13th International Conference on the Crystallization of Biological

Nov 30, 2011 - Macromolecules, a biennial conference organized by the. International Organization for Biological Crystallization. (IOBCr)) demonstrate...
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13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13) Proceedings Overview Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13) Janet Newman† and Joseph R. Luft*,‡ †

CSIRO Materials Science and Engineering, Parkville, VIC 3052, Australia Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203, United States

‡

Improved X-ray techniques have now permitted the determination of the structure of over 70 proteins, only a small fraction of the number of potential interest, and the time for analysis has been reduced considerably due to sophisticated computerized equipment. So declared R. S. Feigelson, who was the editor of the Proceedings of the First International Conference on Protein Crystal Growth, held at Stanford University in 1985.1 Even now, with 3 orders of magnitude more structures, we are still saying that only a small fraction of the proteins of interest have been studied and still stating that the time for analysis is shrinking − due not only to our current computer equipment, but also to the many scientific and technological breakthroughs that have propelled us from 70 structures in 1985 to 75 000 structures in 2010.2 Contemporary structural efforts now routinely include membrane proteins, multiprotein complexes, and other technologically challenging biological systems. Furthermore, X-ray structures are not created in isolation nor are they considered esoteric; they are fully integrated with the biological sciences. We are so familiar with having an atomic structure as the starting point for understanding biological mechanisms that the lack of (or inability to obtain) structural information directly affects our understanding of biological mechanisms. Crystallization has been generally identified as our major point of failure for years, 3 but there are many aspects of crystallization: this is a broad discipline. The 13th ICCBM (International Conference on the Crystallization of Biological Macromolecules, a biennial conference organized by the International Organization for Biological Crystallization (IOBCr)) demonstrated the divide and conquer approach investigators have taken to resolve specific problems within crystallization which included protein preparation and physicochemical characterization, crystallization screening, crystal detection, optimization, and preparation for diffraction experiments. Progress has been made in all aspects of this multifaceted process including breakthroughs in nanocrystallography that have the potential to make crystallization just another step, rather than a restriction, in the structural pipeline. This overview offers a context for the individual articles published in the special ICCBM 13 virtual issue of Crystal Growth & Design, a historical perspective, and our opinions on what the future may hold for biological macromolecular crystallization. Protein Production, Formulation, Modification, and Characterization. Possibly the most profound advance since that first meeting in 1985 has been the development and © 2011 American Chemical Society

routine application of methods to produce large quantities of protein from non-natural sources. Although recombinant DNA technologies were developed in the early 1970s,4 few of the proteins crystallized before that first meeting in 1985 were from recombinant systems. The importance of this is not only in being able to overproduce proteins that are naturally found only in trace amounts, but more to subtly re-engineer the protein: to change its length, to substitute specific amino acid residues, to add purification tags, and to control the posttranslational modifications by site-directed mutagenesis or by selection of the host expression system. These fundamental tools have allowed us to search for − and sometimes find − the right construct for structural studies and have led to many of the lovely success stories of structural determinations presented at the meeting. It is sobering that one of the perennial “beer questions” at a crystallization meeting − “Do purification tags hinder crystallization?” − would not even have made sense during that first crystallization meeting in 1985. In “The Role of Small Molecule Additives and Chemical Modification in Protein Crystallization”,5 McPherson describes his recent work on the specific chemical relationships that can exist between small molecules and proteins and methods to chemically modify proteins to alter their crystallization behavior. Given the importance of small molecules for improving crystallization, we should mention a tool that was unavailable in 1985, differential scanning fluorimetry (DSF). This technology is currently used by many crystallization laboratories to rapidly screen and identify small molecules that increase the protein’s melting temperature (Tm).6 While Tm is not directly correlated with the overall propensity for proteins to crystallize, an increased Tm for a single protein with specific small molecules often indicates an improved stability and thus an increased likelihood for crystallization.7 It must be remembered that the protein and its formulation is an important, arguably the most important, crystallization variable.8 Despite spectacular advances in X-ray technologies, structural investigation of protein complexes remains a formidable challenge. A wonderful retrospective of physicochemical methods that were used to investigate the ribosome prior to the determination of its structure by crystallography9 is Received: September 14, 2011 Revised: November 22, 2011 Published: November 30, 2011 3

