ELECTRON CRYSTALLOGRAPHY:Its role in proteomics,

Present and future

Kenneth H. DowningLawrence Berkeley National Laboratory

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

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…

Tubulin:A cytoskeletal protein of eukaryotic cells that is

essential for many functions

Dimer > protofilament > microtubule

Protofilaments in microtubules, Zn-sheets

Microtubule Zn-sheet 25 nm >1000 nm

Electron diffraction from tubulin crystal

2.7 Å

3.5 Å

2fo - fc map after refinement

Tubulin Structure & Topology

Tubulin dimer

H3

M-loop

GDP

GTP

Taxol

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

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-bindingdomain

Intermediatedomain

M-loop

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

Dimer > protofilament > microtubule

Microtubule image, boxed into segments

Microtubule map at 8 Angstroms

Lateral interactions

H3

H10

M-loop

H1-S2 loop

H2-S3 loop H6

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 bacteriaIntegral 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 elucidatedmechanism 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 stabilizedby interactions with many MAPS

Doublet image at ~10 Å should reveal novel tubulin-tubulininteractions as well as some tubulin MAP interactions

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