystallization trial that includes the presence of a dye useful in the practice of the invention and overlays the crystallization trial with an oil. Such an automated solution can include a microarray for crystallization trials, an automated dispensing mechanism for dispensing the solutions, an automated dispensing mechanism for dispensing the overlaying oil, and an automated means for detecting crystal growth.  
         [0067]    In particularly useful embodiments contemplated, the method can encompass use of an analysis station that detects the color of crystals in samples that are provided to the analysis station in an automated fashion. As will be recognized by those of ordinary skill in the art, detection of crystal color can include the monitoring and detection of selected portions of the spectra of electromagnetic radiation that are absorbed, reflected or transmitted by a crystal, whereby detection of said selected portions of the spectra are indicative of a color.  
         [0068]    Such automated methods can further include the sorting of crystals in regard to their determined characteristics. Such sorting can be of a physical nature (i.e., the samples containing the crystals are segregated according to the nature of crystals contained therein) or can be of an informational nature (i.e., the identity of samples containing crystals of a particular nature and/or the location of crystals of a particular nature within a sample are recorded).  
         [0069]    Such automated methods can also include determination of the number of crystals or objects of specified character or identity within a given sample, set of samples or other groups. Further, the number and identity relating to obtained crystals can also be used as a descriptor of conditions used to obtain crystals. For example, the total number of biomolecule crystals obtained and/or the fraction of crystals obtained that are biomolecule crystals can be used to describe results obtained using specific sets of conditions that can be used to form crystals of a specified character.  
         [0070]    The automated method can monitor samples or crystals within samples. The automated method can operate in response to a predetermined program. The predetermined program can include input or instructions from the user. Input or instructions can be provided prior to the screening process or can be provided during the screening process either in response to queries generated by the predetermined program or by the initiative of the user.  
         [0071]    Data obtained from the method can include images and data sets representing images or data derived from both images or selected portions of images. Images can be acquired automatically, with user action or with a combination of both automated and non-automated processes. Particular details regarding details of data analysis and determination of whether a biomolecule or a nonbiomolecule (i.e., a protein crystal or a salt crystal) will, of course, vary depending on the characteristics of the crystallization conditions and the dye being used. Optimization of such particular details are well understood by those of skill in the art and would be recognized not to rise to the level of undue experimentation.  
         [0072]    The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.  
       EXAMPLE ONE  
       [0073]    Crystallization of Selected Proteins from Protein Solutions Containing Izit. Introduction  
         [0074]    The ability to distinguish between salt and protein crystals has been a problem solved by conventional x-ray crystallographers in a variety of methods. Those methods include the “crush” test whereby a crystal is crushed and determined to be salt if it makes a cracking sound. A second method involves placing the crystal in a x-ray beam and looking for diffraction resolution. A third method includes the addition of a dye composed of methylene blue under the commercial name of Izit to the volume containing a crystal. Theoretically, a crystal of protein contains large channels of solvent and will readily uptake the dye, while a crystal of salt is so well packed that the dye will not be taken up into the salt crystal. Each of these methods has their pros and cons. The crush test con is readily apparent, crush your protein crystal and you&#39;re back to the starting point. Mounting crystals and shooting to ascertain their ability to diffract takes a significant amounts of time and effort. The addition of dye to stain protein crystals is clearly useful, even if not 100% accurate. However, the addition of dye to stain crystals resulting from a crystallization trial cannot be easily used under certain circumstances, particularly those circumstances where small volumes are required.  
         [0075]    The process for high through put screening for protein crystallization conditions has been miniaturized which thus improves efficiency, but results in the growth of unmanageably small crystals. The crystals are too small for either the crush test or x-ray analysis, due to difficulty in handling, indeed, even addition of dye to the small volumes and to the small crystals is difficult. A simple solution to the problem, the scaling up of various trials to determine if crystals were salt or protein, results in the creation of a bottleneck for high flux structure determination projects, thus negating one of the primary advantages sought in the miniaturization of the screening process.  
         [0076]    Herein, an alternative method of discriminating between protein and salt crystals is disclosed. As described in this example for the purpose of illustration, it includes the addition of a blue dye to crystallization trials of approximately one microliter or lower volumes under paraffin oil as an aid to the detection and the characterization of protein crystals and/or the characterization of crystals as protein crystals.  
         [0077]    Materials and Methods:  
         [0078]    The proteins screened in this example were: hen egg white lysozyme (Boehringer Mannheim, 1243004), 30 mg/ml in 0.1 M sodium acetate (pH 4.7); thaumatin (Sigma, T7638), 30 mg/ml in 100 mM Tris-HCl (pH 6.5); bovine catalase (Sigma, C40), 30 mg/ml in 50 mM HEPES (pH 7.0); cellulase from  T viride  (Sigma, C40), 40 mg/ml in 100 mM Tris-HCl (pH 8.5); bovine trypsin (Sigma, T8253), 60 mg/ml in 25 mM HEPES (pH 7.0), 10 mM CaCl 2 , 10 mg/ml benzamidin HCl; porcine pepsin (Sigma, P6887), 60 mg/ml in 100 mM cacodylate buffer (pH 6.5), 0.2 M Ca acetate; equine serum albumin (Sigma, A3434), 50 mg/ml in 50 mM sodium acetate (pH 5.5);  Bacillus lichenformis  α-amylase (Sigma, A4551), 25 mg/ml in 50 mM cacodylate buffer (pH 6.75), 2 mM CaCl 2 ; Barley β-amylase (Sigma, A7130), 8 mg/ml in 100 mM HEPES (pH 7.5); bovine β-lactoglobulin (Sigma, L3908), 10 mg/ml in 100 mM Tris-HCl (pH 6.5). Precipitants were prepared using reagents from Sigma or Hampton Research. Paraffin oil and Izit were purchased from Hampton Research. Izit is a commercial preparation of methylene blue dye, a planar aromatic dye with a molecular weight of 319 daltons. Crystallization trials were performed in Labsystems 384 clinical plates under 40 μl of paraffin oil. Crystallization buffers used for: lysozyme (100 mM sodium acetate (pH 4.7), 7% NaCl); thaumatin (1.5 M sodium potassium tartrate); catalase (Crystal Screen 36 (Hampton Research), 100 mM Tris (pH 8.5), 8% PEG8000)); cellulase (1.4 M ammonium sulfate); trypsin (Crystal Screens 4, 15, 16, 20, 28,30, 31 (Hampton Research), namely for, (4) 0.1 M Tris-HCl (pH 8.5), 2.0 M ammonium sulfate; (15) 0.2 M ammonium sulfate, 0.1 M sodium cacodylate (pH6.5), 30% PEG8000; (16) 0.1 M HEPES (pH7.5), 1.5 M lithium sulfate monohydrate; (20) 0.2 M ammonium sulfate, 0.1 M sodium acetate trihydrate (pH 4.6), 25% PEG4000; (28) 0.2 M sodium acetate trihydrate, 0.1 M sodium cacodylate (pH 6.5), 30% PEG8000; (30) 0.2 M ammonium sulfate, 30% PEG8000;(31) 0.2 M ammonium sulfate 30% PEG4000; pepsin, 18% PEG8000; equine serum albumin, 2.0 M ammonium sulfate; α-amylase, 1 N NaOH; β-amylase, 0.17 M ammonium sulfate, 15% PEG8000; β-lactoglobulin, 2.0 M ammonium sulfate.  
         [0079]    Crystallization trials were initiated by mixing equal portions of protein and precipitant solutions prior to depositing a 0.5 μl protein solution drop in the bottom of a crystallization chamber. Protein solutions were then covered with 40 μl of paraffin oil. The crystallization chambers were left unsealed to incubate at 20° C. Crystal growth was monitored daily.  
         [0080]    Results  
         [0081]    The dye added to, and present in, the protein solutions of the nanoliter volume crystallization trials conducted under oil did not appear to diffuse into the paraffin oil. The dye did not stain salt crystals formed during the trials, nor did the presence of the dye interfere with salt crystal formation. As a further control, a 0.5 μl drop of undiluted Izit under 40 μl of paraffin or Al&#39;s (Hampton Research) oil was allowed to incubate at 20° C. After 72 hours the dye did not appear to have diffused into the surrounding oils. Even after evaporation of the Al&#39;s oil, the blue dye remained in place. As further controls, four 0.5 μl samples of saturated NaCl solution were deposited into four separate containers. In three of these, crystallization trials were initiated with the addition of Izit in quantities of 1 part per 30 parts solution, 1 part per 60 parts solution, and 1 part per 100 parts solutions. Crystals of NaCl were formed under oil in both the absence and the presence of Izit. In none of the trials, under any of the dilutions tested, did the blue dye enter, and thereby color, the salt crystals. As reported, Izit itself will crystallize under conditions of high salt. When this occurs, the crystallized Izit form distinctive networks of blue fibers or sea urchin crystals. (Note: in Example Three, evidence is presented that suggests that other crystalline forms of Izit or dyed crystals of component(s) can occur. In particular, it is noted that sodium thiocyanate might form dyed crystals under particular conditions.)  
