electron crystallography: its role in proteomics, present and future kenneth h. downing lawrence...
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ELECTRON CRYSTALLOGRAPHY:Its role in proteomics,
Present and future
Kenneth H. DowningLawrence Berkeley National Laboratory
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Resolution of present microscopes -- ~1Å,but much worse for biology
Fundamental problem in obtaining biological data by EMis radiation damage
Improve signal-to-noise ratio byaveraging many equivalent images
Exposure ~ 10 electron/Å2,Noise ~ 30% in 1-Å pixel
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Crystals provide a large number of equivalent images in a single shot
-- all in same orientation, so easy to average
Examples of structures solved byElectron crystallography:
Results, limitations, prospects…
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Tubulin:A cytoskeletal protein of eukaryotic cells that is
essential for many functions
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Dimer > protofilament > microtubule
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Protofilaments in microtubules, Zn-sheets
Microtubule Zn-sheet 25 nm >1000 nm
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Electron diffraction from tubulin crystal
2.7 Å
3.5 Å
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2fo - fc map after refinement
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Tubulin Structure & Topology
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Tubulin dimer
H3
M-loop
GDP
GTP
Taxol
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Tubulin - drug interactions
Taxol stabilizes microtubules
-- as do several other drugs:
Drugs that interfere with microtubule dynamics can stop cell division
epothilones
sarcodictyin / eleutherobin
discodermolide
many Taxol (paclitaxel) analogues
• These can be studied by diffraction methods
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Density map with Taxol
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Microtubule-stabilizing drugs
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3-D Electron diffraction data
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Reciprocal Lattice Line Data
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Lattice line data for Taxol, epothilone
Taxol epothilone-A
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Epothilone - Taxol density map
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Taxol, Epothilone-A, Eleutherobin and Discodermolide bound to tubulin
GTP-bindingdomain
Intermediatedomain
M-loop
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Microtubules imaged in 400-kV EM,
Boxed into ~500 Å segments
Segments aligned to reference constructed from
crystal structure -
corrected in- and out-of-plane tilts,
variations in axial twist
Used 89 MT images, ~1200 segments,
~200,000 monomers
Result ~8 Å resolution
3-D Reconstruction of Microtubule
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Dimer > protofilament > microtubule
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Microtubule image, boxed into segments
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Microtubule map at 8 Angstroms
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Lateral interactions
H3
H10
M-loop
H1-S2 loop
H2-S3 loop H6
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Summary -
Tubulin structure solved by electron crystallography
Drug interactions studied with diffraction data
Microtubule structure by cryo-EM shows tubulin-tubulin interactions
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BACTERIORHODOPSIN:
A light-driven proton pump in bacteriaIntegral membrane protein
Structural paradigm for all rhodopsins, G-protein coupled receptors
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First 3-D structure solved by electron crystallography(1990; resolution ~3.5 Å)
Refined structure, high resolution images ~1995
Higher-resolution 3-D structures by EM, x-ray
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BR in projection at 2.6 Å resolution
(Grigorieff, Beckmann, Zemlin 1995)
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Bacteriorhodopsin photocycle
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Summary -
Bacteriorhodopsin structure solved by electron crystallography
Conformational changes studied by electron diffraction
EM resolution extended to ~ 3 Å
High resolution x-ray diffraction finally elucidatedmechanism of proton pumping
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How can EM compete with x-ray diffraction?
• it shouldn’t compete!
New instrumentation, along with continuing methods development --The keys to better and faster structure solutions
Role for EM is mainly structures not amenable to x-ray
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Our latest Electron Microscope
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Energy-loss Filtered Diffraction Patterns
unfiltered filtered
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Energy-loss Filtered Diffraction Patterns
unfiltered
filtered
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Microtubule doublets are tubulin complexes stabilizedby interactions with many MAPS
Doublet image at ~10 Å should reveal novel tubulin-tubulininteractions as well as some tubulin MAP interactions
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The role of electron microscopy in proteomics:
Electron crystallography gives single molecule structureat “atomic” resolution
EM is particularly good at studying large complexes
Ligand interactions and small conformational change can also be studied by crystallographic approaches