Cloning through diffraction: Goals and technologies at the Center for High-throughput Structural Biology (CHTSB). INTRODUCTION: The goal of the Center for High-Throughput Structural Biology (CHTSB) is to overcome the most significant obstacles to structure determination by focusing on technology development in areas related to sample preparation for X-ray diffraction studies. CHTSB is a specialized research center created as part of the NIH Protein Structure Initiative. The center is focusing on several areas; developing yeast (Saccharomyces cerevisiae) as a tool for structural biology, the efficient screening, optimization and production of crystals, and technology for crystal handling and remote data collection. The targets include transmembrane proteins and protein-protein complexes that have traditionally been difficult to study by diffraction methods. Engineering Yeast for Efficient and Robust Incorporation of Selenomethionine into Expressed Proteins: Yeast is a useful system for expressing protein. Its advantages include: 1) It is a eukaryotic system; 2) The ability to produce large amounts of protein; 3) Rapid and inexpensive culturing; 4) Completeness of genetic, genomic, metabolic characterization; 5) History of usefulness for structure determination; 6) Similarities to mammalian cells in post-translational modifications, sub-cellular trafficking, protein folding, biological pathways; 7) Availability of yeast strains with altered protein degradation, unfolded protein response, post-translational modifications, intracellular trafficking; 8) Existence of large, interactive community of yeast laboratories; 9) Ease of genetic manipulation. However, because of the toxicity of selenomethionine to yeast, it was difficult to achieve efficient selenomethionine incorporation into yeast-expressed proteins. Substituting selenomethionine for methionine is a method commonly used for multiwavelength anomalous dispersion (MAD) phasing methods with other expression systems. To overcome the bottleneck of selenomethionine incorporation using a yeast expression system the source of selenomethionine toxicity was identified, and a protocol for selenomethionine incorporation in yeast-expressed protein was developed. These results constitute a general solution to the problem of effective selenomethionine incorporation into proteins expressed in yeast, and remove a major obstacle for the use of yeast as an alternative to E. coli for expression of proteins for structural analysis by X-ray crystallography. Furthermore, these results suggest that efficient selenomethionine incorporation in other organisms might be effected by the employment of similar methods to prevent conversion of selenomethionine into the source of the toxicity, Se-adenosylselenomethionine (Malkowski et al., 2007). TECHNOLOGY DEVELOPMENTS : A summary of CHTSB technologies describing their fit within the target to structure pipeline, and maturity. PRODUCTION - YEAST AS A TOOL established biochemical assays to identify whether the protein is maintained in a native state in the presence of detergents. Genes encoding the target TMPs are transferred via ligation-independent cloning (LIC) procedures to a series of vectors that allow galactose-controlled expression of reading frames fused to C-terminal His6, His10, and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Current efforts are focused on overcoming bottlenecks in protein production and crystallization by introducing the following improvements in the production pipeline: 1) improving overall levels of cellular expression of TMPs by altering protocols for cell growth and induction of expression; 2) increasing efficiency of cell lysis; 3) increasing the efficiency of detergent solubilization; 4) increasing the efficiency of proteolytic removal of affinity tags; 5) developing protocols for obtaining highly concentrated protein preparations that do not contain high detergent concentrations; 6) optimizing the amount of residual lipid purifying with the TMP; 7) reducing the number of steps required for effective purification; 8) testing the use of additives such as lipids and enzyme inhibitors to stabilize purified TMP (White at al., 2006). Development of Methods for Medium-Throughput Cloning, Expression, Analysis, and Purification of Protein:Protein Complexes: To expedite cloning and expression analysis for protein:protein complexes, Drs. Eric Phizicky