         [0082]    For most proteins, the addition of dye to the protein samples tested did not interfere with crystal formation, and furthermore, resulted in the production of colored protein crystals, i.e. the dye was taken up by the protein crystals (Table 1; FIG. 1). For each test protein, equal volumes of sample and crystallization buffer were mixed. These crystallization samples were either taken as is, “neat,” or Izit was added at a ratio of 1:30, 1:60, or 1:100 in respect to the crystallization sample volume. After preparation of the crystallization samples, the samples were deposited into the crystallization chambers as described in the Materials and Methods section. Crystal trial samples were then covered with 40 μl of paraffin oil and the experiments monitored for the formation of protein crystals. Seven out of ten test proteins crystallized in the absence of dye, specifically, lysozyme, thaumatin, cellulase, pepsin, trypsin, equine serum albumin and catalyse crystals formed in the absence of the blue dye. Each of these test proteins also crystallized in the presence of dye, but crystallization of only four definitively resulted in blue crystals, specifically, lysozyme, thaumatin, trypsin and equine serum albumin. Equine serum albumin displayed phase changes in the absence and presence of dye. Crystallization of cellulase resulted in small flat square plates that appeared to display staining intensity, but it was not possible to definitively characterize the cellulase crystals as blue due to the thinness of the resultant crystals and the resulting similarity of the coloration to the background. The color of cellulase crystals was similar to background hence it was not evident if these crystals absorbed the dye. In addition, characterization of the cellulase crystals was further complicated by their small size. The background intensity due to unincorporated dye present in each sample was similar for each protein tested. The amount of dye incorporated into the protein crystals, and therefore the intensity of resulting coloration, depended on the concentration of dye used and the number and size of the crystals (FIG. 1). The reason(s) for the inability of catalase and pepsin to form crystals in the presence of Izit are unknown. It is reasonable to postulate that the failure of catalase and pepsin crystals to form in the presence of Izit is only isolated to the single set of conditions used in this trial. As evidenced by the results when attempting to crystallize trypsin, alteration of the environmental conditions to which a protein solution is subjected can effect changes in the incorporation of dye into a protein crystal.  
         [0083]    The addition of blue dye to micro and even nano crystallization trials under oil, or in a miniaturized crystallization condition screening device, such as a microarray, will provide significant savings in material, time and money. In large part, the savings result from the ability to determine that crystals formed in a miniaturized system, under a specific set of conditions, are protein crystals, without having to replicate the specific set of conditions on a larger scale, thereby negating much of the initial benefit of miniaturization. While this improved method of screening crystallization conditions is by no means a replacement for growing suitable sized crystals for analysis, it offers a significant improvement to the process of determining appropriate conditions to grow the suitable sized crystals required.  
       EXAMPLE TWO  
       [0084]    Screening Crystallization Conditions for Crystallization of Selected Proteins from Protein Solutions Containing Izit.  
         [0085]    Introduction  
         [0086]    The blue dye Izit facilitated the screening of crystallization conditions suitable for producing crystals of lysozyme, pepsin and β-lactoglobulin. The experimental design used in conjunction with the ability to detect nano-scale crystals, facilitated by the use of the dye, allowed 450 different sets of conditions to be screened. This set of trials, performed in duplicate and systematically screening 10 variables, consumed only a few micrograms of protein. Each crystallization variable was represented by several implementations. For example, the variable organic precipitant was represented by five levels: 1,2-methyl-2,4-pentanediol (MPD); 2, polyethylene glycol 400 (PEG400); 3, PEG2000; 4, PEG4000; and 5, PEG8000. This is in stark contrast to classical approaches normally used in crystallography that use tens of milligrams of protein to screen approximately 24-48 sets of conditions. The ease and economy in which protein crystallization conditions can be screened using these disclosed methods allows a thorough sampling of crystallization parameters for any given protein. This allows an investigator to readily identify conditions optimal for crystallization and can be used to determine conditions suitable for the growth of protein crystals of suitable size and quality for analysis and characterization.  
         [0087]    Materials and Methods:  
         [0088]    The proteins used as test proteins for this example were: hen egg white lysozyme (Boehringer Mannheim, 1243004), 30 mg/ml in 0.1 M sodium acetate (pH 4.7); porcine pepsin (Sigma, P6887), 60 mg/ml in 100 mM cacodylate buffer (pH 6.5), 0.2 M Ca acetate; bovine β-lactoglobulin (Sigma, L3908), 10 mg/ml in 100 mM Tris-HCl (pH 6.5). The concentrations and buffer solutions used for the nano crystallization screen are identical to those used in the 0.5 μl scale experiments described earlier. Prior to the initiation of the nano crystallization screen IZIT was added to each sample using a dilution of 1 part dye to 19 parts protein (v/v).  
         [0089]    Paraffin oil and Izit were purchased from Hampton Research. Nanoliter crystallization screens were initiated by deploying various ratios of protein sample containing blue dye and crystallization buffer (described in Appendix One) into a chamber and then overlaying with 40 μl of Al&#39;s oil. The final volume of the experiments were 80 nl. The ratio of protein to precipitating buffer was either 1:2, 1:1, or 2:1. The exact composition of each of the 450 recipes used is recorded in the table of screen conditions (Appendix One). The pH of the recipes can be calculated using the Henderson-Hasselback equation. Unless otherwise indicated, the buffer components included in the crystallization trial condition recipes are the base (HEPES and MES). MPD and β-OG are 2-methyl-2,4-pentanediol and N-octyl-β-d-gluocpyranoside. PEG400, PEG2K, PEG4000 and PEG 8K are polyethylene glycols of indicated size/mass as is known to those of skill in the art.  
         [0090]    The experimental nanoliter crystallization volumes were sealed during the trials. Data were collected four days after the initiation of the experiment and recorded. The results were scored by visual inspection.  
         [0091]    Crystallization trials were conducted using the precipitant solutions described in Appendix one at the ratios indicated. 450 trial conditions, repeated in duplicate, were tested for each of the proteins. The total volume for each trial protein solution was 80 nanoliters at the beginning of the trial. Each volume of protein solution was overlayed with 40 μl of Al&#39;s oil. The crystallization chambers were sealed and left to incubate at 4° C. and 20° C. Crystal growth was recorded on the fourth day.  
         [0092]    Results  
         [0093]    Conditions which promoted crystallization for each of the three proteins tested were identified. The numbers and types of crystals formed in the trials can be found in Table 2. For each of the three proteins, i.e., lysozyme, pepsin and β-lactoglobulin, there were 900 total trial results (450 trial screening conditions performed in duplicate). Results for each trial, i.e.; each separate volume of protein solution, were analyzed and categorized. These categories include: (1) a clear drop, (2) phase separation, (3) precipitate (regular, granular), (4) microcrystals/precipitate, (5) rosettes or spherulites, (6) needles (one-dimensional growth), (7) plates (two-dimensional growth), (8) small crystals (three-dimensional growth of less than 0.2 mm) and (9) crystals (three-dimensional growth of greater than 0.2 mm). The categories or scores used for the nanoliter crystallization trials were adopted from those suggested by Hampton Research. Scoring of individual experiments to assign each to a category was performed by visual inspection. As the titles of the categories suggest, the results of each experiment were scored based upon presence or absence of detectable species and upon the species&#39; morphology. For example, needle-like crystals are those appear to have one axis that is very long compared to the others, while plates have two axes of approximately equivalent size, each significantly larger than the third. A three dimensional crystal appears to have growth in three dimensions and looks like a brick. For the other categories, precipitate looks like sand, while both phase separations and rosettes look like mixtures of oil and water. The rosettes usually appear to have dense staining in their centers. In some instances, there is greater ambiguity as to the proper categorical assignment. In addition, the total number of trials in which blue crystals formed is also indicated. The number of blue crystals is calculated from visual inspection of categories 6-9, i.e., needles, plates, small three dimensional crystals and larger three dimensional crystals. A representative micrograph of blue crystals formed from each of the three proteins screened is shown in FIGS.  2 - 4 . Specifically: FIG. 2 shows a blue lysozyme crystal grown in the presence of Izit using screening condition 361; FIG. 3 shows a blue pepsin crystal grown in the presence of Izit using screening condition 75; and FIG. 4 shows a blue β-lactoglobulin crystal grown in the presence of Izit using screening condition 213.  
       EXAMPLE THREE  
       [0094]    Screening Crystallization Conditions for Crystallization of Selected Proteins from Protein Solutions Containing Izit and Controls.  
         [0095]    Methods similar to those described in Example 2 were used to test the method of the invention using additional proteins and conditions. FIG. 5 shows micrographs of these trials wherein proteins, buffer, conditions and dye were varied in character and quantity. Trials were conducted as described before and using the noted screening Expt. # [Stock] conditions outlined in Appendix One. In the following example; T1=4° C., T2=15° C., and T3=22° C.  
         [0096]    Crystallization conditions using Izit dye w/o any added protein were examined. For example, screening conditions 251 (at T3), 254 (at T3), 285 (at T2), 266 (at T3) and 339 (at T3) were conducted. Results from conditions 251, 285, 266, and 339 are shown in FIGS. 5A, B, C, and D, respectively. Condition 254, results not shown, resulted in no observable precipitation or crystallization. Condition 251 (FIG. 5A) also does not result in any observable crystal formation. However, condition 285, which includes acetate buffer at pH 4.5, 42 mM Ammonium Sulfate, 3.7% MPD, 1% glycerol and 2 mM magnesium chloride, and condition 339, which includes acetate buffer at pH 4.5, 460 mM thiocyanate, 0.8% PEG 8000, 0.5% glycerol and 8 mM arginine-HCl, both result in apparent crystals.  
