BC: Electron cryo microscopy in structural biology

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BC Electron cryo microscopy in structural biology

• Ardan Patwardhan a.patwardhan_at_ic.ac.ukDept. of
  Biological SciencesImperial CollegeNovember,
  2003

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Specimen contrast
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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

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Cryo specimen preparation

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

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Cryo EM grid
Supporting carbon film
Metal grid
Ice holes
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An ice hole

• Particles are randomly positioned and orientated

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EM images

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

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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

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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)

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Translational Alignment

• Requires reference image(s) to align to

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Rotational Alignment

• Requires reference image(s) to align to

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Classification

• Combine like views to improve signal to noise

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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

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Angular reconstitution

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

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Slice through 3D
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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

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Pros and cons

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

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Examples Ribosome
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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)

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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