The Ontario Structural Genomics Initiative

1. The Ontario Structural Genomics Initiative

2. REFERENCES Nature Structural Biology, 7, 903-909, 2000 Journal of Molecular Biology, 302, 189-203, 2000 Nature Structural Biology, SG supplement, Nov 2000 Structure, 6, 265-267, 1998 Nature Structural Biology, 6, 11-12, 1999 Current Opinion in Biotechnology, 11, 25-30, 2000 Nature Genetics, 23, 151-157, 1999

3. STRUCTURAL GENOMICS The determination of the three-dimensional structures of the proteins encoded by the genes from an entire genome. The complete DNA sequences of many organisms are known and there are 100 ongoing genomic sequencing projects. The natural extension of sequencing projects is the determination of the corresponding protein structures. The goals of current genomics projects are to understand the cellular and molecular functions of all the gene products. Ultimately to help in the design of diagnostics and therapeutics.

4. SEQUENCED GENOMESNCBI Genome Database A. aeolicus (1522) M. thermoautotrophicum (1855) A. fulgidus (2407) M. jannaschii (1715) B. subtilis (4100) M. tuberculosis (3918) B. burgdorferi (850) M. genitalium (467) C. elegans (19 099) M. pneumoniae (677) C. trachomatis (1052) P. horikoshii (1979) C. pneumoniae (894) R. prowazekii (834) E. coli (4289) S. cerevisiae (5885) H. influenzae (1709) Synechocystis sp.(3169) H. pylori (1566) T. pallidum (1031) A. thaliana (15 000 ) H. sapiens (?30 000) LEGEND: Archaea Bacteria Eucarya

5. THE PROTEOMICS CHALLENGE Any Genome

6. FUNCTIONAL PROTEOMICS Genome Wide Analysis protein-protein interactions protein expression/localization biochemical assays protein structure

7. BEYOND SEQUENCING PROJECTS

8. THE POST-GENOMIC ERA Functional proteomics currently exploits several complementary technologies DNA Microarray Technology For genome-wide transcription profiling Protein-Ligand Interactions To discover small molecule inhibitors of proteins To discover function Protein-Protein Interactions To define the network of regulatory interactions To discover function

9. PROTEINS WITH 3D HOMOLOGS

10. MAKING STRUCTURAL GENOMICSA REALITY Initially the rate determining step in SG was preparing suitable protein samples. - Need faster methods in protein production - Must overcome bottleneck of growing crystals - Initiated program directed solely at this issue

11. GOALS OF STRUCTURAL GENOMICS to develop improved methods that will result in high-throughput biology and protein structure determination robots, robots, robots cloning expression purification crystallization to determine new protein folds to determine the functions of unknown proteins

12. STRUCTURAL GENOMICS A move away from hypothesis driven research�a system where structures are solved first followed by asking questions about the protein later. A large number of targets are required from which high-throughput methods must be implemented for such a project to be successful Cloning, expression and purification are important!! What targets? What is the priority of targets?

13. STRUCTURAL GENOMICS PROJECTS A. Edwards U of T 20 M. thermoautotrophicum S.H. Kim Berkeley 12 Methanococcus jannaschii S. Yokoyama Tokyo U 10 Thermus thermophilus J. Moult CARB 10 Haemophilus influenzae D. Eisenberg UCLA 8 Pyrobaculum aerophilum A. Sali BNL 3 S. cerevisiae

14. SG CONSORTIUMS The NIH/NIGMS have funded 7 SG centers with each center obtaining about $4 million US per year in funding. New York SG Consortium (www.nysgrc.org) Midwest Center for SG (UHN/UofT) The Berkeley SG Center Northeast SG Consortium (UHN/UofT) (www.nesg.org) Tuberculosis SG Consortium (www.doe-mbi.ucla.edu/TB) The Southeast Collaboratory for SG The Joint Center for SG (www.jcsg.org)

15. SG COMPANIES Integrative Proteomics Inc. Toronto (www.integrativeproteomics.com) Structural Genomix Inc. San Diego (www.stromix.com) Syrrx Inc. La Jolla (www.syrrx.com) Astex Inc. Cambridge (www.astex-technology.com) Structure-Function Genomics Piscataway

16. CRYSTALLOGRAPHIC DEVELOPMENTS Multiwavelength Anomalous Dispersion Synchrotron Radiation Cryocrystallography CCD Detectors and Image Plates Software

18. Overview of Structural Proteomics Genome Analysis and Target Selection Cloning, Expression and Purification Crystallography NMR Structure Fold and Functional Analysis

19. STRUCTURE SHOW AND TELL The structure will reveal the fold of the protein. TIM barrel, Rossmann fold

20. STRUCTURE SHOW AND TELL The structure will reveal the active site. protease (Ser-His-Asp)

21. STRUCTURE SHOW AND TELL The structure may reveal evolutionary links between proteins lacking sequence similarity.

22. STRUCTURE SHOW AND TELL The structure may reveal the function of the protein.

23. TARGET SELECTION Groups are focusing on complete organisms; thermophilic, mesophilic or halophilic eukaryotic or prokaryotic classes of proteins from different organisms There isn�t a coordinated international group that assigns targets (yet!). Some groups may solve the same structures (redundant). two SG pilot projects solved factor 5A first!!! Membrane proteins and proteins whose structures are already solved are eliminated.