         [0097]    Crystallization conditions with various proteins were also examined. FIGS. 5E and 5F show results from β-lactoglobulin under conditions with phosphate buffer at pH 8 and with PEG 400 (at T1) and with phosphate buffer at pH 6, PEG 8000, 10% glycerol, 0.008M Arg-HCl (at T3), respectively. FIGS. 5G, 5H, and  5 I show results from myristol transferase under conditions 291 (at T3), 197 (at T2) and 259 (at T3), respectively. FIG. 5J shows results from pepsin under condition 347 (at T3). FIGS. 5K and 5L show results from Renac2 under conditions 41 (at Ti) and 339 (at T3), respectively. FIGS. 5M and 5N show results from Rob1 under conditions 212 (at T2) and 337 (at T3), respectively. FIG. 5O shows results from B10 in acetate buffer at pH 4.5, with 450 mM thiocyanate, 0.8% PEG 8000, 0.5% glycerol and 8 mM arginine HCl (at T3).  
         [0098]    Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.  
         [0099]    It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.  
                                             TABLE 1                           Summary of IZIT Crystallization Results            Protein   Neat   1:30   1:60   1:100   Condition   Initial pH               Lysozyme   Xtl   Bxtl   Bxtl   Bxtl       4.7       Thaumatin   Xtl   Bxtl   Bxtl   Bxtl   HR   6.5       Catalase   Xtl   —   —   —   CS36   8.0       Cellulase   Xtl   Xtl   Xtl   Xtl       8.5       Pepsin   Xtl   —   —   —       6.5       Equine serum   Pc   BPc   BPc   BPc       5.5       albumin       α-amylase   —   —   —   —       7.0       β-amylase   —   —   —   —       7.5       β-lactoglobulin   —   —   —   —       6.5       Trypsin   Xtl   Bxtl   Bxtl   Bxtl   CS4   7.0       Trypsin   Xtl   Xtl   Xtl   Xtl   CS15   7.0       Typsin   Xtl   Bxtl   Bxtl   Bxtl   CS16   7.0       Trypsin   Xtl   Xtl   Xtl   Xtl   CS20   7.0       Trypsin   Xtl   Xtl   Xtl   Xtl   CS28   7.0       Trypsin   Xtl   Xtl   Xtl   Xtl   CS30   7.0       Trypsin   Xtl   Xtl   Xtl   Xtl   31   7.0                          
 
         [0100]    [0100]                                           TABLE 2A                           Lysozyme                Score 1     Frequency                            1   795           2   58           3   17           4   3           5   1           6   4           7   1           8   21           Number of blue crystals   10                        
         [0101]    [0101]                                           TABLE 2B                           Pepsin                Score 1     Frequency                            1   591           2   24           3   30           4   9           5   0           6   25           7   164           8   57           Number of blue crystals   2                        
         [0102]    [0102]                                           TABLE 2C                           β-Lactoglobulin                Score 1     Frequency                            1   807           2   58           3   8           4   13           5   1           6   5           7   7           8   1           Number of blue crystals   5                        
         [0103]    [0103]                                                   1 Score Definitions            Score   Score Descriptor                    1   Clear Drop       2   Phase Separation       3   Precipitate (Regular Granular)       4   MicroCrystals / Precipitate       5   Rosettes or Spherulites       6   Needles (1D Growth)       7   Plates (2D Growth)       8   Crystals (3D Growth &lt; 0.2 mm)       9   Crystals (3D Growth &gt; 0.2 mm)                    
         [0104]    [0104]                                                                                                                                                                                                                                           Code                                            5                                                                           1   1           4   Mono-                   So-   So-           Am-   so-                               2       1                   dium   dium   2   3   mo-   dium       2   3   4   5           Argi-   3   Gly-       Expt   1   4   Hydro-   Chlo-   Citric   Acetic   nium   Phos-   1   PEG   PEG   PEG   PEG   2   3   nine   Beta-   cerol           Recipe   Protein       #   MES   HEPES   xide   ride   Acid   Acid   Sulfate   phate   MPD   400   2K   4000   6K   MgCl2   CaCl2   HCl   OG   [75%   1       dis-   dis-       (Stock)   [0.5M]   [0 5M]   [2M]   [2M]   [2M]   [2M]   [2M]   [2M]   [30%]   [30%]   [30%]   [30%]   [30%]   [0 2M]   [0 2M]   [0 5M]   [0 5M]   w/v]   Water   Total   pensed   pensed                    1   62   0   9   0   0   0   0   40   203   0   0   0   0   0   0   0   31   41   614   1000   3   1       2   0   62   83   0   0   0   0   114   0   0   0   125   0   0   0   31   0   0   585   1000   3   1       3   62   0   11   48   0   0   0   0   0   0   0   0   240   15   0   0   31   41   552   1000   3   1       4   0   62   215   0   75   0   0   0   0   0   0   83   0   0   15   31   0   21   499   1000   3   1       5   62   0   151   0   0   152   0   0   14   0   0   0   0   0   15   0   3   41   566   1000   3   1       6   62   0   204   0   75   0   0   0   0   83   0   0   0   0   15   0   31   21   509   1000   3   1       7   62   0   7   121   0   0   0   0   0   0   0   0   11   0   0   0   31   41   729   1000   3   1       8   62   0   11   40   0   0   0   0   0   0   0   196   0   0   0   0   31   21   641   1000   3   1       9   0   62   4   115   0   0   0   0   0   0   0   0   127   15   0   31   0   21   626   1000   3   1       10   62   0   301   0   152   0   0   0   0   13   0   0   0   15   0   0   31   41   385   1000   3   1       11   0   62   51   0   0   39   0   0   0   0   0   194   0   15   0   0   31   0   608   1000   3   1       12   0   62   8   0   0   0   149   0   0   13   0   0   0   0   15   0   0   0   753   1000   3   1       13   62   0   11   182   0   0   0   0   0   0   0   0   16   15   0   0   0   21   694   1000   3   1       14   0   62   98   0   0   95   0   0   0   105   0   0   0   15   0   0   31   21   573   1000   3   1       15   0   62   4   0   0   0   182   0   0   0   16   0   0   0   15   0   0   21   701   1000   3   1       16   0   62   39   0   0   0   0   48   244   0   0   0   0   0   0   31   0   21   556   1000   3   1       17   0   62   94   0   0   0   0   95   0   0   108   0   0   0   0   0   31   21   591   1000   3   1       18   62   0   40   0   0   0   0   181   16   0   0   0   0   0   0   0   31   0   670   1000   3   1       19   62   0   43   0   0   48   0   0   0   0   0   235   0   0   15   0   31   0   566   1000   3   1       20   0   62   47   0   0   0   0   114   0   0   129   0   0   0   0   31   0   0   617   1000   3   1       21   62   0   7   0   0   0   0   183   0   16   0   0   0   0   0   0   31   41   661   1000   3   1       22   0   62   164   0   0   152   0   0   0   0   14   0   0   15   0   31   0   41   522   1000   3   1       23   62   0   3   0   0   0   96   0   0   106   0   0   0   0   0   31   0   41   661   1000   3   1       24   62   0   37   0   0   0   0   0   0   197   0   0   0   15   0   31   0   21   598   1000   3   1       26   62   0   3   40   0   0   0   0   0   0   0   197   0   0   15   0   31   41   611   1000   3   1       26   62   0   124   0   0   115   0   0   0   0   0   0   127   0   15   0   0   21   536   1000   3   1       27   62   0   105   0   0   96   0   0   0   0   0   0   106   0   0   0   0   41   590   1000   3   1       28   62   0   7   96   0   0   0   0   0   106   0   0   0   0   15   31   0   41   642   1000   3   1       29   62   0   17   0   0   0   0   182   0   0   0   16   0   0   0   31   0   21   672   1000   3   1       30   0   62   4   116   0   0   0   0   131   0   0   0   0   0   0   0   0   41   647   1000   3   1       31   0   62   4   0   0   0   181   0   0   0   0   0   16   0   0   0   0   0   738   1000   3   1       32   62   0   11   48   0   0   0   0   0   237   0   0   0   0   15   0   0   0   628   1000   3   1       33   62   0   7   0   0   0   115   0   0   0   0   126   0   0   0   0   31   21   639   1000   3   1       34   0   62   104   0   0   96   0   0   0   0   0   0   106   15   0   0   31   41   546   1000   3   1       35   0   62   127   0   0   0   0   181   0   0   0   16   0   0   0   0   31   0   584   1000   3   1       36   0   62   142   0   0   0   0   152   0   13   0   0   0   0   0   0   0   41   590   1000   3   1       37   62   0   130   0   0   149   0   0   0   0   0   0   13   0   0   0   31   0   615   1000   3   1       38   0   62   8   0   0   0   118   0   0   0   11   0   0   0   15   31   0   0   756   1000   3   1       39   0   62   87   0   0   75   0   0   85   0   0   0   0   15   0   0   0   21   655   1000   3   1       40   0   62   107   0   0   0   0   151   0   0   0   0   13   0   0   31   0   21   