BC: Electron cryo microscopy in structural biology Ardan Patwardhan Dept. of Biological Sciences Imperial College November, 2003

Specimen 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 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 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), (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