macromolecular electron microscopy michael stowell mcdb b231 [email protected]
TRANSCRIPT
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References and other useful material Texts
Biophysical Electron Microscopy: Basic Concepts and Modern Techniquesby U. Valdre (Editor), Peter W. Hawkes (Editor)
Three-Dimensional Electron Microscopy of Macromolecular Assemblies by Joachim Frank
Negative Staining and Cryoelectron Microscopy: The Thin Film Techniquesby Robin J. Harris, James R. Harris
Reviews Henderson, R. The potential and limitations of neutrons, electrons and
X-rays for atomic resolution microscopy of unstained biological molecules. Q Rev Biophys 28, 171-93 (1995).
Glaeser, R. M. Review: electron crystallography: present excitement, a nod to the past, anticipating the future. J Struct Biol 128, 3-14 (1999).
Stowell, M. H., Miyazawa, A. & Unwin, N. Macromolecular structure determination by electron microscopy: new advances and recent results. Curr Opin Struct Biol 8, 595-600 (1998).
Web http://em-outreach.sdsc.edu/web-course/toc.html http://ncmi.bcm.tmc.edu/%7Estevel/spintro/siframes.htm http://cryoem.berkeley.edu/~nieder/em_for_dummies/
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Topics Why electrons Sample preparation Types of Samples Data collection Data processing
Data processing 1: single particle Data processing 2: 2D xtal
Data analysis (statistics, resolution criteria) Interpretation Examples and other
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Why use electrons…part IIElectrons
80-500keV
X-rays
1.5 A
Ratio inelastic/elastic 3 10
Mechanism of damage 2nd e- emission
Photoelectric e- emission
Energy per inelastic event 20 eV 8 keV
Energy per elastic event 60 eV 80 eV
Energy relative to electrons
inelastic (Compton) 1 400
elastic (Rayleigh) 1 1000
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Negative Stain and Cryo
Negative stain (usually 0.5% uranyl acetate) Easy to prepare Good contrast Preservation Sample distortion Resolution limited to about 20 angstroms
Cryo Difficult sample prep Low contrast Best preservation and therefore resolution
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Negative staining
Bob Horne (Cambridge)
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Cryo prep using holey filmH. Fernandez-moranB. GlaeserK. TaylorJ. Dubochet
Aaron Klug
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Flash freeze in liquid ethane
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Samples Single Particles (Proteins, Ribosome)
No crystallization Weak amplitude, no diffraction, alignment ambiguity, particle
flexibility ~7 angstroms
Fibers and filaments (tubulin, collagen) No crystallization, 2D distortion corrections, phase restrictions Weak amplitude, no diffraction ~9 angstroms
2D crystals (BR, AQP, LHCII) Diffraction amplitudes, 2D distortion corrections,
crystallographic methods Crystallization, many tilts required, anisotropic data ~3 angstroms
Tubular crystals (AchR, Ca++-ATPase) Crystallization, No diffraction Isotropic data, 3D distortion corrections, phase restrictions ~5 angstroms
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Single particles
Applicable to any protein or protein complex > 50kD
Most common sample Number of software
suites available Resolution ~9A (<7
with symmetry)
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Fibers and filaments
DNA, collagen, etc
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2D XtalsHenderson and Unwin
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Tubular crystals
2D xtal
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Tubular xtal versus 2D or 3D xtal
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Data collection
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Image recording Film
High density content (~20kx16k pixels)Slow (development time, drying) Requires digitization (scanning takes hours)
CCD Low density content (4kx4k pixels)Fast (ms to sec)Direct digital
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Processing data Single Particles (Proteins, Ribosome)
Pick particles Align Classify, average and reconstruction
Fibers and filaments (tubulin, collagen) Pick segments determine symmetry Align/rotate Average
2D crystals (BR, AQP, LHCII) Process images to achieve phases Process diffraction data for amplitudes Combine and refine as in X-ray
Tubular crystals (AchR, Ca++-ATPase) Determine tube symmetry Pick segments and distortion correction Average and sum segments
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Data processing 1: single particleMostly swiped from Steve Ludtke’s web site http://ncmi.bcm.tmc.edu/~stevel/EMAN/doc/
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Pick particles (manual or semiauto)
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Looking for astigmatism, drift, charging etc….
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Now on to the first model
First rule of thumb…be cautious… How to classify particles
Reference free classification and alignmentMSA
Application of symmetry Random conical tilt
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Reference free classification MSA
Can we tell the symmetry a priori???
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MSA…….variance….(SD)2
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Random conical tilt
Image pairs taken of the same sample with an angular tilt applied between them
Determine particle pairs and construct reference model
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Use of common lines to align different orientations
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Refinement
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Multiple rounds of refinement…
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Convergence when no improvement in the alignment statistics
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Try different symmetries
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Data processing 2: 2D xtal
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Why lattice lines?Z dimension has an effective real space D of infinityHence in reciprocal space the lattice spacing is 0
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A well refined EM map
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Resolution and Resolvability
Single particles, filaments, tubesFSC
Which criteria to use (0.5 or 3 sigma)
2D xtals diffraction (like X-ray)But anisotropy or point spread function
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FSC
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Point spread function
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Resolution vs Resolvability
Resolution is a calculated valueFSC or measured amplitudes above a certain
sigma value. Resolvability is a perceived value
What can a see in the map Is a 4 angstroms map really 4 angstroms is one
cannot discern beta sheet structure?