Workshop Structural Proteomics of Biological Complexes

1. Workshop Structural Proteomics of Biological Complexes

2. Questions Why is it timely to study complexes ? What are the most appropriate model organisms ? How to predict relevant complexes ? How to purify complexes ? How to validate their biological functions ? What are the necessary technologies ? What are the realistic goals and timetables ? What are the necessary resources to accomplish the goals ? What are the funding mechanisms for such an initiative ?

3. Workshop Agenda Session I Cell Biology and Complexes Session II Biological Processes and Protein Machines Session III Genetic and Chemical Approaches Session IV Computational Approaches Session V Electron Cryomicroscopy Session VI Crystallography Session VII Proteomics Centers Session VIII Recommendations

5. Baylor College of Medicine Wah Chiu wah@bcm.tmc.edu

6. Electron Cryomicroscopy: A Structural Biology Tool Instrument resolution beyond 2.2 Å

7. Structural Features Revealed by Electron Cryomicroscopy • Crystalline arrays : 3.7-3.0 Å • polypeptide traced • Helical arrays: 9 - 4.0 Å •  helices and ß strands resolved • Single machines: 9 – 6.8 Å •  helices and ß sheets visualized • Single machines: 9 – 6.8 Å •  helices and ß sheets visualized • Single machines: 9 – 6.8 Å •  helices and ß sheets visualized • Subcellular assemblies and whole cell: 50 - 15 Å • identify components and domains

8. GroEL D Chen

9. 9 Å Structure of GroEL S Ludtke

10. 9 Å Structure of GroEL

11. 6.8 Å Structure of Rice Dwarf Virus Zhou et al. Nature SB (2001) 8:868-73

12. Rice Dwarf Virus Outer Shell Protein P8

13. 4 9 6 1 7 2 3 Pseudo Atomic Model of RDV

14. 500Å 500Å Images of P22 Phage Mature phage Procapsid

15. Structural Transitions in Phage Maturation Jiang et al (2003) Nature SB 10:131-5

16. Issues in CryoEM for Studying Single Particles of Complexes • High structural purity • Chemical vs computational • High resolution • Now 7-9 Å • Future 3-4 Å • High throughput analysis • Now 5-10 months • Future 1- few weeks

17. Experimental and Computational Processes • Biochemical Purification • Cryo-Specimen Preparation • Data Collection • Data Digitization • Pre-processing • Initial Model • Structure Refinement • Structure Visualization • Structure Analysis • Biochemical Purification • Cryo-Specimen Preparation • Data Collection • Data Digitization • Pre-processing • Initial Model • Structure Refinement • Structure Visualization • Structure Analysis

18. Resources Needed • Manpower • Chemists or Biochemists • Physicists or Engineers • Computational Scientists • New Instrumentation • EM • CCD Camera • Freezing Apparatus • High Performance Computers