STRUCTURAL
PROTEMICS and its Impact on the LIFE SCIENCES
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STRUCTURAL
PROTEMICS and its Impact on the
LIFE SCIENCES
Joel L. Sussman, Israel Silman Weizmann Institute of Science, Israel
editors
World Scientific NEW JERSEY
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Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
Library of Congress Cataloging-in-Publication Data Structural proteomics and its impact on the life sciences / editors, Joel L. Sussman, Israel Silman. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-981-277-204-6 (hardcover : alk. paper) ISBN-10: 981-277-204-9 (hardcover : alk. paper) 1. Proteomics. 2. Proteins--Structure. I. Sussman, Joel. II. Silman, Israel. [DNLM: 1. Proteomics. QU 58.5 S932 2008] QP551.S817 2008 572'.6--dc22 2008009897
British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.
Copyright © 2008 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.
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Preface Joel L. Sussman* and Israel Silman†
The concept of structural genomics (SG) arose in the mid-to-late 1990s in both the USA and Japan, triggered by the success achieved in applying high-throughput (HTP) sequencing methods to whole genomes. It was envisaged that application of a similar HTP approach to obtaining the three-dimensional structures of a substantial fraction of the entire set of proteins of a given organism, the “proteome,” would be an efficient way of filling in the gaps in observed “foldspace.” The decision to adopt such an approach resulted in the investment of large sums of money, i.e. hundreds of millions of dollars, in large-scale structural genomics projects in both countries. Thus, in Japan, the Protein Research Group was established at the RIKEN Genomic Sciences Center in 1998, and in the USA, the Protein Structure Initiative (PSI), funded by the NIH/NIGMS, commenced at nine major centers in 2000. These projects were characterized by concentration of resources in a small number of large centers, by development of novel, automated technologies which would allow a HTP pipeline approach to structure determination, and a focus on novel folds as a major target criteria. Europe was slower in implementing HTP approaches to structural biology. Various national efforts such as the Protein Structure Factory Departments of *Structural Biology and †Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel v
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(PSF) in Berlin, the Oxford Protein Production Facility (OPPF), and the Genopoles in France, led the way. But it was only towards the end of 2002 that the first Europe-wide project began, an EC-funded Integrated Project entitled Structural Proteomics in Europe (SPINE). While benefiting from the technological achievements of the US and Japanese programs, and itself also concerned with developing cutting edge technologies as a means of achieving its objectives, SPINE from the outset focused these technologies on biomedically relevant targets. Indeed, as implied by its name, it aimed to establish a pan-European biomedically oriented structural proteomics program, placing significant emphasis on functional aspects of the target proteins studied. SPINE was followed by a series of EC-funded programs of various scopes and funding, some placing an emphasis on technological development, and others on attacking various classes of targets. A similar emphasis on the use of the emerging HTP technologies to solve structures of biomedical relevance was adopted by the Structural Genomics Consortium (SGC), established in 2003 with the support of Canadian and British sponsors from both the public and private sectors, with laboratories in Oxford, Toronto and, subsequently, in Stockholm. From the outset, the scale of funding of the PSI met with considerable criticism, especially in the USA, which has been going through a period during which funding for research by individual PIs has been hard to come by. Many critics have argued that the $270M spent on funding the Pilot Phase of the PSI, over a period of five years, could have been more effectively utilized to fund hypothesis-driven research directed towards targets of fundamental or applied interest. Nevertheless, the achievements of the various SG/SP consortia, viewed in aggregate, and achieved in less than a decade, are impressive. In aggregate, the US PSI centers (September 2000 to June 2005) have determined over 1100 structures (http://www.nigms. nih.gov/Initiatives/PSI/Background/PilotFacts.htm). During PSI-2, which is still ongoing, ~1200 structures have already been solved, of which the vast majority share less than 30% sequence identity with any structure already deposited in the PDB, and many represent novel folds. As a result of the efforts of all the consortia (US, Japanese and
