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BMII: Electron cryo microscopy in structural biology

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... Orlova, R. Finn, T. Pape, D. Cohen, H. Stark, R. Schmidt, M. Schatz ... Dr. Tillman Pape. Dr. Elena Orlova. Alexis Rohou. David Carpentier. Martin Bommer ... – PowerPoint PPT presentation

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Title: BMII: Electron cryo microscopy in structural biology


1
BMII Electron cryo microscopy in structural
biology
  • Ardan Patwardhan a.patwardhan_at_ic.ac.ukDept. of
    Biological SciencesImperial CollegeMay, 2002

2
Transmission electron microscopy
  • Gun emits electrons
  • Electric field accelerate
  • Magnetic (and electric) field control path of
    electrons
  • Electron wavelength _at_ 200KeV ? 2x10-12 m
  • Resolution normally achievable _at_ 200KeV ? 2 x
    10-10 m ? 2Å

3
Specimen contrast
4
Phase Contrast
  • Is not directly observable
  • Converted to amplitude contrast by defocusing
    specimen
  • Limited to study of thin specimens (lt1000Å)
  • Same technique used in light microscopy to study
    unstained specimens
  • Why not use stain?- May affect macromolecular
    structure

5
Cryo specimen preparation
  • Preserve native environment
  • High vacuum? need frozen specimens!
  • Snap freezing for amorphous ice phase, not
    crystalline ice phase

6
Cryo EM grid
Supporting carbon film
Metal grid
Ice holes
7
An ice hole
  • Particles are randomly positioned and orientated

8
EM images
  • 2D projections of 3D objects
  • Similar to x-ray images

9
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

10
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)

11
Translational Alignment
  • Requires reference image(s) to align to

12
Rotational Alignment
  • Requires reference image(s) to align to

13
Classification
  • Combine like views to improve signal to noise

14
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

15
Angular reconstitution
  • Determine angles of projections relative to each
    other in 3D
  • Find common line projections to determine
    relative angles

16
Slice through 3D
17
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

18
Pros and cons
  • Excellent tool for difference studies
  • Resolution not yet as good as for x-ray
    crystallography and NMR

19
Examples Ribosome
20
References
  • M. van Heel, B. Gowen, R. Matadeen, E. Orlova, R.
    Finn, T. Pape, D. Cohen, H. Stark, R. Schmidt, M.
    Schatz and A. PatwardhanSingle-particle
    electron cryo-microscopy towards atomic
    resolution.Quarterly Review of Biophysics 33(4),
    307 - 369(2000)

21
Credits
  • Prof. Marin Van Heel
  • Dr. Tillman Pape
  • Dr. Elena Orlova
  • Alexis Rohou
  • David Carpentier
  • Martin Bommer
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