616   1000   3   1       41   0   62   8   0   0   0   151   0   0   13   0   0   0   15   0   0   31   21   700   1000   3   1       42   62   0   7   149   0   0   0   0   0   0   0   13   0   0   15   0   0   0   754   1000   3   1       43   0   62   92   0   31   0   0   0   0   158   0   0   0   0   15   0   31   21   592   1000   3   1       44   0   62   84   0   0   76   0   0   0   84   0   0   0   0   15   0   0   41   638   1000   3   1       45   62   0   11   0   0   0   75   0   0   0   0   0   83   15   0   31   0   21   702   1000   3   1       46   62   0   354   0   152   0   0   0   0   0   0   13   0   0   0   31   0   41   347   1000   3   1       47   0   62   12   0   0   0   48   0   0   0   245   0   0   15   0   0   31   41   546   1000   3   1       48   62   0   164   0   76   0   0   0   0   0   86   0   0   15   0   31   0   41   525   1000   3   1       49   62   0   9   0   0   0   0   32   0   158   0   0   0   0   0   31   0   41   668   1000   3   1       50   62   0   112   0   40   0   0   0   202   0   0   0   0   15   0   31   0   21   518   1000   3   1       51   0   73   151   0   49   0   0   0   0   242   0   0   0   0   0   0   36   0   449   1000   3   1       52   73   0   75   0   30   0   0   0   0   0   150   0   0   0   0   0   36   24   611   1000   3   1       53   73   0   197   0   72   0   0   0   0   0   0   0   79   18   0   0   36   48   477   1000   3   1       54   0   73   193   0   0   185   0   0   0   0   0   16   0   0   18   0   36   0   479   1000   3   1       55   0   73   9   30   0   0   0   0   0   149   0   0   0   0   0   36   0   48   655   1000   3   1       56   0   73   4   49   0   0   0   0   251   0   0   0   0   0   0   36   0   48   537   1000   3   1       57   0   73   9   0   0   0   29   0   0   0   0   0   145   0   18   0   36   0   690   1000   3   1       58   73   0   8   95   0   0   0   0   0   0   108   0   0   0   18   36   0   48   613   1000   3   1       59   73   0   18   0   0   0   0   29   0   0   148   0   0   0   0   0   36   0   696   1000   3   1       60   73   0   223   0   94   0   0   0   106   0   0   0   0   0   18   0   36   24   425   1000   3   1       61   0   73   87   0   30   0   0   0   0   0   0   0   147   0   0   0   0   24   640   1000   3   1       62   0   73   127   0   0   114   0   0   0   10   0   0   0   0   0   0   36   48   591   1000   3   1       63   0   73   4   151   0   0   0   0   0   0   0   13   0   0   0   36   0   48   674   1000   3   1       64   0   73   161   0   49   0   0   0   0   0   0   244   0   0   0   36   0   48   389   1000   3   1       65   73   0   13   188   0   0   0   0   0   16   0   0   0   0   0   0   0   48   652   1000   3   1       66   0   73   9   0   0   0   119   0   0   0   0   130   0   0   0   0   36   48   585   1000   3   1       67   0   73   53   0   0   49   0   0   0   0   249   0   0   18   0   0   0   24   533   1000   3   1       68   0   73   143   0   49   0   0   0   0   0   0   0   246   18   0   0   0   48   423   1000   3   1       69   0   73   4   0   0   0   148   0   0   13   0   0   0   18   0   0   0   0   744   1000   3   1       70   73   0   13   0   0   0   112   0   0   0   0   0   10   0   18   36   0   24   714   1000   3   1       71   73   0   8   149   0   0   0   0   0   0   0   0   13   0   18   0   36   24   678   1000   3   1       72   0   73   49   0   0   0   0   118   0   0   0   129   0   0   0   0   0   24   607   1000   3   1       73   0   73   132   0   40   0   0   0   0   0   0   196   0   18   0   0   36   48   457   1000   3   1       74   0   73   120   0   0   111   0   0   0   0   10   0   0   18   0   0   0   0   669   1000   3   1       75   0   73   4   94   0   0   0   0   0   104   0   0   0   18   0   0   36   24   646   1000   3   1       76   0   73   14   0   0   0   70   0   0   0   0   0   77   0   0   36   0   0   730   1000   3   1       77   73   0   132   0   0   151   0   0   13   0   0   0   0   18   0   0   36   48   529   1000   3   1       78   0   73   116   0   0   112   0   0   0   10   0   0   0   0   18   36   0   24   611   1000   3   1       79   73   0   6   0   0   0   0   118   0   0   0   0   130   0   0   0   0   24   649   1000   3   1       80   73   0   14   0   0   0   0   94   0   0   107   0   0   0   0   0   0   24   689   1000   3   1       81   0   73   9   0   0   0   70   0   79   0   0   0   0   0   0   0   36   0   733   1000   3   1       82   0   73   4   188   0   0   0   0   17   0   0   0   0   18   0   0   0   24   678   1000   3   1       83   73   0   129   0   0   148   0   0   0   0   0   0   13   0   0   36   0   0   601   1000   3   1       84   0   73   4   0   0   0   39   0   0   0   200   0   0   18   0   0   36   24   605   1000   3   1       85   0   73   14   30   0   0   0   0   0   149   0   0   0   0   0   36   0   48   650   1000   3   1       86   73   0   75   0   30   0   0   0   0   0   0   0   147   0   0   0   0   24   651   1000   3   1       87   73   0   19   0   0   0   0   39   198   0   0   0   0   0   0   0   0   0   671   1000   3   1       88   73   0   42   0   0   30   0   0   0   0   0   148   0   18   0   36   0   48   604   1000   3   1       89   0   73   158   0   49   0   0   0   0   0   0   0   242   0   0   36   0   0   442   1000   3   1       90   73   0   89   0   30   0   0   0   152   0   0   0   0   0   18   36   0   48   553   1000   3   1       91   0   73   14   119   0   0   0   0   0   0   134   0   0   0   0   0   36   48   576   1000   3   1       92   73   0   65   0   0   72   0   0   81   0   0   0   0   0   0   0   36   48   625   1000   3   1       93   0   73   152   0   49   0   0   0   0   0   0   0   244   0   18   36   0   24   403   1000   3   1       94   0   73   9   29   0   0   0   0   0   145   0   0   0   0   0   0   0   0   744   1000   3   1       95   73   0   203   0   94   0   0   0   106   0   0   0   0   0   0   0   36   24   463   1000   3   1       96   73   0   43   0   0   0   0   186   0   0   17   0   0   0   0   0   36   24   620   1000   3   1       97   0   73   9   0   0   0   117   0   0   0   0   0   129   0   18   36   0   0   618   1000   3   1       98   0   73   14   0   0   0   148   0   13   0   0   0   0   0   0   0   0   0   753   1000   3   1       99   73   0   13   0   0   0   49   0   0   246   0   0   0   0   18   36   0   48   516   1000   3   1       100   73   0   148   0   0   148   0   0   13   0   0   0   0   0   0   36   0   0   582   1000   3   1       101   0   100   203   0   0   0   0   214   0   19   0   0   0   0   0   0   0   0   464   1000   2   2       102   0   100   6   167   0   0   0   0   0   0   15   0   0   25   0   50   0   67   570   1000   2   2       103   0   100   33   0   0   0   0   31   0   0   156   0   0   0   0   50   0   67   554   1000   2   2       104   0   100   50   0   0   0   0   114   0   0   0   10   0   0   0   0   50   33   643   1000   2   2       105   100   0   21   0   0   0   0   167   0   0   15   0   0   0   0   0   50   67   580   1000   2   2       106   0   100   322   0   106   0   0   0   0   0   0   0   117   0   0   0   0   67   289   1000   2   2       107   100   0   54   0   0   57   0   0   292   0   0   0   0   25   0   0   0   67   405   1000   2   2       108   0   100   76   0   0   71   0   0   0   78   0   0   0   0   25   0   0   0   649   1000   2   2       109   100   0   7   0   0   0   0   106   0   0   0   116   0   0   0   50   0   67   555   1000   2   2       110   100   0   61   0   0   44   0   0   224   0   0   0   0   0   0   0   50   67   454   1000   2   2       111   100   0   18   0   0   0   135   0   0   0   153   0   0   0   0   50   0   0   544   1000   2   2       112   0   100   6   0   0   0   31   0   0   0   156   0   0   0   0   0   0   67   641   1000   2   2       113   100   0   27   0   0   0   0   57   289   0   0   0   0   0   0   0   50   33   443   1000   2   2       114   100   0   296   0   138   0   0   0   156   0   0   0   0   0   0   0   50   67   194   1000   2   2       115   100   0   129   0   43   0   0   0   0   0   0   0   216   25   0   50   0   33   403   1000   2   2       116   100   0   18   0   0   0   167   0   0   0   0   0   15   0   25   50   0   67   559   1000   2   2       117   100   0   11   114   0   0   0   0   0   0   0   10   0   25   0   50   0   33   655   1000   2   2       118   100   0   5   104   0   0   0   0   0   115   0   0   0   0   25   0   50   33   567   1000   2   2       119   0   100   6   218   0   0   0   0   19   0   0   0   0   0   25   0   0   67   565   1000   2   2       120   0   100   41   0   0   0   0   43   221   0   0   0   0   0   0   50   0   33   511   1000   2   2       121   100   0   18   0   0   0   136   0   0   0   154   0   0   0   25   50   0   33   483   1000   2   2       122   100   0   163   0   57   0   0   0   289   0   0   0   0   25   0   50   0   33   282   1000   2   2       123   100   0   232   