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European), 5968 protein structures had been deposited in the Protein Data Bank as of 11-Dec-2007 (http://www.rcsb.org/pdb/ static.do?p=general_information/pdb_statistics). Although some of these structures may be redundant, or even appear uninteresting at first sight, many are of the highest technical quality, of fundamental and/or medical importance and, taken overall, provide a valuable database. Moreover, it has been reported that, in 2005, structures arising out of structural genomics and structural proteomics efforts accounted for 44% of the total number of novel structures reported. Although many of the novel structures solved by the SG/SP centers were, on the surface, low-hanging fruit, which filled gaps either in a given proteome or in fold space without yielding novel functional information, other targets have been, as already mentioned, of great fundamental and/or medical importance. Furthermore, filling in fold space provides a robust body of templates for homology modeling, which can rapidly take advantage of these templates as computational techniques become more sophisticated, and computing power increases. Thus, in our view, whatever policy decisions are taken with respect to funding of large-scale SG/SP projects, what has been achieved so far will have a lasting impact on biological and biomedical research. Indeed, the EC, through a Specific Support Action (SSA), established the Forum for European Structural Proteomics (FESP, see http://www.ec-fesp.org) to assess the current status and make recommendations for future European infrastructure requirements in the SG/SP area. We feel, therefore, that it is timely to publish a book in which these achievements are presented with a look to their potential impact on biological and biomedical research in general. In this volume, we have tried to bring together experts capable of addressing all aspects of the SG/SP effort, from target selection, through the various techniques for expressing and purifying proteins and protein complexes and the methodologies for solving their structures, to their impact on drug design and on coping with emerging diseases. In view of the ongoing debate on SG/SP funding, we have also included a special chapter dealing with policy, which includes sections written by several scientists and officials who have been closely associated with the decision-making processes.
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Contents
Preface
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List of Contributors
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Chapter 1 The Importance of Target Selection Strategies in Structural Biology Enrique E. Abola and Raymond C. Stevens Chapter 2 The Impact of Structural Proteomics on Macromolecular Structure Databases James D. Watson and Janet M. Thornton Chapter 3 The Impact of 3D Structures on a Protein Knowledgebase: From Proteins to Systems Ursula Hinz and Amos Bairoch Chapter 4 Bioinformatics of Protein Function Arthur M. Lesk, Vineet Sangar, Helen Parkinson and James C. Whisstock Chapter 5 Comparative Modeling in Structural Genomics John Moult ix
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Chapter 6 The Contribution of Structural Proteomics to Understanding the Function of Hypothetical Proteins Michael D. Suits, Allan Matte, Zongchao Jia and Miroslaw Cygler Chapter 7 Intrinsically Disordered Proteins Peter Tompa Chapter 8 Metalloproteins: Structure, Conservation and Prediction of Metal Binding Sites Marvin Edelman, Mariana Babor, Ronen Levy and Vladimir Sobolev Chapter 9 The Impact of Protein Expression Methodologies on Structural Proteomics A. Chesneau, H. Yumerefendi and D. J. Hart Chapter 10 Protein Complexes Assembly by Multi-Expression in Bacterial and Eukaryotic Hosts Christophe Romier Chapter 11 The Impact of Structural Proteomics on the Prediction of Protein–Protein Interactions Christina Kiel and Luis Serrano Chapter 12 Cryo-Electron Microscopy in the Era of Structural Proteomics Alasdair C. Steven and David M. Belnap
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Chapter 13 On NMR-based Structural Proteomics Thomas Szyperski
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Chapter 14 Structural Proteomics in Relation to Signaling Pathways Florence Bedez, Arnaud Poterszman and Dino Moras
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Chapter 15 The Impact of Structural Proteomics on Drug Design Yuan-Ping Pang
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Chapter 16 Structural Proteomics of Emerging Viruses: The Examples of SARS-CoV and Other Coronaviruses Rolf Hilgenfeld, Jinzhi Tan, Shuai Chen, Xu Shen and Hualiang Jiang Chapter 17 High-throughput Technologies for Structural Biology: The Protein Structure Initiative Perspective Andrzej Joachimiak Chapter 18 European Structural Proteomics — A Perspective Susan Daenke, E. Yvonne Jones and David I. Stuart Chapter 19 Structural Genomics and Structural Proteomics: A Global Perspective Lucia Banci, Wolfgang Baumeister, Udo Heinemann, Gunter Schneider, Israel Silman and Joel L. Sussman
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Chapter 20 Policies in Structural Genomics/Structural Proteomics
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A.
