BC: Electron cryo microscopy in structural biology
Ardan Patwardhan [email protected]. of Biological SciencesImperial CollegeNovember, 2003
Specimen contrastAmplitude Contrast
Phase Contrast
Phase Contrast
• Is not directly observable
• Converted to amplitude contrast by defocusing specimen
• Limited to study of thin specimens (<1000Å)
• Same technique used in light microscopy to study unstained specimens
• Why not use stain?- May affect macromolecular structure
Cryo specimen preparation• Preserve native environment
• High vacuum need frozen specimens!
• Snap freezing for amorphous ice phase, not crystalline ice phase
Cryo EM “grid”
Supportingcarbon film
Ice holes
Metal grid
An ice hole• Particles are randomly positioned and
orientated
EM images
• 2D projections of 3D objects
• Similar to x-ray images
EM images are very noisy!!
• Beam damage limits exposure
• At our disposal: Thousands of randomly oriented macromolecular images with very poor signal to noise ratio
• Image processing techniques used to combine thousands of 2D images into a 3D reconstruction of the particle
Particle Picking
• Objective: identify particles in micrograph and cut out patches containing one particle each
• Can be done automatically, in some cases, especially if the molecule possesses icosahedral symmetry
• Most cases still done manually- tedious, difficult and boring
• Need to collect between 1000 and 10000 particles to get going (the more the better)
Translational Alignment• Requires reference image(s) to align to
Rotational Alignment• Requires reference image(s) to align to
Classification• Combine like views to improve signal to
noise
Chicken and egg problem
• The class “sum” images can be used as references for alignment
• The quality of the classification depends on how well aligned the data is
• In general, steps of alignment and classification have to be repeated several times
Angular reconstitution• Determine angles of projections relative to each other in
3D
• Find common line projections to determine relative angles
Slice through
3D
Reprojection
• 3D density map can be used to generate projections that can be used to realign the raw images
• Process may have to be repeated several times
Pros and cons
• Excellent tool for difference studies
• Resolution not yet as good as for x-ray crystallography and NMR
Examples: Ribosome
References
• M. van Heel, B. Gowen, R. Matadeen, E. Orlova, R. Finn, T. Pape, D. Cohen, H. Stark, R. Schmidt, M. Schatz and A. Patwardhan:Single-particle electron cryo-microscopy: towards atomic resolution.Quarterly Review of Biophysics 33(4), 307 - 369(2000)
CreditsBiological Sciences
• Prof. Marin Van Heel
• Dr. Tillman Pape
• Dr. Elena Orlova
• Alexis Rohou
• David Carpentier
• Martin Bommer
• Richard Hall
• Dr. Pampa Ray
Division of Medicine
• Dr. Edward P. Morris• Danielle Paul
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