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presented by Peter B. Moore “Structure Determination without Crystals: The Ribosome 1970−2000”.10 The methods are not simply of historical interest, but are of practical use, offering insight and complementary tools for the analysis and characterization of large protein complexes. One of the thought-provoking messages from this paper is that the intrinsic noise of biophysical characterizations can make a clean, unambiguous interpretation very difficult and thus highlights the importance of simultaneously looking at complementary methods, techniques, and strategies. Structural biology as a whole has become much more open to collaborative research and to the application of disparate methods to solve increasingly complex structural problems. We no longer expect crystallographic structures to provide the answers to all of our biological inquiries in isolation. Multidisciplinary approaches are embraced by the crystallographic community to better understand biological problems that no individual technique can fully decipher. Structural methods that once vied with each other for “best in field” status are now considered complementary: crystallography, NMR spectroscopy, cryo-electron microscopy, and small angle X-ray scattering (SAXS) each bring a unique viewpoint to answer the most challenging biological questions. The whole is much greater than the sum of the parts, and these structural tools, in conjunction with biochemical and biophysical tools, tremendously enhance our understanding of the structural and functional relationships that drive biology. Cocktails and Crystallization Screening Methods. Despite fundamental advances in crystallization screens, we are frequently stymied by a failure to identify initial crystallization conditions. The original sparse-matrix screen, developed by Jancarik and Kim11 two decades ago, is an effective screen that many of us routinely employ. Now we have thousands of commercially available crystallization cocktails, but many of these conditions are chemically very similar, or identical, and can be traced back to a common ancestor.12 Rather than using repetitive screens, a subset of chemically diverse screens will maximize the value of each experiment as described in the article “PICKScreens, A New Database for the Comparison of Crystallization Screens for Biological Macromolecules”.13 We have yet to explore many classes of chemicals that could play important roles in crystallization screening. The paper “Polysaccharides as Sole Precipitants in Protein Crystallization for X-ray Diffraction Studies”14considers one alternative to the usual choices for precipitants, while “Practical Use of Glycerol in Protein Crystallization”15 shows a number of uses for a more familiar chemical. Related to this topic, “Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization”16 describes physicochemical approaches that were used to monitor the formation of a stable oligomer for crystallization. A method using fluorescence anisotropy to indicate conditions where a protein is more likely to crystallize and expands the range of crystallization conditions is described in “Developing a Fluorescence-based Approach to Screening for Macromolecule Crystallization Conditions”.17 Membrane Protein Crystallization. The progress being made in the crystallographic study of membrane proteins was a focus and highlight of ICCBM13. Membrane proteins remain one of our most technically challenging macromolecular crystallographic pursuits. Currently, there are fewer than 400 unique structures of membrane proteins in the PDB, less than 1% of all structures. This despite the fact that they account for