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The Protein Structure Initiative: Policies and Update John Norvell and Jeremy Berg B. Structural Genomics in European Framework Programs Josefina Enfedaque, Saša Jenko Kokalj and Jacques Remacle C. Policy Aspects in Structural Genomics/Proteomics Barbara Skene D. Policies and Updates of the RIKEN Structural Genomics/Proteomics Initiative Shigeyuki Yokoyama E. The International Structural Genomics Organization: Policies for Structural Genomics Thomas C Terwilliger, Shigeyuki Yokoyama, Udo Heinemann, Ian Wilson, Dino Moras, David Stuart, Seiki Kuramitsu, Edward N. Baker, Stephen Burley and Joel Sussman Index
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554 559
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Enrique E. Abola Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla California 92037 USA Mariana Babor Department of Plant Sciences Weizmann Institute of Science Rehovot 76100 Israel Amos Bairoch Swiss Institute of Bioinformatics Centre Medical Universitaire 1, rue Michel-Servet 1211 Geneve 4 Switzerland Edward N. Baker School of Biological Sciences University of Auckland Private Bag 92019 Auckland New Zealand xiii
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Lucia Banci Centro Risonanze Magnetiche University of Florence Via Luigi Sacconi 6 Sesto Fiorentino Florence 50019 Italy Wolfgang Baumeister Max Planck Institute of Biochemistry Am Klopferspitz 18a Martinsried D-82152 Germany Florence Bedez Institut de Génétique et de Biologie Moléculaire et Cellulaire UMR 7104, 1 rue Laurent Fries BP 10142, 67404 Illkirch Cedex France David M. Belnap Department of Chemistry and Biochemistry Brigham Young University Provo, UT 84602 USA Jeremy Berg National Institute of General Medical Sciences National Institutes Health Bethesda, MD 20B92-6200 USA Stephen Burley SGX Pharmaceuticals Inc 10505 Roselle Street San Diego, CA 92121 USA
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Shuai Chen Institute of Biochemistry Center for Structural and Cell Biology in Medicine University of Lübeck Ratzeburger Allee 160 23538 Lübeck Germany A. Chesneau EMBL Grenoble Outstation 6 rue Jules Horowitz BP181, 38042 Grenoble Cedex 9 France Miroslaw Cygler Department of Biochemistry McGill University 845 Sherbrooke Street West Montreal, Quebec H3A 2T5 Canada Susan Daenke Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive Oxford, OX3 7BN UK Marvin Edelman Department of Plant Sciences Weizmann Institute of Science Rehovot 76100 Israel
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Josefina Enfedaque European Commission Research Directorate General BE-1049 Brussels Belgium D. J. Hart EMBL Grenoble Outstation 6 rue Jules Horowitz BP181, 38042 Grenoble Cedex 9 France Udo Heinemann Max-Delbruck-Center for Molecular Medicine Robert-Roessle-Str 10 Berlin D-13125 Germany Rolf Hilgenfeld Institute of Biochemistry Center for Structural and Cell Biology in Medicine University of Lübeck Ratzeburger Allee 160 23538 Lübeck Germany Ursula Hinz Swiss Institute of Bioinformatics Centre Medical Universitaire 1, rue Michel-Servet 1211 Geneve 4 Switzerland
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Zongchao Jia Department of Biochemistry Queen’s University Kingston, ON K7L 3N6 Canada Hualiang Jiang Drug Discovery and Design Center State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Zuchongzhi Rd. 555, Shanghai 201203 China Andrzej Joachimiak Biosciences Division Midwest Center for Structural Genomics and Structural Biology Center Argonne National Laboratory 9700 S Cass Ave. Argonne IL 60439 E. Yvonne Jones Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive Oxford, OX3 7BN UK
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Christina Kiel EMBL-CRG Systems Biology partnership Unit Centre de Regulacio Genomica (CRG) Dr Aiguader 88 08003 Barcelona Spain Saša Jenko Kokalj Department of Biochemistry and Molecular Biology Jozef Stefan Institute Jamova 39 1000 Lijubljana Solvenija Seiki Kuramitsu Department of Biological Sciences Graduate School of Science Osaka University 1–1 Machikaneyama-cho Toyonaka, Osaka 560-0043 Japan Arthur M. Lesk Department of Biochemistry and Molecular Biology, and the Huck Institute for Genomics, Proteomics and Bioinformatics The Pennsylvania State University University Park, PA 16802 USA Ronen Levy Department of Plant Sciences Weizmann Institute of Science Rehovot 76100 Israel
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Allan Matte Biotechnology Research Institute 6100 Royalmount Ave. Montreal QC H4P 2R2 Canada Dino Moras Institut de Génétique et de Biologie Moléculaire et Cellulaire UMR 7104, 1 rue Laurent Fries BP 10142, 67404 Illkirch Cedex France John Moult Center for Advanced Research in Biotechnology University of Maryland Biotechnology Institute 9600 Gudelsky Drive Rockville, MD 20850 USA John Norvell National Institute of General Medical Sciences National Institutes Health Bethesda, MD USA Yuan-Ping Pang Computer-Aided Molecular Design Laboratory Mayo Clinic Rochester, Minnesota USA Helen Parkinson European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton Cambridgeshire CB10 1SD United Kingdom
