Workshop

Structural Proteomics of Biological Complexes

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 ?

Workshop Agenda

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

Baylor College of Medicine

Wah Chiu

wah@bcm.tmc.edu

Electron Cryomicroscopy: A Structural Biology Tool

Instrument resolution beyond 2.2 Å

• Single machines: 9 – 6.8 Å helices and ß sheets visualized

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

• Subcellular assemblies and whole cell: 50 - 15 Å

identify components and domains

• Single machines: 9 – 6.8 Å helices and ß sheets visualized

• Single machines: 9 – 6.8 Å helices and ß sheets visualized

GroEL

D Chen

9 Å Structure of GroEL

S Ludtke

9 Å Structure of GroEL

6.8 Å Structure of Rice Dwarf Virus

Zhou et al. Nature SB (2001) 8:868-73

Rice Dwarf Virus Outer Shell Protein P8

1

3

2

4

69

7

Pseudo Atomic Model of RDV

Images of P22 Phage

500Å500Å

Procapsid Mature phage

Structural Transitions in Phage Maturation

Jiang et al (2003) Nature SB 10:131-5

Issues in CryoEM for Studying Single Particles of Complexes

• High structural purityChemical vs computational

• High resolutionNow 7-9 ÅFuture 3-4 Å

• High throughput analysisNow 5-10 monthsFuture 1- few weeks

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

• ManpowerChemists or

BiochemistsPhysicists or EngineersComputational

Scientists

• New InstrumentationEMCCD CameraFreezing Apparatus

• High Performance Computers

Resources Needed