BC: Electron cryo microscopy in
structural biology
Ardan Patwardhan
a.patwardhan@ic.ac.uk
Dept. of Biological Sciences
Imperial College
November, 2003
Specimen contrast
Amplitude 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”
Supporting
carbon film
Metal grid
Ice holes
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)
Credits
Biological 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