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Arnaud Poterszman Institut de Génétique et de Biologie Moléculaire et Cellulaire UMR 7104, 1 rue Laurent Fries BP 10142, 67404 Illkirch Cedex France Jacques Remacle European Commission BE-1049 Brussels Belgium Christophe Romier IGBMC 1 rue Laurent Fries B.P. 10142, 67404 Illkirch Cedex Vineet Sangar Department of Biochemistry and Molecular Biology, and the Huck Institute for Genomics, Proteomics and Bioinformatics The Pennsylvania State University University Park, PA 16802 USA Gunter Schneider Karolinska Institutet Scheelevägen 2 SE-171 77 Stockholm Sweden Luis Serrano EMBL-CRG Systems Biology partnership Unit Centre de Regulacio Genomica (CRG) Dr Aiguader 88 08003 Barcelona Spain
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Xu Shen Drug Discovery and Design Center State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Zuchongzhi Rd. 555, Shanghai 201203 China Israel Silman Department of Neurobiology Weizmann Institute of Sceince Rehovot 76100 Isreal Barbara Skene Formerly Head of Department, Molecules, Genes and Cells The Welcome Trust London NW1 2BE UK Vladimir Sobolev Department of Plant Sciences Weizmann Institute of Science Rehovot 76100 Israel Alasdair C. Steven Laboratory of Structural Biology National Institute of Arthritis, Musculoskeletal, and Skin Diseases National Institutes of Health Bethesda, MD 20892 USA
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Raymond C. Stevens Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla California 92037 USA David I. Stuart Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive Oxford, OX3 7BN UK Michael D. Suits Department of Biochemistry Queen’s University Kingston, ON K7L 3N6 Canada Joel L. Sussman Department of Structural Biology Weizmann Institute of Science Rehovot 76100 Israel Thomas Szyperski 816 Natural Sciences Complex Chemistry Department State University of New York at Buffalo Buffalo, NY 14260 USA
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Jinzhi Tan Institute of Biochemistry Center for Structural and Cell Biology in Medicine University of Lübeck Ratzeburger Allee 160 23538 Lübeck Germany Thomas C Terwilliger Los Alamos National Laboratory Los Alamos, NM 87545 USA Janet M Thornton EMBL — European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton Cambridge, CB10 1SD UK Peter Tompa Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O. Box 7 Hungary James D Watson EMBL — European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton Cambridge, CB10 1SD UK
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James C. Whisstock Department of Biochemistry and Molecular Biology Victorian Bioinformatics Consortium Monash University Clayton Campus Melbourne, Victoria 3168 Australia Ian Wilson The Scripps Research Institute 10550 N. Torrey Pines Rd La Jolla, CA 92037 USA Shigeyuki Yokoyama Protein Research Group Genomic Sciences Center RIKEN Yokohama Institute 1-7-22 Suehiro-cho Tsurumi, Yokohama 230-0045 Japan H. Yumerefendi EMBL Grenoble Outstation 6 rue Jules Horowitz BP181, 38042 Grenoble Cedex 9 France
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Chapter 1
The Importance of Target Selection Strategies in Structural Biology Enrique E. Abola and Raymond C. Stevens*
Introduction The industrialization of biology — the large-scale acquisition of biological data — has been pioneered by the sequencing of entire genomes and is being applied to the characterization of other important biological molecules such as the proteome, the interactome (arguably a subset of the proteome), the glycome, and the metabolome. As of October 2007, the complete genomic sequences of 676 organisms have been published and more projects are underway. The DNA sequence data alone is insufficient to generate the level of understanding of biological systems that most biologists seek. Understanding how biological systems operate from the level of single proteins and enzymes, to the level of protein-protein interactions, and finally at the level of intact cellular physiological pathways, a goal of systems biology, will require detailed, quantitative characterization of cellular proteins and their interactions, which is facilitated by access to protein structural information. Thus, the number and types of questions that can now be addressed by structural biologists has increased dramatically. The scope of protein structure space is still too immense for a *Corresponding author:
[email protected]. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. 1
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completely unfocused approach to data acquisition. Therefore, target selection is still a critical step in establishing an industrial-scale protein structure project. About 50 years after the publication of the first 3-dimensional structure of a protein, that of sperm whale myoglobin completed in John Kendrew’s laboratory1 (a structural proteomics (SP) project in itself as myoglobin from multiple species were pursued), structural biologists have started to explore the possibility of conducting highthroughput (HT) structural studies to permit the structural characterization of proteomes. HT approaches, such as parallel studies of multiple protein targets, are expected to revolutionize the way structures are determined by moving away from one-by-one structural studies. These new approaches are expected to produce important scientific results at reduced costs by using economy of scales and by generating standardized and more