BC: Electron cryo microscopy in structural biology

Ardan Patwardhan a.patwardhan@ic.ac.ukDept. 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