30% of all proteins in sequenced genomes and are the single largest class of drug targets.18 Obtaining the structures of these biomedically important molecules is hampered by difficulties in their expression, purification, and the production of sufficiently well-ordered crystals. Since 1985, which was also the year the first integral membrane protein structure was described, significant progress has made in the tools for working with integral membrane and membrane-associated proteins, including the availability of highly purified detergents. When crystallizing a membrane protein, the sample is not just the purified protein or protein assembly, but rather a heterogeneous system that includes protein, detergent, and other chemical components such as lipids. An experimental approach to optimize these conditions is captured in the paper “HiLiDe A Systematic Approach to Membrane Protein Crystallization by Screening for Optimal Lipid and Detergent to Protein Ratios”,19 where the protein/lipid/detergent complex is the crystallizable entity. Another major advance has been the development of crystallization methods reliant upon the lipidic cubic phase,20 as described in “Crystallizing Membrane Proteins in Lipidic Mesophases. A Host Lipid Screen,”21 “Membrane Protein Crystallization in Lipidic Mesophases. Hosting Lipid Effects on the Crystallization and Structure of a Transmembrane Peptide”,22 and “Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases”.23 While we have not achieved routine structural investigations of membrane proteins, we have certainly made incredible progress. Seeding. We’ve all seen crystals with a fiber at their heart. The crystallization community has long recognized that the difference between a clear drop and one having a crystal can be dependent upon the serendipitous landing of a stray piece of dust in the experiment drop. Deliberate seeding of crystallization experiments continues to be an active area of research with ever-evolving technologies. Evidence for this can be found in the four papers dedicated to seeding technologies presented in this special issue, “Growth of Protein Crystals by SyringeType Top-Seeded Solution Growth”,24 “Combining Counterdiffusion and Microseeding Techniques to Increase the Success Rate in Protein Crystallization”,25 “A Straightforward and Robust Method for Introducing Human Hair as a Nucleant into High Throughput Crystallization Trials”,26 and “Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization”.27 A related paper, “Hetero- vs Homogeneous Nucleation of Protein Crystals Discriminated by Supersaturation”,28 uses functionalized surfaces to investigate nucleation processes. For many of us, “to seed or not to seed” is no longer the question; it has been replaced by, “Which method should I use?” Seeding seems to be one of the most effective tools available to increase the percentage of crystallization hits when a protein provides few leads,29 and a small effort using simple tools can produce crystal hits from outcomes that are otherwise unsuitable for diffraction studies.30 Optimization Strategies. While the role of screening is to identify chemical regions where a protein shows a propensity to crystallize, optimization is guided toward the fine-tuning of these conditions. “What’s in a Drop? Correlating Observations and Outcomes to Guide Macromolecular Crystallization Experiments”31 offers a rational, generally applicable strategy for optimization. A very nice demonstration of the well-known optimization concept, small changes can make a big dif ference, is 4

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presented in “Development of a Crystallization Protocol for the DbeA1 Variant of Novel Haloalkane Dehalogenase from Bradyrhizobium elkani USDA94”.32 The support for microgravity as a crystallization variable has waxed and waned over the years. “High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity”33 reports a specific case where microgravity-grown crystals led to a structure of biological significance. “Exploiting Protein Engineering and Crystal Polymorphism for Successful X-ray Structure Determination”34 describes four interesting case studies, specifying the critical parameters that led to their crystallographic structures. Automation has had an interesting effect on both screening and optimization; it seems to have nudged the crystallization method used by most laboratories to the automation-friendly sitting-drop vapor diffusion35 and microbatch-under-oil36 techniques. Crystallization methods significantly affect the path and kinetics to supersaturation,37 yet most laboratories do not stray from a single, preferred method. Perhaps improvements in efficiency might come from less limited approaches, for example, by applying well-known, alternative crystallization methods such as counter-diffusion38 and microdialysis, 39 both of which offer different kinetic and physicochemcal pathways to supersaturation. Crystal Detection, Mounting, and Diffraction. There is the familiar reciprocal relationship for diffracted X-rays, and there is the less well-postulated, but often experienced, reciprocal correlation between crystal size and the relative importance of the macromolecule to the investigator’s research. Intense and readily available microfocus beamlines now enable crystals to be harvested directly from the initial, sub-microliter screening experiments for data collection leading to structural solutions. Advances in obtaining diffraction information from membrane protein nanocrystals at the Stanford Linac Coherent Light Source were presented by John Spence during ICCBM13 and hint at what the future may hold for structural biologists. Crystals large enough for diffraction studies may be so small that they are difficult to observe and manipulate, which has led to the development of innovative microcrystal handling systems. Another promising development, in situ data collection, is described in the article, “The SLS Crystallization Platform at Beamline X06DA - A Fully Automated Pipeline Enabling in Situ X-ray Diffraction Screening”.40 This highlights a technology that has the potential to eliminate the all too often “spine-tingling experience” and increasingly difficult problem of mounting microcrystals for X-ray diffraction analysis. Another major breakthrough since 1985 has been the development of cryocrystallography − it was in 1988 that Hope reported protein crystals could be routinely cooled to liquid nitrogen temperatures to increase their lifetime in the beam.41 Efforts to make this process more efficient continue to this day with the article, “Flash Cooling Protein Crystals: Estimate of Cryoprotectant Concentration using Thermal Properties”.42 Data-Mining. The articles “Image Annotation and Database Mining to Create a Novel Screen for the Chemotype Dependent Crystallization of HCV NS3 Protease”43 and “Optimization of Protein Crystallization: The OptiCryst Project”44 highlight efforts to set up and analyze significant numbers of crystallization trials. This article also points to an issue that exists with contemporary efforts that affects independent laboratories and disparate structural genomics efforts. An ontology − or any other way of capturing domain knowledge − for the active and direct communication of

