Download Downing

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Microtubule wikipedia , lookup

X-ray crystallography wikipedia , lookup

Transcript
ELECTRON CRYSTALLOGRAPHY:
Its role in proteomics,
Present and future
Kenneth H. Downing
Lawrence Berkeley National Laboratory
Resolution of present microscopes -- ~1Å,
but much worse for biology
Fundamental problem in obtaining biological data by EM
is radiation damage
Exposure ~ 10 electron/Å2,
Noise ~ 30% in 1-Å pixel
Improve signal-to-noise ratio by
averaging many equivalent images
Crystals provide a large number of equivalent images in a
single shot
-- all in same orientation, so easy to average
Examples of structures solved by
Electron crystallography:
Results, limitations, prospects…
Tubulin:
A cytoskeletal protein of eukaryotic cells that is
essential for many functions
Dimer > protofilament > microtubule
Protofilaments in microtubules, Zn-sheets
Microtubule
25 nm
Zn-sheet
>1000 nm
Electron diffraction from tubulin crystal
2.7 Å
3.5 Å
2fo - fc map after refinement
Tubulin Structure & Topology
Tubulin dimer
GDP
Taxol
H3
b
M-loop
GTP
a
Tubulin - drug interactions
Drugs that interfere with microtubule dynamics can stop cell division
Taxol stabilizes microtubules
-- as do several other drugs:
epothilones
sarcodictyin / eleutherobin
discodermolide
many Taxol (paclitaxel) analogues
• These can be studied by diffraction methods
Density map with Taxol
Microtubule-stabilizing drugs
3-D Electron diffraction data
Reciprocal Lattice Line Data
Lattice line data for Taxol, epothilone
Taxol
epothilone-A
Epothilone - Taxol density map
Taxol, Epothilone-A, Eleutherobin
and Discodermolide bound to tubulin
GTP-binding
domain
M-loop
Intermediate
domain
3-D Reconstruction of Microtubule
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
Dimer > protofilament > microtubule
Microtubule image, boxed into segments
Microtubule map at 8 Angstroms
Lateral interactions
H2-S3 loop
H6
M-loop
H3
H1-S2 loop
H10
Summary -
Tubulin structure solved by electron crystallography
Drug interactions studied with diffraction data
Microtubule structure by cryo-EM shows
tubulin-tubulin interactions
BACTERIORHODOPSIN:
A light-driven proton pump in bacteria
Integral membrane protein
Structural paradigm for all rhodopsins, G-protein coupled receptors
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
BR in projection at 2.6 Å resolution
(Grigorieff, Beckmann, Zemlin 1995)
Bacteriorhodopsin photocycle
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 elucidated
mechanism of proton pumping
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
Our latest Electron Microscope
Energy-loss Filtered Diffraction Patterns
unfiltered
filtered
Energy-loss Filtered Diffraction Patterns
unfiltered
filtered
Microtubule doublets are tubulin complexes stabilized
by interactions with many MAPS
Doublet image at ~10 Å should
reveal novel tubulin-tubulin
interactions as well as some
tubulin MAP interactions
The role of electron microscopy in proteomics:
Electron crystallography gives single molecule structure
at “atomic” resolution
Ligand interactions and small conformational change
can also be studied by crystallographic approaches
EM is particularly good at studying large complexes