and Elizabeth Grayhack have developed a suite of vectors for use in yeast strains with commonly found genetic markers. Each vector features high-throughput, ligation-independent cloning and is designed to express genes under control of the strong, regulatable galactose promoter (PGAL), thereby permitting the cloning of genes that are otherwise toxic as well as achieving high-level expression of the corresponding proteins. An initial set of vectors express individual ORFs (Open Reading Frames) with a tri-partite affinity tag on their C-terminus. This tag is comprised of a protease 3C site, an HA epitope, a His6 tag for immobilized metal ion affinity chromatography (IMAC) purification, and the ZZ domain of protein A for purification on IgG Sepharose. Binary protein complexes are made using two otherwise identical vectors (one with a LEU2 marker and one with a URA3 marker), each expressing different genes of the complex. More recently developed vectors allow for expression of two proteins simultaneously, using a bi-directional PGAL promoter, with different tags on each ORF of a pair. Three such sets of vectors, containing either a LEU2 marker or a URA3 marker, have been constructed for expression of an ORF- 3C-HA-His6-ZZ fusion together with an untagged ORF, a His6-tagged ORF, or a His10-tagged ORF. These vectors have three important uses. First, vectors with different tags can be used to unequivocally demonstrate the existence of a complex, by co-purification of ORFs using only one tag on one ORF. Second, use of a vector with a non-cleavable His6 or His10-tag on one ORF, and a cleavable ZZ tag on the other ORF, allows for efficient purification using two affinity steps, each directed at one ORF of the complex. Third, use of pairs of vectors, each with a bi-directional PGAL promoter, allows for purification of complexes comprised of as many as four protein subunits. Expression with these vectors can be as high as 15 mg/L in the best cases. Several medium-throughput methods for analysis of expression and purification of protein complexes have also developed. These employ the use of small cultures, rapid analysis of expression using stick-and-strip binding to either IgG Sepharose or IMAC followed by analysis of proteins on SDS-PAGE, and rapid analysis of authentic complexes employing differentially tagged proteins. Major ongoing research efforts in the lab are focused on the development of methods to further enhance expression of proteins in yeast, on further documentation of the utility of yeast for expression of exogenous proteins and complexes of proteins, and on development of medium-throughput methods for detection of complexes suitable for purification and subsequent structural analysis (Phizicky et al., 2006). After setting up screening experiments, the outcomes are digitally recorded 1 day after the addition of protein and weekly thereafter for a period of 4 weeks using 3 custom-designed imaging systems. Each has the capacity to hold 28 plates and image at a rate of 3 plates (4500 experiments) per hour. Images are stored on RAID arrays and archived on offline media. Outcomes are reviewed to identify combinations of proteins and cocktails that demonstrate a propensity to crystallize. Successful combinations advance through the structure- determination pipeline to the crystal-growth optimization stage. Samples that fail to produce any screening outcomes suitable for optimization are analyzed for solubility data that can be used to optimize the protein formulation. Figure 2. (a) A 1536 well plate with enlargements of four crystallization hits and (b) two of the three imaging tables. Two of the tables are at 23 o C while the third is in a temperature-controlled room to maintain the temperature used for crystallization during imaging. (a) (b) Figure 1. A breakdown of specific areas within the target to structure pipeline that are being developed by groups making up the CHTSB. All of these technologies are being developed as tools for the biology, structural biology, and structural genomics communities. The color-coding used in this chart corresponds to sections of similarly color-coded, detailed descriptions of the technologies in the main text. Development of a Membrane Protein Crystallization Screen based upon Detergent Phase Boundaries: Crystallization conditions for a protein- detergent complex are often near the detergent phase boundary (Weiner 2001). Using dye-partitioning, the phase behavior of 10 detergents, 7 PEGs, and 9 salts were sampled at both 4 and 23 o C. These phase boundaries serve as a guide to formulate detergent-specific cocktails that are less likely to produce an immediate detergent phase separation and subsequent TMP precipitation during crystallization screening. The cocktails target those regions of the phase diagram that is most likely to promote favorable interactions for crystallization. Silver Bullets to Promote Macromolecular Crystallization : A fundamentally different approach was used to develop a set of ‘silver bullet’ cocktails. 