1. Structural Genomics South East Asian Training Course on Bioinformatics Applied to Tropical Diseases R. Natesh, ICGEB, INDIA 1-October-2004 Structural Genomics

2. Organization of the talk Structural genomics and Bioinformatics Tools used in structural genomics

3. Structural Genomics - Buzz word With the draft release of Human Genome. Human Genome contains of 3 billion base pairs. Human Genome is estimated to contain 30 to 40 thousand genes that encode proteins. Structures of only ~10 % of proteins known to human are known. (27321 structures, ~4K NMR structures), (24536 proteins, 7278 unique structures with < 70 % identity. 5037 Homo Sapiens, ~2000 structures with < 70 % identity)

4. Structure to function with structural basis for therapeutics is the main goal of the Structural Genomics. Three Dimensional structures can yield knowledge to discover newer and efficient drug design to cure diseases. High throughput mode. Structure  understand function

6. Classification based on Sequence & Structure similarities would enable to identify related proteins eg. SCOP, CATH. Structural Genomics/biology uses Bioinformatics as tool to setup such databases. Example of one such consortium is “Mycobacterium tuberculosis Structural Genomics Consortium” http://www.doe-mbi.ucla.edu/TB/

7. Computational molecular biology created the rationale for structural genomics by deriving the general principles of protein structure organization and by providing a tentative upper boundary for the total number of existing protein folds. (As the number of structure increases the estimated fold increases but with a damping factor since the number of folds is assumed to be limited) Efficient ways of their prediction and classification. Comparative protein sequence and structure analysis is a major cost-saving factor in high-throughput structure determination leading to optimal, most economic selection of targets for X-ray crystallography or NMR studies. Structural Genomics and Bioinformatics

8. Bioinformatics also plays a crucial role in assessment and classification of the new structural data obtained. Bioinformatics research, in its turn, directly benefits from the flood of data generated by structural genomics projects, resulting in improved algorithms, software, and databanks.

9. PDB has search tools

10. Search Lite LinkLink

11. Example

12. PDB Search fields http://www.rcsb.org/pdb/cgi/queryForm.cgi

13. Status of unreleased entries

14. One of the major tool used is Crystallography apart from NMR and electron microscopy. Structural Genomics is almost always address with X-ray crystallography. Robotics in Crystallography – For crystallisation, crystal mounting etc. Automation in Data collection..etc. Bioinformatics Tools – Blast, Fasta etc., to identify the proteins of interest for particular disease eg. tropical diseases like Malaria and Leishmaniasis. SCOP, CATH, DALI Tools used in structural genomics

15. Gene clone Over expression Purification Crystallisation

16. Agincourt Take the next step. Agincourt™, Syrrx’s high-throughput Nanovolume Crystallization® robot. This robot has set up more crystallization experiments than any single organization in the world (over 5 million experiments to date) and has crystallized over 300 different proteins. Syrrx's high-throughput structural biology (HTSB) technology platform enables the determination of protein structures more quickly, reliably, and economically than has been previously possible. Syrrx then uses these protein structures to discover new drugs to treat unmet medical needs.

17. The Diamond ESRF Synchrotron light sources

18. Resolution:R-merge I/σ(I) 1.8 Å (1.86-1.8 Å)0.05 (0.11)31.06 (11.73) Beamline Automation

19. More than 50 heavy atom soaking experiments conducted. An example of the Data sets used in structure solution.

20. 'P 21 21 21' ORTHORHOMBIC X,Y,Z 1/2-X,-Y,1/2+Z -X,1/2+Y,1/2-Z 1/2+X,1/2-Y,-Z

22. MR

25. Advantages of Ultrahigh or Atomic resolution Phase Problems can be solved directly. They allow comprehensive least square refinement of the structure with anisotropic ADP. The final R factor can be < 10 %. Multiple (Dual) Conformations can be seen. Unrestrained refinement (Coordinate Error Estimates) The position of many hydrogen atoms can be seen in density maps.

28. Substrate Specificity

29. Steric hindrance between Arg276 and Trp 275 (seen in 0.89 A T. aurantiacus xylanase structure) may play a key role in substrate specificity

33. Themophile Mesophile PEPTIDE SORT

34. Natesh et al., Nature, 421,551-554 (30 th Jan 2003).

40. Inhibitors Captopril (Classic first ACE inhibitors) Lisinopril, Enalapril, Ramipril…. Most common is persistant dry cough. Big fall in BP 1st, Kidney and liver problem, a type of swelling (angioedema), rash, inflammation of the pancreas, hay fever-like symptoms, sinusitis, sore throat, nausea, vomiting, indigestion, diarrhoea, constipation and blood cell changes. SIDE EFFECT

42. Captopril  Carboxypeptidase * Ondetti and Cushmann Lisinopril & Enalapril and their relatives  Thermolysin * Patchett et al., (side effects). Present ACE Stucture enables  2 nd generation SBDD.

43. N- and C-terminal selectivity of known (current) ACE inhibitors

44. HICUP Molecular Modeling

45. DALI

46. Structural Genomix - Structure to Function to Drug discovery. High throughput regime. Tools include X-ray crystallography, electron microscopy, NMR molecular biology Bioinformatics - select target proteins. Summary