crystallization results does not exist. We lack a common language to describe our experiments and their outcomes. Experiments that fail to produce a structure are generally not reported and essentially lost to the community at large. The absence of such a resource has a marked impact the effectiveness and broad application of crystallization-based data mining strategies and ignores a valuable data set that we all know to be vast: the crystallization experiments that failed to yield harvestable crystals. Summary. Crystallization of biological macromolecules and their structural investigation by X-ray crystallography remain a significant challenge in 2011. This is despite all of the significant advances in every aspect of the science and the associated technologies. Even so, our current structural investigations and their associated methods would have been deemed science fiction/fantasy − or at least wishful thinking − if they were discussed during the 1985 Stanford meeting. As the toolbox of technologies and methodologies develops, we continue to push the envelope of what systems are considered amenable to structural investigation. We want to understand biological systems of increasing size and complexity in the detail that atomic resolution X-ray structures allow. We realize that advances in crystallization and associated technologies are intimately tied to advances in our structural and functional understanding of biology. The state-of-the-art in X-ray crystallography encompasses both the very large and the very small; this special issue discusses the structures of very large complexes of biological macromolecules (up to and including the ribosome) and structural data collected from very small crystals − just tens of unit cells, whose diffraction could be only be measured with the phenomenal power of the free electron laser.45 It may be that microcrystalline precipitates, one of the outcomes of a crystallization experiment that is so obviously unsuitable for the collection of single-crystal diffraction data, may well be all that we need to produce experimentally derived crystallographic structures in the not so distant future. Nanocrystallography has the potential to become the event horizon of the crystallization bottleneck. In closing, we praise the organization of ICCBM13 as it was simply outstanding. We thank the Chairman, Dr. Martin Caffrey, and his staff, including Siobhan O’Hare, for their exceptional planning and efforts to make ICCBM13 so enjoyable for all who attended. The meeting agenda unfolded in the beautiful venue of Trinity College in the heart of Dublin, Ireland. Participants were treated to lectures and poster presentations that were of exceptional quality. For a detailed report on the scientific presentations we refer you to the article “Overview of the 13th International Conference on the Crystallization of Biological Macromolecules”.46 The true measure of quality came in the presentations’ aftermath; we watched their catalytic effect, promoting very active scientific discussions in the dozens of pubs and restaurants that surrounded Trinity College. We wish to thank the journal Crystal Growth & Design, for agreeing to publish this special issue dedicated to ICCBM13, most especially to the founding Editor-in-Chief Dr. Robin D. Rogers and Coordinating Editor Mihaela Rogers for their professionalism, patience, and wealth of information that they so kindly bestowed upon their guest coeditors. Finally, we thank all of the participants in the very successful 13th ICCBM for continuing to make the crystallization of biological macromolecules such a relevant and exciting scientific endeavor. 5

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Corresponding Author *E-mail: [email protected].

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