200 small molecules were combined to form reagent mixes containing 3 to 20 different chemicals. These cocktails are formulated with one of two precipitating agents; 30% PEG 3350, or 50% Tacsimate TM, both buffered at pH 7.0. The small molecules within the cocktails promote macromolecular contacts. The macromolecule selects a small molecule from the reagent. This small molecule will often appear in the crystal structure at a location that promotes structural stability. These cocktails are commercially available (Hampton Research, HR2-096) (McPherson et al., 2006; Larson et al., 2007a,b) Figure 3. (a) Dye-partitioning highlights the phase behavior of a detergent in the presence of gradually increasing concentrations of salt and PEG. Wells that are uniform in color are not sufficiently supersaturated to cause phase separation of the detergent. Wells that display a marked contrast indicate phase-separated detergent. The crystallization cocktails designed from this data target the border region just below the phase boundary. (b) The phase-boundary data for one detergent in the presence of gradually increasing concentrations of a single salt and five chemically distinct PEGs. (a)(b) Figure 4. Mellitic acid ‘grappling hook’ with 6 carboxylate groups in a unique bovine trypsin structure Yeast Trans Membrane Protein Production: To address the severe lack of three-dimensional structural information for eukaryotic transmembrane proteins (TMPs), Dr. Mark Dumont and his colleagues at the University of Rochester are developing protocols for the expression and purification of TMPs in the yeast Saccharomyces cerevisiae. Initial efforts have been focused on a set of endogenous yeast TMPs that are the highest expressing reading frames in a previously constructed genomic collection of Saccharomyces cerevisiae expression clones. This collection targets reading frames for which there are CRYSTALLIZATION High-Throughput Crystallization Screening: Purified, soluble proteins are processed through a mature, high-throughput screening facility (located at the Hauptman-Woodward Medical Research Institute) to identify crystallization conditions. Syringe-based liquid-handling systems are used to prepare microbatch-under-oil crystallization experiments in 1536-well microassay plates. Each experiment plate holds a single macromolecule combined with 1536 chemical solutions (cocktails). Individual experiments are composed of 200 nanoliters of protein solution and 200 nanoliters of cocktail solution. A screen of 1536 experiments is set up with 400 nL of protein solution (at a concentration of ~10 mg/mL). The high-throughput laboratory currently screens as many as 200 different macromolecules each month from structural genomics centers and the general scientific community. As of March 2008, 852 investigators sent 9855 samples to the laboratory for screening, resulting in 15 million crystallization experiments. In addition to the CHTSB efforts this facility is used by other structural genomics groups and serves the general biomedical community (Luft et al., 2003). Optimization of Crystallization Conditions: Combinations of protein and cocktail solutions that produced crystalline outcomes during screening are formatted into 96 well source plates without reformulation. Solutions are dispensed to set up a series of 16 experiment drops with a percentage composition of decreasing protein solution and increasing cocktail solution. Replicate experiment plates are prepared and separately incubated at different temperatures. In cases where DVR/T fails to produce crystals of sufficient quality for X-ray diffraction analysis, it still provides considerable insight into the effects of temperature and chemistry on the sample’s solubility (Luft et al., 2007).