0   0   218   0   0   0   0   0   0   19   25   0   0   0   67   339   1000   2   2       124   100   0   111   0   0   108   0   0   0   0   0   116   0   0   25   0   50   67   426   1000   2   2       125   0   100   163   0   0   0   0   167   15   0   0   0   0   0   0   50   0   67   438   1000   2   2       126   0   100   41   0   0   0   0   43   218   0   0   0   0   0   0   0   50   0   548   1000   2   2       127   100   0   53   0   0   44   0   0   0   0   224   0   0   25   0   50   0   67   437   1000   2   2       128   0   100   19   0   0   0   56   0   0   0   287   0   0   25   0   0   0   0   513   1000   2   2       129   100   0   37   0   0   0   0   116   0   0   0   10   0   0   0   50   0   67   620   1000   2   2       130   100   0   142   0   57   0   0   0   0   0   292   0   0   0   25   0   50   67   266   1000   2   2       131   0   100   429   0   138   0   0   0   0   0   0   151   0   25   0   0   50   67   40   1000   2   2       132   100   0   96   0   31   0   0   0   0   152   0   0   0   25   0   50   0   67   479   1000   2   2       133   0   100   294   0   103   0   0   0   0   0   117   0   0   0   25   0   50   0   311   1000   2   2       134   100   0   18   138   0   0   0   0   0   0   156   0   0   0   25   0   0   67   497   1000   2   2       135   0   100   6   57   0   0   0   0   292   0   0   0   0   0   0   50   0   67   428   1000   2   2       136   0   100   88   0   0   0   0   214   19   0   0   0   0   0   0   0   0   0   579   1000   2   2       137   100   0   11   0   0   0   73   0   0   0   0   81   0   25   0   50   0   67   593   1000   2   2       138   0   100   172   0   0   167   0   0   0   0   15   0   0   0   0   0   50   67   429   1000   2   2       139   100   0   24   0   0   0   0   216   0   19   0   0   0   0   0   50   0   33   558   1000   2   2       140   100   0   11   116   0   0   0   0   0   0   0   10   0   25   0   50   0   67   621   1000   2   2       141   100   0   52   0   0   43   0   0   0   218   0   0   0   25   0   0   50   33   480   1000   2   2       142   0   100   662   0   216   0   0   0   19   0   0   0   0   0   0   50   0   33   81   1000   2   2       143   100   0   11   0   0   0   116   0   0   0   10   0   0   25   0   50   0   67   621   1000   2   2       144   100   0   140   0   0   136   0   0   0   0   0   150   0   0   25   0   50   33   365   1000   2   2       145   0   100   347   0   114   0   0   0   0   0   0   10   0   0   0   0   50   33   345   1000   2   2       146   0   100   6   0   0   0   112   0   0   0   0   10   0   25   0   0   50   0   697   1000   2   2       147   100   0   5   0   0   0   138   0   0   0   0   0   152   0   0   50   0   67   488   1000   2   2       148   0   100   49   0   0   30   0   0   0   0   0   0   147   0   0   0   0   0   675   1000   2   2       149   0   100   129   0   0   116   0   0   0   0   0   10   0   25   0   0   0   67   553   1000   2   2       150   100   0   60   0   0   43   0   0   0   0   219   0   0   0   0   50   0   0   528   1000   2   2       151   0   62   51   0   0   48   0   0   0   238   0   0   0   0   15   0   31   21   534   1000   3   1       152   62   0   4   0   0   0   0   40   0   0   202   0   0   0   0   0   31   21   642   1000   3   1       153   62   0   3   0   0   0   118   0   0   0   0   10   0   0   0   31   0   0   776   1000   3   1       154   0   62   191   0   0   183   0   0   0   0   16   0   0   15   0   0   0   41   492   1000   3   1       155   62   0   7   0   0   0   118   0   131   0   0   0   0   0   0   31   0   41   613   1000   3   1       156   0   62   12   0   0   0   40   0   0   197   0   0   0   0   15   31   0   21   623   1000   3   1       157   62   0   11   151   0   0   0   0   0   0   0   0   13   15   0   0   0   21   728   1000   3   1       158   0   62   12   0   0   0   121   0   11   0   0   0   0   15   0   0   31   41   708   1000   3   1       159   0   62   361   0   121   0   0   0   0   0   11   0   0   0   15   0   31   41   359   1000   3   1       160   62   0   7   0   0   0   181   0   0   0   0   16   0   15   0   31   0   0   689   1000   3   1       161   0   62   4   0   0   0   48   0   245   0   0   0   0   0   0   0   0   41   600   1000   3   1       162   0   62   236   0   75   0   0   0   0   0   0   83   0   0   15   31   0   21   478   1000   3   1       163   62   0   202   0   75   0   0   0   84   0   0   0   0   15   0   31   0   0   532   1000   3   1       164   0   62   12   116   0   0   0   0   131   0   0   0   0   0   15   0   0   41   624   1000   3   1       165   0   62   101   0   32   0   0   0   0   158   0   0   0   0   0   0   31   41   578   1000   3   1       166   62   0   16   0   0   0   0   31   0   156   0   0   0   0   0   0   0   21   714   1000   3   1       167   0   62   4   114   0   0   0   0   0   0   0   125   0   0   0   0   31   0   665   1000   3   1       168   0   62   123   0   0   116   0   0   0   128   0   0   0   0   15   0   0   41   515   1000   3   1       169   62   0   11   31   0   0   0   0   0   0   158   0   0   0   15   0   31   0   692   1000   3   1       170   0   62   12   0   0   0   94   0   0   0   0   104   0   15   0   31   0   0   683   1000   3   1       171   0   62   12   95   0   0   0   0   0   0   0   0   105   15   0   0   31   21   660   1000   3   1       172   0   62   12   0   0   0   75   0   0   83   0   0   0   0   0   0   0   0   770   1000   3   1       173   0   62   55   0   0   48   0   0   242   0   0   0   0   15   0   0   31   0   548   1000   3   1       174   0   62   357   0   119   0   0   0   11   0   0   0   0   0   0   0   0   21   431   1000   3   1       175   0   62   8   119   0   0   0   0   11   0   0   0   0   15   0   0   31   21   734   1000   3   1       176   62   0   104   0   48   0   0   0   0   0   0   0   237   0   0   0   31   0   520   1000   3   1       177   0   62   8   0   0   0   152   0   14   0   0   0   0   0   0   0   31   41   693   1000   3   1       178   62   0   179   0   75   0   0   0   85   0   0   0   0   0   15   0   0   21   563   1000   3   1       179   0   62   8   75   0   0   0   0   0   0   84   0   0   15   0   0   31   0   726   1000   3   1       180   62   0   296   0   149   0   0   0   0   0   0   0   13   15   0   0   31   0   434   1000   3   1       181   0   62   157   0   0   149   0   0   0   0   0   13   0   0   0   0   31   0   588   1000   3   1       182   62   0   97   0   40   0   0   0   0   0   0   0   197   0   15   31   0   21   537   1000   3   1       183   0   62   28   0   0   0   0   31   0   0   0   0   155   0   0   0   31   0   694   1000   3   1       184   0   62   46   0   0   0   0   40   203   0   0   0   0   0   0   0   31   41   577   1000   3   1       185   62   0   204   0   95   0   0   0   0   0   0   0   105   15   0   31   0   21   467   1000   3   1       186   62   0   85   0   0   75   0   0   0   83   0   0   0   0   0   0   31   21   643   1000   3   1       187   0   62   12   95   0   0   0   0   108   0   0   0   0   0   15   31   0   21   657   1000   3   1       188   62   0   307   0   116   0   0   0   131   0   0   0   0   0   0   31   0   41   313   1000   3   1       189   0   62   94   0   0   0   0   96   0   0   0   0   106   0   0   0   0   41   601   1000   3   1       190   62   0   3   116   0   0   0   0   131   0   0   0   0   0   15   0   0   41   632   1000   3   1       191   62   0   5   0   0   0   0   75   0   0   85   0   0   0   0   31   0   21   722   1000   3   1       192   0   62   87   0   0   0   0   121   11   0   0   0   0   0   0   0   0   41   679   1000   3   1       193   0   62   98   0   0   94   0   0   0   0   107   0   0   0   15   31   0   0   594   1000   3   1       194   62   0   7   0   0   0   0   181   0   0   0   0   16   0   0   0   31   0   705   1000   3   1       195   0   62   62   0   0   0   0   152   14   0   0   0   0   0   0   0   0   41   670   1000   3   1       196   0   62   4   182   0   0   0   0   0   0   0   16   0   15   0   0   0   21   701   1000   3   1       197   0   62   8   0   0   0   39   0   200   0   0   0   0   0   0   0   0   0   691   1000   3   1       198   62   0   131   0   0   151   0   0   0   0   0   0   13   0   15   0   0   21   608   1000   3   1       199   0   62   154   0   48   0   0   0   0   0   0   237   0   0   15   31   0   21   433   1000   3   1       200   0   62   87   0   0   0   0   121   0   0   0   0   11   0   0   0   0   41   679   1000   3   1       201   0   73   14   118   0   0   0   0   133   0   0   0   0   0   18   36   0   24   584   1000   3   1       202   0   73   32   0   0   0   0   72   0   0   0   0   79   0   0   36   0   48   650   1000   3   1       203   73   0   29   0   0   0   0   95   108   0   0   0   0   0   0   0   0   48   648   1000   3   1       204   0   73   56   0   0   0   0   49   0   0   0   0   242   0   0   0   0   0   581   1000   3   1       205   