25. DRUG DISCOVERYANTIBIOTICS Targets in this area of structural genomics are bacterial proteins that are essential for growth and survival. cell wall biosynthesis aromatic amino acid biosynthesis The development of a broad spectrum antibiotic would encompass the structures of a single protein from different bacterial organisms.

26. DRUG DISCOVERYHUMAN DISEASE Targets in this area of structural genomics are G-protein coupled receptors, ion channels and kinases etc. -GPCRs and ion channels are membrane proteins and are more difficult to purify and crystallize The development of techniques to allow over-expression, purification and crystallization of these targets is required and in progress.

27. AIMS OF PILOT PROJECT determine feasibility of a Structural Genomics Project develop technologies necessary for large-scale initiatives develop high-throughput (HTP) cloning develop high-throughput expression develop high-throughput purification

28. Methanobacterium thermoautotrophicum isolated in 1971 thermophile (optimal growth T is 65�C) methanogen (grows on methane as a carbon source) sequenced (Smith, DL et al., 1997, J. Bact., 179, 7135) 1 751 377 bp and 1855 orfs 13% are similar to eucaryal sequences proteins in DNA metabolism, transcription and translation archaeal proteins are smaller and more stable than bacterial and eukaryal homologs

29. PROTEIN FUNCTION

30. CLONING OF MT GENES PCR amplification of gene of interest purification of PCR product ligation into pET15b expression vector T7 promoter induced with IPTG cleavable hexahistidine fusion tag transformation into DH5? E. coli cells plasmid prep transformation into BL21(DE3) E. coli cells expression and purification

31. LIMITED PROTEOLYSIS single domain proteins and proteins less than 40 kDa can be expressed in E. coli multi-domain proteins and proteins greater than 40 kDa are quite difficult to express in E. coli these proteins may be expressed in yeast or baculo OR these proteins must be broken down into domains

32. PROTEINS DESTINED FOR NMR

33. COMPARISON OF N15 NMR SPECTRA

35. PROTEINS FOR CRYSTALLOGRAPHY

36. STRUCTURE DETERMINATION STEPS Clone Gene Purify Protein Crystallize Protein Collect X-Ray Diffraction Data Identify Selenium Sites Calculate Phases using MAD Calculate Electron Density Map Build Model of Protein in Electron Density Refine and Rebuild Protein Model

37. PROTEIN CRYSTALLIZATION A crystal is an ordered three-dimensional array of molecules in the same orientation held together by non-covalent interactions. Crystals are grown by slow-controlled precipitation from crystallization conditions that do not denature the protein. These conditions can contain precipitants such as salts (NaCl, AmSO4), organic solvents (EtOH, MPD) or polymers (PEG), buffers, additives and ions.

38. PROTEIN CRYSTALLIZATION cont�d Each protein has its own empirically determined crystallization condition. pH ionic strength protein concentration temperature ions precipitant We cannot sample complete crystallization matrices. We start off with approximately 200 different crystallization solutions and hope for the best.

39. PROTEIN CRYSTALLIZATION cont�d

40. PROTEIN CRYSTAL

41. X-Ray DIFFRACTION

42. X-RAY DIFFRACTION IMAGE

43. PROGRESS TOWARDS HTP CLONING Initial Rate 24 clones per person per week Current rate 96 clones per person per week

44. PROGRESS TOWARDS HTP PROTEIN EXPRESSION Established conditions to maximize number of soluble clones bacterial strain induction conditions �magic� plasmid

45. PROGRESS TOWARDS HTP PURIFICATION Initial Rate 1 protein/person/week Current Rate 8 proteins/person/week Target Rate 16 proteins/person/week

46. ACHIEVEMENTS We have optimized HTP cloning. We have optimized HTP expression and purification. We are in the process of automating cloning and purification.

47. SUMMARY OF MT PROTEINS

51. CONCLUSIONS FROM FEASIBILITY STUDY

52. STRATEGIES FOR TACKLING RECALCITRANT PROTEINS

53. TAKE HOME LESSON think about biology on a genomic scale

54. PROTEINS: Structure, Function and Genetics has inaugurated a new short format of �Structure Notes� designed to provide brief accounts of structures that contain �too little new information to warrant a full length article� what can you expect from robots!!! - Bill L Duax

57. 2000 OCI SUMMER STUDENTS Ashleigh Tuite Fred Cheung Laura Faye Toni Davidson

58. COLLABORATORS Lawrence McIntosh (UBC) Cameron Mackereth Mike Kennedy (PNNL) John Cort Mark Gerstein (Yale) Yuval Kluger Kalle Gehring (McGill) G. Kozlov