Mary Rosenblum Meriem Said Edward Snell Elizabeth Snell Max Thayer Christina Veatch Charles Weeks Jennifer Wolfley Kathy Clark Sara Connelly Mark Dumont Nadia Fedoriw Elizabeth Grayhack Yoshiko Kon Maryann Mikucki Eric Phizicky Erin Quartley Katrina Robinson 83% correct 98% correct Figure 6. Tip/capillary development showing (a) a schematic of the plastic capillary/tip, (b) the tip positioned within a uni- puck illustrating how the form factor is compatible with many systems that utilize a standard magnetic cap, (c) an early test example with a crystal within the tip (and an enlarged image of the crystal) (d) diffraction from a crystal within one of the plastic tips cryocooled under pressure (see later section). Figure 5. (a) Cocktails and proteins are arranged into two separate 96 well source plates. A 96 syringe liquid –handling system is used to aspirate from the source plate of cocktail and protein and deliver the solutions at 16 different volume ratios into a 1536 well plate containing USP grade mineral oil. The experiment plates are prepared in replicate for incubation at different temperatures. (b) Illustrates the impact of temperature on the solubility of a protein sample from the structural biology community. This is the same protein, P6306 set up with two different cocktail solutions. Note the cocktail dependent inverse relationship of the temperature-dependent solubility displayed by these experiments. Regions of clear drops that border on drops having phase separation (crystals, precipitate) are likely to be at or near metastable supersaturation, ideal for seeding. Image Analysis: There are over 90 million images generated by the screening laboratory that remain unclassified. A training set of images to guide software- controlled classifiers was compiled. Crystallization outcomes from 96 macromolecular samples that underwent 1536 cocktail screening (MW range of 10 – 2000 kDa) were manually classified by 8 reviewers (each image classified by 3 reviewers, with an even distribution of reviewers/image). Of the 147,456 images selected for this study 70, 465 were unanimously classified. Images fell into ten main categories. The distribution of precipitates (41.7%) and clear drops (41.2%) was nearly identical; 49 of the 96 samples had 1 or more outcomes classified as crystals; crystals were a rare occurrence appearing in 0.38% of the images. Machine classification is taking place at OCI using the human-classified ‘truth data’. Feature generation that will lead to the development of more accurate classifiers is progressing on IBM Blue Gene, and The World Community Grid. Currently over 7,500 years of computer processing time has been used for the distributed project. Table 1. Performance of current classifier. Precipitated and clear drops on average constitute 83% (1275 out of 1536) outcomes of the HT crystal screen. If we can focus our efforts on the 17% (261 out of 1536) images that are most likely to contain crystals, we can eliminate time-consuming manual review of the less- interesting outcomes. By reordering the images, placing clear and precipitated drops after the more-interesting outcomes, we can significantly improve our efficiency and throughput. Crystal Production, Cryopreservation, and Diffraction in Capillaries : We are developing pipette tips as vessels for crystal growth cryopreservation, and in situ X-ray diffraction. This will improve efficiency and eliminate potential damage caused by physical manipulation. This work is a collaboration between SSRL, HWI, and Cornell. Our collaborators at SSRL designed a pipette tip for compatibility with HWI liquid-handling systems and the automated sample handling system at SSRL. Tips produced from a number of different plastics were tested for X-ray diffraction properties (SSRL), compatibility with robotics (HWI, SSRL) (Cohen et al. 2002), crystallization (HWI), and cryo-compatibility (Cornell). Large scale production of tips is now underway. (a)(b) (c) (d) Software Developments for Crystallization: Software has been developed to enhance the capabilities, and improve the efficiency of the crystallization laboratory (Figure 7a-f). (a)“Slide ‘n Shoot Pro” controls imaging hardware, with auto-centering plate definition routines. (b)“CrystalWriter”, controls optical disc hardware to back up image data. (c)“DVR/T Setup” & “WellPacker” creates DVR/T experiment documents and packages DVR/T results/images, (d)“AutoSherlock” for presenting