0   73   113   0   39   0   0   0   198   0   0   0   0   18   0   36   0   0   523   1000   3   1       206   73   0   21   0   0   0   0   49   0   242   0   0   0   0   0   36   0   0   580   1000   3   1       207   73   0   128   0   0   117   0   0   0   0   0   0   129   18   0   36   0   0   500   1000   3   1       208   0   73   61   0   0   0   0   149   0   0   0   13   0   0   0   0   0   24   680   1000   3   1       209   0   73   563   0   185   0   0   0   0   0   0   16   0   0   0   36   0   0   127   1000   3   1       210   73   0   138   0   49   0   0   0   0   0   249   0   0   18   0   0   0   24   448   1000   3   1       211   0   73   9   185   0   0   0   0   0   0   16   0   0   0   0   0   0   0   717   1000   3   1       212   0   73   75   0   0   0   0   185   0   0   0   0   16   0   0   0   36   0   615   1000   3   1       213   0   73   214   0   70   0   0   0   0   0   79   0   0   18   0   36   0   0   510   1000   3   1       214   73   0   8   186   0   0   0   0   0   0   17   0   0   0   18   36   0   24   638   1000   3   1       215   0   73   14   0   0   0   111   0   0   10   0   0   0   0   18   0   36   0   739   1000   3   1       216   0   73   40   0   0   0   0   94   0   0   0   0   104   0   0   0   36   24   628   1000   3   1       217   73   0   10   0   0   0   0   40   0   0   0   196   0   0   0   0   0   48   633   1000   3   1       218   0   73   482   0   151   0   0   0   13   0   0   0   0   0   0   0   36   48   215   1000   3   1       219   73   0   4   0   0   0   0   30   0   0   150   0   0   0   0   36   0   24   683   1000   3   1       220   73   0   120   0   0   119   0   0   0   0   0   0   131   18   0   0   36   48   454   1000   3   1       221   73   0   299   0   151   0   0   0   0   0   0   0   13   18   0   0   0   48   398   1000   3   1       222   0   73   14   30   0   0   0   0   0   0   0   148   0   0   0   36   0   48   651   1000   3   1       223   73   0   8   0   0   0   95   0   0   0   0   104   0   18   0   0   0   48   653   1000   3   1       224   73   0   10   0   0   0   0   29   0   0   0   0   145   0   0   0   0   0   744   1000   3   1       225   0   73   462   0   151   0   0   0   0   0   13   0   0   0   0   36   0   48   216   1000   3   1       226   73   0   197   0   72   0   0   0   0   0   0   79   0   0   18   0   0   48   514   1000   3   1       227   0   73   549   0   185   0   0   0   0   16   0   0   0   0   0   0   36   0   141   1000   3   1       228   0   73   9   0   0   0   185   0   0   0   0   16   0   0   18   0   36   0   663   1000   3   1       229   0   73   446   0   149   0   0   0   0   0   0   0   13   0   0   36   0   24   259   1000   3   1       230   0   73   19   0   0   0   0   39   0   0   200   0   0   0   0   36   0   24   606   1000   3   1       231   73   0   112   0   39   0   0   0   198   0   0   0   0   18   0   0   0   0   560   1000   3   1       232   0   73   189   0   0   186   0   0   17   0   0   0   0   0   18   0   36   24   456   1000   3   1       233   0   73   124   0   0   111   0   0   0   10   0   0   0   0   18   0   0   0   664   1000   3   1       234   73   0   43   0   0   0   0   186   0   16   0   0   0   0   0   36   0   24   621   1000   3   1       235   73   0   311   0   117   0   0   0   132   0   0   0   0   0   18   36   0   0   313   1000   3   1       236   0   73   100   0   29   0   0   0   0   0   0   144   0   18   0   0   0   0   635   1000   3   1       237   0   73   9   0   0   0   93   0   0   0   0   102   0   0   18   36   0   0   668   1000   3   1       238   0   73   282   0   93   0   0   0   0   103   0   0   0   0   18   0   0   0   431   1000   3   1       239   73   0   66   0   30   0   0   0   0   0   0   146   0   0   18   0   0   24   644   1000   3   1       240   0   73   140   0   49   0   0   0   0   242   0   0   0   0   0   0   36   0   460   1000   3   1       241   0   73   4   71   0   0   0   0   0   0   0   78   0   16   0   0   0   24   732   1000   3   1       242   0   73   94   0   0   0   0   93   0   103   0   0   0   0   0   0   36   0   600   1000   3   1       243   0   73   23   0   0   0   0   49   0   0   0   240   0   0   0   0   0   0   616   1000   3   1       244   73   0   252   0   118   0   0   0   0   0   0   129   0   0   18   0   0   24   386   1000   3   1       245   73   0   46   0   0   40   0   0   0   197   0   0   0   0   18   36   0   48   542   1000   3   1       246   73   0   28   0   0   0   0   93   0   0   0   102   0   0   0   0   0   0   704   1000   3   1       247   0   73   29   0   0   0   0   30   0   0   0   0   149   0   0   0   0   48   671   1000   3   1       248   0   73   74   0   0   70   0   0   0   0   79   0   0   18   0   36   0   0   650   1000   3   1       249   73   0   4   0   0   0   118   0   0   0   0   129   0   18   0   0   36   24   598   1000   3   1       250   0   73   74   0   0   70   0   0   0   77   0   0   0   0   18   0   36   0   652   1000   3   1       251   100   0   120   0   0   104   0   0   118   0   0   0   0   0   0   50   0   33   474   1000   2   2       252   100   0   11   0   0   0   116   0   0   0   0   10   0   0   0   0   0   67   696   1000   2   2       253   100   0   11   112   0   0   0   0   0   0   0   10   0   0   0   0   50   0   717   1000   2   2       254   100   0   119   0   0   103   0   0   0   114   0   0   0   0   0   50   0   0   514   1000   2   2       255   100   0   18   0   0   0   57   0   0   0   290   0   0   25   0   0   0   33   477   1000   2   2       256   100   0   5   0   0   0   112   0   0   0   0   0   10   0   0   0   50   0   723   1000   2   2       257   0   100   6   0   0   0   214   0   0   0   19   0   0   25   0   50   0   0   586   1000   2   2       258   100   0   5   73   0   0   0   0   0   81   0   0   0   0   25   50   0   67   599   1000   2   2       259   100   0   5   0   0   0   57   0   0   0   0   0   283   25   0   0   0   33   497   1000   2   2       260   100   0   14   0   0   0   0   44   0   219   0   0   0   0   0   0   0   67   657   1000   2   2       261   0   100   135   0   0   0   0   135   153   0   0   0   0   0   0   50   0   0   427   1000   2   2       262   100   0   11   0   0   0   114   0   0   10   0   0   0   0   0   0   50   33   681   1000   2   2       263   100   0   19   0   0   0   0   138   0   0   0   0   152   0   0   50   0   67   474   1000   2   2       264   100   0   11   0   0   0   112   0   0   0   0   10   0   25   0   0   50   0   692   1000   2   2       265   100   0   39   0   0   30   0   0   0   0   0   0   147   0   25   50   0   0   609   1000   2   2       266   0   100   12   114   0   0   0   0   10   0   0   0   0   0   25   50   0   33   655   1000   2   2       267   100   0   11   30   0   0   0   0   0   0   150   0   0   0   25   0   50   0   634   1000   2   2       268   0   100   91   0   0   72   0   0   82   0   0   0   0   0   0   0   50   33   572   1000   2   2       269   100   0   61   0   0   43   0   0   0   216   0   0   0   0   0   0   0   33   546   1000   2   2       270   100   0   140   0   56   0   0   0   0   0   0   278   0   0   0   0   50   0   375   1000   2   2       271   0   100   302   0   108   0   0   0   0   0   120   0   0   0   0   50   0   67   256   1000   2   2       272   100   0   5   43   0   0   0   0   0   216   0   0   0   0   25   0   50   33   527   1000   2   2       273   0   100   12   72   0   0   0   0   0   0   82   0   0   0   0   0   50   33   650   1000   2   2       274   100   0   40   0   0   0   0   135   0   0   0   148   0   0   0   50   0   0   527   1000   2   2       275   100   0   11   31   0   0   0   0   0   152   0   0   0   0   25   0   0   67   514   1000   2   2       276   100   0   14   0   0   0   0   57   0   0   0   0   285   0   0   50   0   67   426   1000   2   2       277   100   0   5   0   0   0   135   0   0   150   0   0   0   25   0   0   0   0   585   1000   2   2       278   100   0   18   0   0   0   43   0   0   216   0   0   0   0   25   0   50   33   514   1000   2   2       279   0   100   106   0   0   0   0   103   0   0   117   0   0   0   0   0   0   0   573   1000   2   2       280   100   0   11   0   0   0   71   0   0   0   80   0   0   0   25   50   0   0   663   1000   2   2       281   0   100   19   0   0   0   167   0   0   0   0   14   0   0   25   0   0   67   608   1000   2   2       282   100   0   65   0   0   57   0   0   0   0   290   0   0   0   0   50   0   33   405   1000   2   2       283   0   100   353   0   116   0   0   0   0   0   0   10   0   25   0   50   0   67   279   1000   2   2       284   0   100   170   0   0   165   0   0   0   0   15   0   0   0   0   50   0   33   466   1000   2   2       285   0   100   12   167   0   0   0   0   0   0   0   0   15   0   0   0   0   67   639   1000   2   2       286   100   0   18   0   0   0   0   114   0   0   10   0   0   0   0   0   0   33   724   1000   2   