Electron Cryomicroscopy is a Structural Biology Tool

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Workshop Structural Proteomics of Biological
Complexes
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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 ?
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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
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Baylor College of Medicine Wah
Chiu wah_at_bcm.tmc.edu
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Electron Cryomicroscopy A Structural Biology
Tool
Instrument resolution beyond 2.2 Å
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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

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GroEL
D Chen
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9 Å Structure of GroEL
S Ludtke
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9 Å Structure of GroEL
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6.8 Å Structure of Rice Dwarf Virus
Zhou et al. Nature SB (2001) 8868-73
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Rice Dwarf Virus Outer Shell Protein P8
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Pseudo Atomic Model of RDV
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Images of P22 Phage
Mature phage
Procapsid
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Structural Transitions in Phage Maturation
Jiang et al (2003) Nature SB 10131-5
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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

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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

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Resources Needed

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

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