generalizable protocols and evaluation metrics for protein expression, purification and structural characterization. Over the past 10 years, the results of successful pilot studies have been reported by the various SP programs. This leads to the important question of what target selection strategies should then be used in the future by both SP and non-SP laboratories in the light of what has been learned from these pilot studies. This chapter summarizes the strategies in target selection and prioritization used by various SP groups and provides a brief summary of their recent results. We explore the potential of small and medium sized laboratories as well as larger collaborative efforts to make use of the new technologies, protocols and approaches developed by these initial SP initiatives, with a special emphasis on studying biological systems through class-directed target selection approaches.
Global Structural Efforts and their Target Selection Strategies By 2000, several groups, both academic and for-profit companies, were being setup to establish HT structure determination production lines. Initial efforts were focused on developing new technologies and protocols for each step in the process, from initial cloning to final
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deposition of coordinate data sets to public databases. The immediate aim was to convert the one-by-one structure determination process, using both single-crystal X-ray diffraction and solution NMR techniques, to work in a HT mode. Although the initial mandate was technology development, there was also a requirement to solve a relatively large number of protein structures to serve both as proof of concept and as a justification of the approach adopted by each center. Major government-sponsored consortium efforts were formed: the Protein Structure Initiative (PSI-1) in the USA, the Structural Proteomics (SPINE) integrated project in Europe, and Project 3000 in Japan, and smaller efforts in other countries. Another major effort was a joint venture between government and industry. The Structural Genomics Consortium, an international project funded by Canada, Sweden, the Wellcome Trust in the UK and industry, with laboratories in Oxford, Stockholm and Toronto. For-profit companies, such as SYRRX, SGX, and ASTEX, were setup with the goal of improving the drug discovery and development process by reducing the risks and cost of getting at the structures of drug targets and their complexes. Each major consortium had an overarching target selection and prioritization strategy. By and large, all the SP groups mentioned above pursued targets based on general principles which followed the classdirected target strategies outlined in papers published as part of the dialogue on definition and implementation of structural proteomics.2,3 This is exemplified by the paper by Terwilliger et al. (see Table 1; Ref. 3), which put forward a list of protein classes and a scientific rationale for selecting them, and also suggested a protocol for implementation of the target selection strategy. Four classes were suggested: 1) the construction of a database of structural motifs; 2) the study of proteins from microorganisms, including pathogens and thermophiles; 3) a largescale target class including human targets of biomedical interest, protein assemblies, proteins from plants or animals; and 4) a small-scale target class that is the study of important protein families (e.g. protein kinases, transcription factors). Class 1 attempts to generate structural annotations, while the rest are motivated by the goal of generating functional annotation. These two goals are somewhat related (i.e. fold may
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Target Classes for a Protein Structure Initiative†
Class of Proteins (1) Database of protein structure motifs (2) Proteins from a microorganism Proteins from a pathogen Proteins from a thermophile (3) Large-scale targets Human proteins Plant or animal proteins Protein assemblies (4) Small-scale targets Groups of structurally-similiar proteins Proteins from a metabolic pathway †
Importance Prediction of protein structure Potential drug targets Robust enzyme Medical applications Biotechnology Protein interactions Predicting protein evolution Biocatalysis
From Terwilleger et al.3
provide clues to function), and one can argue that the target lists generated by some of the PSI-1 centers are more focused on an attempt to functionally annotate an organism’s proteome (e.g. the Joint Center for Structural Gemomics (JCSG)’s studies of Thermotoga maritima). Below, we summarize the activities of the various SP centers, their initial target selection strategies, and the outcomes. Although the Japanese effort, Protein 3000, has produced more than 2500 structures, accounting for half of the 5000 structures solved in all SP centers worldwide, we have not included an extensive discussion of their efforts. At this time, their target selection strategies remain unpublished, although early descriptions4 indicate that their main effort was geared towards the generation of structural annotations, viz. looking for new folds. Final statistics indicate that only 34% of their structures have novel sequences and the list of their solved structures appears to indicate that secondary target selection criteria were used.