screening experiment results in chemical space. (e) This “AutoSherlock” screenshot displays outcomes of crystallization results plotted on a grid showing the conditions sampled and more importantly those not. In this case PEG and pH play a major role in optimization. The “Grid” shows parameters that should be sampled by a grid screen for optimization. (f) An example of how populating the crystallization results in chemical space (using AutoSherlock) can show a potential area for crystallization occurring between a precipitate and clear conditions. Macroscope, a software for viewing screening results and manual scoring, has been available at no cost to users of the crystallization screening laboratory for the past 8 years. 20% PEG 20K 20% PEG 8K 20% PEG 4K 20% PEG 1K pH Small to large Bad Grid Unknown 6578 PEG 4K PEG 8K PEG 1K PEG 0.4K pH Precipitate Crystals Clear (a)(b)(c) (d) (e) (f) Figure 7. Software developed by CHTSB includes: (a)“Slide ‘n Shoot Pro”; (b) “CrystalWriter”; (c) “DVR/T Setup” & “WellPacker”; and (d-f) “AutoSherlock”. The first two developments improve efficiency in the laboratroy, DVR/T Setup and WellPacker are used to present optimization data and AutoSherlock is in beta testing with the general biomedical community to analyze their crystallization screening results. High Pressure Cryocooling and Heavy Atom Derivatives: Successful protein crystallography typically requires that crystals be cryocooled in order to reduce radiation damage at the data collection stage. So far, protein crystals have most commonly been frozen by flash cryocooling at ambient pressure, which often requires a time-consuming search for cryoprotection conditions. These conditions have been established for all the crystallization screening cocktails in the HWI screen (Kempkes et al., 2008). Recently, the CHTSB-affiliated team (led by Prof. Sol Gruner) at the Cornell High Energy Synchrotron Source (CHESS) has developed an alternative procedure, high-pressure cryocooling, which often does not require the addition of chemical cryoprotectants. The result of high-pressure cryocooling is very often a dramatic improvement in the quality and resolution of the diffraction data. The Cornell team is currently investigating the basic underlying principles and experimental parameters important for the optimization of the high pressure cooling method (Kim et al., 2007; 2008). DIFFRACTION DATA Since the high-pressure method involves the use of helium gas as a pressurizing medium, attention has been focused on its extension to diffraction phasing by incorporating heavy noble gases such as krypton or xenon. In the test case of Porcine Pancreatic Elastase (PPE, 240 residues, 26kDa), very high quality diffraction was obtained by the modified high-pressure cyrocooling method without the help of cryoprotectants. Furthermore, a single krypton site with an occupancy of 0.31 could be used successfully for SAD phasing at 1.3Å resolution. The Cornell team is currently working on equipment modifications that will be compatible with a high throughput crystallography pipeline (Kim et al., 2006). Remote Data Collection: By working closely with crystallographers at the Hauptman-Woodward Institute (HWI), the Structural Molecular Biology (SMB) group at the Stanford Synchrotron Radiation Laboratory (SSRL) is perfecting the technology for remote access data collection. The SSRL SMB group operates six beam lines (BL1-5, BL7-1, BL9-1, BL9-2, BL11-1, and BL11-3) for macromolecular crystallography experiments. Additionally, a new undulator station optimized for microcrystal data collection, BL12-2, is being commissioned. The users of these beam lines have the option to collect data remotely and, during the 2007 run period, about 70% of experiments have been scheduled for remote access thereby saving travel time and expenses. While remaining at their home institutions, remote users conduct experiments by means of advanced software tools enabling network-based systems monitoring and control. Remote users have the capability to mount, center (Song et al., 2007), and screen samples as well as to collect, analyze, and backup diffraction data. Automated sample mounting is accomplished with the Stanford Auto-Mounting System (SAM). In this way, users can screen crystals 2-3 times more rapidly with less human error. For rapid crystal ranking and data analysis, the diffraction images collected during screening are