2       287   0   100   12   136   0   0   0   0   0   0   154   0   0   0   25   0   50   33   488   1000   2   2       288   0   100   19   0   0   0   71   0   0   78   0   0   0   25   0   0   50   0   657   1000   2   2       289   0   100   6   0   0   0   116   0   0   0   0   0   10   0   25   0   50   67   626   1000   2   2       290   100   0   9   0   0   0   0   214   19   0   0   0   0   0   0   0   0   0   658   1000   2   2       291   0   100   6   103   0   0   0   0   0   0   0   0   114   25   0   50   0   0   602   1000   2   2       292   100   0   5   0   0   0   57   0   0   286   0   0   0   0   25   50   0   67   410   1000   2   2       293   100   0   44   0   0   0   0   163   0   0   15   0   0   0   0   0   50   0   628   1000   2   2       294   0   100   89   0   30   0   0   0   0   0   150   0   0   25   0   0   0   0   608   1000   2   2       295   0   100   233   0   0   214   0   0   19   0   0   0   0   25   0   0   50   0   350   1000   2   2       296   0   100   147   0   43   0   0   0   0   214   0   0   0   0   0   50   0   0   447   1000   2   2       297   100   0   18   135   0   0   0   0   0   0   0   0   150   25   0   50   0   0   522   1000   2   2       298   100   0   18   104   0   0   0   0   0   0   118   0   0   25   0   0   50   33   551   1000   2   2       299   0   100   645   0   216   0   0   0   0   0   19   0   0   0   25   0   0   33   39   1000   2   2       300   100   0   5   72   0   0   0   0   0   80   0   0   0   0   0   0   0   3   710   1000   2   2       301   0   62   4   0   0   0   32   0   0   0   0   0   158   15   0   0   31   41   658   1000   3   1       302   62   0   3   39   0   0   0   0   0   0   0   194   0   0   15   0   31   0   656   1000   3   1       303   0   62   8   40   0   0   0   0   0   197   0   0   0   0   0   0   0   21   673   1000   3   1       304   62   0   3   32   0   0   0   0   162   0   0   0   0   0   0   0   31   41   670   1000   3   1       305   0   62   117   0   0   114   0   0   0   0   0   125   0   0   15   0   0   0   565   1000   3   1       306   62   0   5   0   0   0   0   114   0   126   0   0   0   0   0   0   31   0   662   1000   3   1       307   0   62   127   0   0   0   0   181   16   0   0   0   0   0   0   0   0   0   614   1000   3   1       308   0   62   141   0   0   0   0   151   0   0   0   0   13   0   0   0   31   21   582   1000   3   1       309   0   62   8   119   0   0   0   0   0   10   0   0   0   0   15   31   0   21   734   1000   3   1       310   62   0   30   0   0   31   0   0   160   0   0   0   0   0   15   31   0   21   651   1000   3   1       311   0   62   12   0   0   0   48   0   242   0   0   0   0   0   15   0   31   0   591   1000   3   1       312   62   0   44   0   0   40   0   0   0   0   0   196   0   0   15   31   0   21   592   1000   3   1       313   62   0   4   0   0   0   0   39   0   0   200   0   0   0   0   0   0   0   695   1000   3   1       314   62   0   5   0   0   0   0   75   0   0   0   82   0   0   0   0   0   0   777   1000   3   1       315   0   62   12   140   0   0   0   0   0   0   0   0   13   0   0   31   0   0   734   1000   3   1       316   62   0   66   0   0   75   0   0   0   0   84   0   0   15   0   31   0   0   667   1000   3   1       317   0   62   46   0   0   0   0   39   0   0   0   194   0   0   0   0   0   0   659   1000   3   1       318   62   0   206   0   76   0   0   0   0   0   85   0   0   15   0   31   0   41   483   1000   3   1       319   0   62   6   0   0   0   121   0   11   0   0   0   0   15   0   0   31   41   712   1000   3   1       320   62   0   11   118   0   0   0   0   0   0   11   0   0   15   0   31   0   0   753   1000   3   1       321   62   0   98   0   40   0   0   0   0   199   0   0   0   15   0   0   0   41   545   1000   3   1       322   62   0   30   0   0   0   0   119   0   0   0   0   10   0   0   31   0   21   727   1000   3   1       323   0   62   110   0   0   0   0   114   0   0   120   0   0   0   0   0   31   0   554   1000   3   1       324   0   62   12   31   0   0   0   0   0   0   0   0   155   15   0   31   0   0   695   1000   3   1       325   62   0   7   0   0   0   114   0   0   0   129   0   0   15   0   0   0   0   673   1000   3   1       326   0   62   43   0   0   31   0   0   0   0   0   155   0   0   15   31   0   21   642   1000   3   1       327   62   0   11   48   0   0   0   0   244   0   0   0   0   0   0   0   31   21   585   1000   3   1       328   0   62   4   0   0   0   181   0   16   0   0   0   0   0   0   31   0   0   707   1000   3   1       329   0   62   28   0   0   0   0   31   0   155   0   0   0   0   0   31   0   0   694   1000   3   1       330   0   62   106   0   32   0   0   0   0   158   0   0   0   15   0   0   31   41   555   1000   3   1       331   62   0   3   119   0   0   0   0   0   0   0   0   10   0   15   0   31   21   739   1000   3   1       332   62   0   301   0   152   0   0   0   14   0   0   0   0   0   15   0   31   41   385   1000   3   1       333   0   62   12   31   0   0   0   0   0   0   160   0   0   0   0   0   0   21   715   1000   3   1       334   62   0   5   0   0   0   0   118   0   0   0   0   10   0   0   31   0   0   774   1000   3   1       335   0   62   87   0   0   75   0   0   85   0   0   0   0   0   0   0   31   21   639   1000   3   1       336   62   0   116   0   0   116   0   0   0   0   0   127   0   0   15   0   31   41   492   1000   3   1       337   62   0   30   0   0   0   0   119   0   0   0   10   0   0   0   0   31   21   727   1000   3   1       338   0   62   74   0   0   0   0   183   0   0   15   0   0   0   0   0   31   41   593   1000   3   1       339   62   0   11   182   0   0   0   0   0   0   0   0   16   0   15   31   0   21   663   1000   3   1       340   0   62   39   0   0   32   0   0   0   0   0   0   158   0   15   0   31   41   622   1000   3   1       341   62   0   14   0   0   0   0   116   0   0   0   127   0   0   0   0   0   41   641   1000   3   1       342   0   62   189   0   0   182   0   0   0   0   0   16   0   0   0   0   0   21   531   1000   3   1       343   0   62   8   48   0   0   0   0   0   240   0   0   0   15   0   0   0   41   566   1000   3   1       344   62   0   9   0   0   0   0   40   0   199   0   0   0   0   0   0   31   41   618   1000   3   1       345   62   0   3   116   0   0   0   0   0   0   0   0   128   0   15   0   31   41   604   1000   3   1       346   62   0   68   0   0   75   0   0   0   0   0   84   0   0   15   0   31   41   624   1000   3   1       347   0   62   4   0   0   0   31   0   0   156   0   0   0   0   15   31   0   21   680   1000   3   1       348   0   62   22   0   0   0   0   48   0   0   0   237   0   0   0   31   0   21   581   1000   3   1       349   62   0   7   96   0   0   0   0   0   0   0   0   106   0   0   0   31   41   658   1000   3   1       350   0   62   168   0   0   0   0   182   15   0   0   0   0   0   0   0   31   21   521   1000   3   1       351   0   73   9   0   0   0   70   0   79   0   0   0   0   0   0   0   0   0   769   1000   3   1       352   73   0   120   0   0   148   0   0   0   0   0   13   0   0   0   36   0   0   602   1000   3   1       353   73   0   184   0   0   186   0   0   0   0   0   0   16   0   18   36   0   24   462   1000   3   1       354   73   0   296   0   149   0   0   0   0   0   0   13   0   0   18   0   36   24   391   1000   3   1       355   0   73   222   0   70   0   0   0   0   0   79   0   0   0   0   0   0   0   557   1000   3   1       356   0   73   109   0   0   85   0   0   0   105   0   0   0   18   0   0   0   48   551   1000   3   1       357   0   73   14   71   0   0   0   0   0   0   80   0   0   0   18   36   0   24   684   1000   3   1       358   73   0   86   0   39   0   0   0   0   0   0   0   193   0   18   0   0   0   591   1000   3   1       359   73   0   13   0   0   0   29   0   0   0   0   144   0   18   0   0   36   0   587   1000   3   1       360   0   73   158   0   0   149   0   0   0   0   13   0   0   18   0   0   0   24   564   1000   3   1       361   73   0   172   0   72   0   0   0   0   0   0   79   0   18   0   36   0   48   502   1000   3   1       362   73   0   205   0   95   0   0   0   0   0   0   0   105   18   0   0   0   48   456   1000   3   1       363   73   0   4   0   0   0   49   0   0   0   0   0   242   0   18   0   0   0   615   1000   3   1       364   0   73   71   0   0   0   0   94   0   104   0   0   0   0   0   0   36   24   597   1000   3   1       365   73   0   4   151   0   0   0   0   0   13   0   0   0   18   0   0   0   48   693   1000   3   1       366   0   73   86   0   0   72   0   0   0   0   0   79   0   0   0   36   0   48   606   1000   3   1       367   0   73   318   0   112   0   0   0   0   10   0   0   0   0   0   0   0   24   463   1000   3   1       368   0   73   446   0   149   0   0   0   0   13   0   0   0   0   16   0   0   24   277   1000   3   1       369   