Selecting and Prioritizing Targets Initially, most target selection processes centered on choosing proteins based on primary and secondary objectives, which were later
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supplemented by additional target prioritization schemes. For example, projects that aimed at exploring fold space focused on methodology and approaches to find clusters of related sequences for which 3-dimensional structures of any of its members were not available in the PDB. There was also an interest in developing and using tools for the identification of domains to facilitate the production of expression constructs. This was particularly important for proteins anticipated to be difficult to crystallize and thus more amenable to NMR structure determination. Targets were further prioritized based on the predicted novelty of sequences or on the prediction that structures of new folds would be obtained. Halfway through the first phase of PSI, it became possible to select and prioritize targets utilizing databases created from the results of the large number of SP experiments carried out in the various PSI laboratories that used different target selection strategies on a number of classes of proteins.5–7 Data on diverse data sets were, therefore, available to attempt to understand successes (e.g. produces soluble constructs or crystallizes) and failures. Two papers from the JCSG exemplify what can be done. Canaves et al.,6 analyzing the JCSG data for T. maritima proteins, and Slabinski, et al.7 analyzing all the available SP data, developed a number of sequence-based metrics, which provides a measure of the difficulty/ease of crystallizing protein (http://ffas.burnham.org/XtalPred-cgi/xtal.pl). Both studies use 12 parameters to arrive at an index. Once crystals are obtained, statistics indicate a 32–38% chance of completing the structure (see Table 2). Interestingly, the Barton laboratory8 used PDB entries to develop a normalized score, OB_SCORE, based on just two parameters, pI and the Gravy index. This z-score estimates the chances of producing diffraction-quality crystals.8 These metrics, along with bioinformatics resources established by the various SP centers, were then used to construct prioritized target lists; in the case of the PSI, prioritization was assigned based primarily on the novelty of the sequence. A more integrated approach to target selection is now provided by a webbased system, sgTarget (http://www.ysbl.york.ac.uk/sgTarget/), that produces homology information that measures the uniqueness of the sequence, as well as the calculated physiochemical properties that
(%) Relative to “Purified” Targets
(%) Relative to “Crystallized” Targets
102306 64773 27886 25659 9357 4863 4055 1727 2950 3746 1642 5195 25278 3 1
100 63.3 27.3 25.1 9.1 4.8 4 1.7 2.9 3.7 1.6 5.1 — — —
— 100 43.1 39.6 14.4 7.5 6.3 2.7 4.6 5.8 2.5 8 — — —
— — — 100 36.5 19 15.8 6.7 11.5 14.6 6.4 20.2 — — —
— — — — 100 52 43.3 — — 40 — 38 — — —
Table downloaded from http://sg.pdb.org/target_centers.html on October 2007.