automatically analyzed. Beamline Emulator: An automated set up that provides rapid X-ray based feedback to the crystal optimization has been constructed at HWI. The system mimics the SSRL Stanford Automated Mounting Systems (SAM) (Cohen et al. 2002). Based on typical data collection times 2 cassettes of samples, 192 in total, will be screened in the laboratory and sorted in ~70 hours. This pre- screening will guide crystal optimization (based on diffraction quality) and maximize productive use of beamtime at SSRL. Figure 8. SAM system installed at HWI with Mar 345 imaging plate detector. DATA MINING Databases and Information Systems: Data mining in all areas is under development. Data mining on a subset of crystallization results is underway. The aim is to expand the analysis developed on this subset to analyze to the full archive of results. SUMMARY People ACKNOWLEDGEMENTS Figure 9. Early data mining results from scored crystallization trials. REFERENCES CHTSB has been focused on technology developments. These developments are now bearing fruit in terms of production, identification of crystallization leads, optimization and diffraction. The tools developed feed into the corresponding pipeline from target to structure but individually are applicable and available to the wider general biomedical community. Cohen et al., (2003). An automated system to mount cryo-cooled protein crystals on a synchrotron beamline, using compact sample cassettes and a small scale robot. J. Appl. Cryst. 35, Collins, M.C., et al. (2007). Structural Rigidity of a Large Cavity-Containing Protein Revealed by High-Pressure Crystallography. J. Mol. Biol. 367: Kempkes, et al. (2008). Glycerol Concentrations Required for the Successful Vitrification of Cocktail Conditions in a High-Throughput Crystallization Screen. Acta Cryst. D64: Kim, C., et al. (2006). Solution of Protein Crystallographic Structures by High-Pressure Cryocooling and Noble-Gas Phasing. Acta Cryst. D62 : Kim, C., Hao, Q., and Gruner, S.M. (2007). High Pressure Cryocooling for Capillary Sample Cryoprotection and Diffraction Phasing at Long Wavelengths. Acta Cryst. D63: Kim, C.U., et al. (2008). Pressure Induced High-density Amorphous Ice in Protein Crystals. J. Appl. Cryst. 41:1-7. Luft et al., (2003).A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol. 142, Luft, J.R., et al. (2007). Efficient Optimization of Crystallization Conditions by Manipulation of Drop Volume Ratio and Temperature. Protein Science 16: Larson, S.B., et al. (2007). A Novel Strategy for the Crystallization of Proteins: X-ray Diffraction Validation. Acta Cryst. D63: Larson, S.B., et al. (2007). A new crystal form of bovine pancreatic RNase A in complex with 2'-deoxyguanosine-5'-monophosphate. Acta Cryst F63: Malkowski, M.G., et al. (2007). Blocking Conversion of Methionine to S-Adenosylmethionine in S. cerevisiae Allows Selenomethionine Incorporation and Multiwavelength Anomalous Dispersion Phasing. Proc. Natl. Acad. Sci., USA, 104: McPherson, A. & Cudney, R. (2006). An Alternative Strategy for Crystallizing Macromolecules. J. Structural Biology 156: McPherson, A., et al. (2007). Development of an Alternative Approach to Protein Crystallization. J. Struct. Funct. Genomics. 8: Phizicky, E.M. & Grayhack, E.J. (2006). Proteome-Scale Analysis of Biochemical Activity. Crit. Rev. Biochem. Mol. Biol. 41: Song, J., et al. (2007). Diffraction-based Automated Crystal Centering. J. Synchrotron Rad. 14: White, M.A., et al. (2006). Characteristics Affecting Expression and Solubilization of Yeast Membrane Proteins. J. Mol. Biol. 365 : Eleanor Cook George DeTitta Christopher Goulah Angela Lauricella Joseph Luft Michael Malkowski Raymond Nagel Walter Pangborn Stephen Potter Work supported by NIH U54 GM074899, the John R. Oishei Foundation, Margaret L. Wendt Foundation, Erie County and the James H. Cummings Foundation. Yi-Fan Chen Sol Gruner Chae Un Kim Joseph Chang Aina Cohen Tzanko Doukov Keith Hodgson Michael Soltis Christian Cumbaa Igor Jurisica John Day Aaron Greenwood Steven Larson Yri Kuznetsov Alexander McPherson Department of Molecular Biology and Biochemistry, University of California, 560 SH, Mail Code 3900 Irvine, CA Department of Physics/CHESS, 162 Clark Hall, Cornell University, Ithaca NY Division of Signaling Biology, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, ON, M5G 2M9 Building 120, SLAC, 2575 Sand Hill Road, Menlo Park, CA 94025