73   0   10   0   0   0   0   30   0   0   0   0   149   0   0   36   0   48   654   1000   3   1       370   0   73   4   0   0   0   185   0   0   0   0   16   0   0   18   0   0   0   704   1000   3   1       371   0   73   9   0   0   0   49   0   249   0   0   0   0   0   18   0   0   24   578   1000   3   1       372   0   73   34   0   0   30   0   0   0   0   0   0   149   0   0   0   36   48   630   1000   3   1       373   73   0   160   0   0   185   0   0   16   0   0   0   0   0   18   0   0   0   548   1000   3   1       374   0   73   189   0   0   186   0   0   0   16   0   0   0   0   0   0   0   24   511   1000   3   1       375   73   0   13   0   0   0   49   0   249   0   0   0   0   0   18   36   0   24   537   1000   3   1       376   0   73   4   0   0   0   95   0   0   0   0   0   105   0   0   0   0   48   574   1000   3   1       377   0   73   333   0   118   0   0   0   0   0   133   0   0   0   0   0   0   24   319   1000   3   1       378   73   0   13   30   0   0   0   0   0   0   0   148   0   0   0   0   36   48   652   1000   3   1       379   73   0   4   0   0   0   72   0   0   0   0   0   79   18   0   0   0   48   706   1000   3   1       380   73   0   125   0   0   114   0   0   0   0   0   10   0   0   0   36   0   48   594   1000   3   1       381   0   73   58   0   0   49   0   0   247   0   0   0   0   0   0   36   0   0   538   1000   3   1       382   0   73   9   40   0   0   0   0   0   0   0   0   197   0   0   0   0   48   633   1000   3   1       383   0   73   32   0   0   0   0   72   0   0   0   79   0   0   0   0   0   48   697   1000   3   1       384   73   0   8   29   0   0   0   0   0   0   0   144   0   18   0   0   0   0   728   1000   3   1       385   73   0   4   49   0   0   0   0   0   246   0   0   0   0   0   0   36   48   544   1000   3   1       386   73   0   13   0   0   0   49   0   0   0   0   0   244   18   0   36   0   24   543   1000   3   1       387   0   73   14   0   0   0   49   0   0   246   0   0   0   18   0   0   0   48   552   1000   3   1       388   73   0   8   0   0   0   117   0   132   0   0   0   0   0   0   36   0   0   634   1000   3   1       389   73   0   4   0   0   0   39   0   200   0   0   0   0   18   0   0   36   24   606   1000   3   1       390   73   0   52   0   0   39   0   0   200   0   0   0   0   0   18   0   0   24   584   1000   3   1       391   73   0   98   0   39   0   0   0   0   0   200   0   0   18   0   0   36   24   512   1000   3   1       392   0   73   14   0   0   0   114   0   0   0   0   10   0   0   0   0   36   46   705   1000   3   1       393   0   73   53   0   0   39   0   0   0   0   198   0   0   0   0   0   0   0   638   1000   3   1       394   73   0   13   0   0   0   114   0   10   0   0   0   0   18   0   36   0   48   687   1000   3   1       395   73   0   8   0   0   0   49   0   0   0   0   242   0   0   0   0   0   24   604   1000   3   1       396   0   73   56   0   0   0   0   49   0   244   0   0   0   0   0   36   0   24   518   1000   3   1       397   73   0   8   0   0   0   94   0   0   0   107   0   0   0   18   0   0   24   676   1000   3   1       398   73   0   99   0   40   0   0   0   0   0   0   0   197   18   0   0   0   48   525   1000   3   1       399   0   73   14   114   0   0   0   0   10   0   0   0   0   0   0   0   36   48   705   1000   3   1       400   73   0   4   0   0   0   0   29   0   0   148   0   0   0   0   0   36   0   709   1000   3   1       401   100   0   18   0   0   0   115   0   0   10   0   0   0   25   0   0   0   67   664   1000   2   2       402   0   100   19   0   0   0   165   0   0   0   15   0   0   25   0   0   0   33   643   1000   2   2       403   0   100   421   0   135   0   0   0   153   0   0   0   0   0   25   0   0   0   166   1000   2   2       404   0   100   155   0   0   136   0   0   0   0   154   0   0   0   0   0   0   33   420   1000   2   2       405   100   0   94   0   0   104   0   0   0   0   0   0   115   0   25   50   0   33   478   1000   2   2       406   0   100   12   0   0   0   216   0   0   19   0   0   0   0   0   0   0   33   619   1000   2   2       407   100   0   80   0   30   0   0   0   153   0   0   0   0   0   25   0   50   33   529   1000   2   2       408   100   0   110   0   0   104   0   0   0   0   118   0   0   0   0   50   0   33   484   1000   2   2       409   0   100   19   72   0   0   0   0   0   0   0   79   0   0   25   0   50   33   621   1000   2   2       410   0   100   384   0   135   0   0   0   0   150   0   0   0   0   25   0   0   0   206   1000   2   2       411   0   100   122   0   0   116   0   0   0   0   0   0   10   0   25   0   0   67   560   1000   2   2       412   100   0   5   44   0   0   0   0   224   0   0   0   0   25   0   50   0   67   485   1000   2   2       413   0   100   179   0   0   167   0   0   0   0   15   0   0   25   0   0   50   67   397   1000   2   2       414   100   0   120   0   0   104   0   0   0   0   0   0   115   0   0   0   0   33   526   1000   2   2       415   0   100   56   0   0   44   0   0   224   0   0   0   0   0   25   0   50   67   434   1000   2   2       416   100   0   140   0   56   0   0   0   0   0   287   0   0   25   0   0   0   0   392   1000   2   2       417   100   0   5   0   0   0   214   0   19   0   0   0   0   0   25   0   0   0   637   1000   2   2       418   100   0   5   0   0   0   103   0   0   0   0   0   114   0   0   50   0   0   628   1000   2   2       419   100   0   11   0   0   0   135   0   0   0   153   0   0   0   25   50   0   0   526   1000   2   2       420   0   100   88   0   0   0   0   114   0   10   0   0   0   0   0   0   0   33   654   1000   2   2       421   0   100   12   73   0   0   0   0   83   0   0   0   0   0   25   0   50   67   589   1000   2   2       422   100   0   67   0   30   0   0   0   0   0   0   146   0   0   0   0   50   0   607   1000   2   2       423   100   0   78   0   0   71   0   0   0   0   0   78   0   0   25   50   0   0   598   1000   2   2       424   0   100   6   153   0   0   0   0   0   0   0   14   0   0   25   50   0   0   642   1000   2   2       425   0   100   208   0   72   0   0   0   0   80   0   0   0   0   0   0   0   33   505   1000   2   2       426   100   0   9   0   0   0   0   214   0   19   0   0   0   0   0   0   0   0   658   1000   2   2       427   0   100   50   0   0   0   0   114   0   10   0   0   0   0   0   0   50   33   643   1000   2   2       428   0   100   19   30   0   0   0   0   0   0   0   0   147   0   0   50   0   0   654   1000   2   2       429   100   0   5   115   0   0   0   0   0   0   10   0   0   0   25   0   0   67   677   1000   2   2       430   0   100   75   0   0   57   0   0   0   0   290   0   0   0   0   0   50   33   394   1000   2   2       431   100   0   7   0   0   0   0   104   118   0   0   0   0   0   0   0   0   33   637   1000   2   2       432   100   0   18   0   0   0   73   0   0   0   83   0   0   0   25   50   0   67   584   1000   2   2       433   100   0   5   0   0   0   104   0   0   115   0   0   0   0   0   0   50   33   592   1000   2   2       434   100   0   11   135   0   0   0   0   0   0   0   148   0   25   0   50   0   0   530   1000   2   2       435   100   0   272   0   114   0   0   0   0   0   10   0   0   0   25   0   0   33   445   1000   2   2       436   100   0   5   0   0   0   43   0   218   0   0   0   0   25   0   0   50   0   559   1000   2   2       437   0   100   126   0   43   0   0   0   0   0   219   0   0   0   0   50   0   0   482   1000   2   2       438   100   0   54   0   0   57   0   0   0   256   0   0   0   0   0   0   0   67   436   1000   2   2       439   0   100   480   0   163   0   0   0   0   14   0   0   0   0   25   0   0   0   208   1000   2   2       440   100   0   18   0   0   0   214   0   0   0   19   0   0   0   0   0   50   0   599   1000   2   2       441   100   0   5   30   0   0   0   0   0   147   0   0   0   25   0   50   0   0   643   1000   2   2       442   100   0   18   103   0   0   0   0   116   0   0   0   0   0   25   0   50   0   588   1000   2   2       443   0   100   6   0   0   0   72   0   0   0   0   0   80   0   0   50   0   33   659   1000   2   2       444   0   100   19   103   0   0   0   0   0   0   117   0   0   0   0   50   0   0   611   1000   2   2       445   100   0   180   0   0   165   0   0   0   0   0   0   14   0   0   0   0   33   507   1000   2   2       446   100   0   11   116   0   0   0   0   10   0   0   0   0   25   0   0   0   67   671   1000   2   2       447   100   0   435   0   163   0   0   0   0   0   0   0   14   0   0   0   50   0   238   1000   2   2       448   0   100   12   214   0   0   0   0   0   0   19   0   0   0   25   0   50   0   579   1000   2   2       449   0   100   294   0   103   0   0   0   0   114   0   0   0   25   0   0   0   0   364   1000   2   2       450   0   100   6   138   0   0   0   0   0   0   0   151   0   0   0   0   50   67   488   1000   2   2