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(%) Relative to “Cloned” Targets
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Cloned Expressed Soluble Purified Crystallized Diffraction-quality crystals Diffraction NMR assigned HSQC Crystal structure NMR structure In PDB Work stopped Test target Other
Total Number of Targets
Structural Proteomics
Status
Success Rates for All Structural Proteomics Centers (October 2007)†
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Table 2
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may affect expression, solubility, and the likelihood that crystals will be obtained.9 In addition to the target selection activities, statistics derived from data-mining activities on production databases (e.g. TargetDB, PepCDB), as well as process and technology evaluation that measured the performance of the various pipelines, are now able to provide a robust estimate and a better understanding of the risks involved in structural studies which had previously been estimated using anecdotal and ad hoc approaches.10
The Protein Structure Initiative (PSI) The PSI project was established by the National Institutes of General Medical Sciences (NIGMS) at the U.S. National Institutes of Health. PSI phase I (PSI-1) studies, initiated in year 2000 and 2001, were conducted in nine centers (Table 3), and were completed in 2005, with over 1100 structures having been deposited in the PDB. Phase II (PSI-2) studies were immediately started, and involved four largescale production centers, six specialized centers for development, two homology modeling centers, and a research grants program focusing on improving the accuracy of the comparative protein structure modeling. Production centers in PSI-2 were required to produce 4000 new structures within five years, while the specialized centers were given the mission of developing new tools and approaches to handle challenging targets, including eukaryotic proteins, integral membrane proteins, and large macromolecular complexes. Within two years of operation, the four PSI-2 production centers had deposited about 1200 structures in the PDB, thus exceeding the five-year combined production output of the PSI-1 centers. The focus of the PSI-1 pilot centers was primarily the development of new tools, technologies, and methodology to increase the success rates and lower the costs of structure determination. Each center was responsible for automating protein sample production and the structure determination pipelines, and for meeting production goals. The final production numbers for the PSI-1 centers are presented in Table 4. As the initial goal of the consortium was to set up the pipelines and test their scalability, about 40% of the total number of
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Structural Proteomics Table 3
List of Major Structural Genomics Organizatons
Center/Consortium 1.
2.
3.
4.
5.
6.
7.
8.
9.
Berkeley Structural Genomics Center (BSGC), USA http://www.strgen.org Center for Eukaryotic Structural Genomics (CESG), USA http://www. uwstructuralgenomics.org Joint Center for Structural Genomics (JCSG), USA http://www.jcsg.org Midwest Center for Structural Genomics (MCSG), USA http://www.mcsg.anl.gov Mycobacterium Tuberculosis Structural Genomics Consortium (TBSGC), USA http://www.doe-mbi.ucla.edu/TB/ New York Structural Genomics Consortium (NYSGC), USA http://www.nysgrc.org Northeast Structural Genomics Consortium (NESG), USA http://www.nesg.org Southeast Collaboratory for Structural Genomics (SECSG), USA http://www.scsg.org Structural Genomics of Pathogenic Protozoa (SGPP), USA http://www.sgpp.org Medical Structural Genomics of Pathogenic Protozoa (MSGPP)
Target Selection Criteria and Target Organism(s) Novel sequences Minimal organisms — M. genitalium, M. pneumoniae Novel sequences Arabidopsis thaliana
Novel sequences Thermatoga Maritima, mouse Novel sequences Proteins from all three kingdoms of life Novel sequences, Mycobacterium tuberculosis
Novel sequences Disease-related proteins from eukaryotes and bacteria Novel sequences Eukaryotic domain families from D. melanogaster, S. cerevisiae, C. elegans, mouse, human Novel sequences P. furiosus, C. elegans, human
Novel sequences, Pathogenic protozoans — Leishmania major, Trypanosoma brucei, Trypnasoma cruzi, Plasmodium falciparum, Entemoeba hystolitica, Gardia, Lamblia, Toxomplasma gondii, Cryptosporidium parvum (Continued )
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The Importance of Target Selection Strategies in Structural Biology Table 3
Center/Consortium 10
Structural Proteomics in Europe (SPINE), UK http://www.spineeurope.org
11.
Structural Genomics Centers, Canada, Sweden, UK http://www.thesgc.com Project 3000, Japan http://www.rsgi.riken.jp
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(Continued ) Target Selection Criteria and Target Organism(s) Bacterial and viral pathogens — B. antrhacis, M. tuberculosis, SARS-CoV, Herpes virus Cancer-related proteins Immune defense, neuronal development and neurodegenerative diseases Human proteins related to diseases and human pathogens Novel sequences and biologically important or human health related
structures were determined in the last year of operation. Overall, these studies now show that, based on the results of PSI-1 activities, there is a 5–10% probability of success for a given target in the class of targets included in the PSI-1 list.
Summary of Target Selection and Results from PSI Centers The overall scientific goal of the PSI effort was to determine enough structures to completely populate a database which could then be used to construct homology-based models covering most of protein space. Thus, the PSI’s primary targets were Class 1 proteins of Table 1. However, target selection efforts were not centralized in PSI-1. All that the centers were required to do was to ensure that a significant number of their targets were unique, i.e. have
