1. ELECTRON CRYSTALLOGRAPHY:
Its role in proteomics,
Present and future
Kenneth H. Downing
Lawrence Berkeley National Laboratory

2. 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

3. 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…

4. Tubulin: A cytoskeletal protein of eukaryotic cells that is
essential for many functions

5. Dimer > protofilament > microtubule

6. Protofilaments in microtubules, Zn-sheets
Microtubule Zn-sheet
25 nm >1000 nm

7. Electron diffraction from tubulin crystal
2.7 Å
3.5 Å

8. 2fo - fc map after refinement

9. Tubulin Structure & Topology

10. Tubulin dimer H3
M-loop
GDP
GTP
Taxol a b

11. 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

12. Density map with Taxol

13. Microtubule-stabilizing drugs

14. 3-D Electron diffraction data

15. Reciprocal Lattice Line Data

16. Lattice line data for Taxol, epothilone
Taxol epothilone-A

17. Epothilone - Taxol density map

18. Taxol, Epothilone-A, Eleutherobin and Discodermolide bound to tubulin
GTP-binding
domain
M-loop
Intermediate
domain

19. 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

20. Dimer > protofilament > microtubule

21. Microtubule image, boxed into segments

22. Microtubule map at 8 Angstroms

23. Lateral interactions H6
H2-S3 loop
M-loop H3
H1-S2 loop
H10

24. Tubulin structure solved by electron crystallography
Summary -
Tubulin structure solved by electron crystallography
Drug interactions studied with diffraction data
Microtubule structure by cryo-EM shows
tubulin-tubulin interactions

25. BACTERIORHODOPSIN: A light-driven proton pump in bacteria
Integral membrane protein
Structural paradigm for all rhodopsins, G-protein coupled receptors

26. 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

27. BR in projection at 2.6 Å resolution
(Grigorieff, Beckmann, Zemlin 1995)

28. Bacteriorhodopsin photocycle

29. Bacteriorhodopsin structure solved by electron crystallography
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

30. 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

31. Our latest Electron Microscope

32. Energy-loss Filtered Diffraction Patterns
unfiltered
filtered

33. Energy-loss Filtered Diffraction Patterns
unfiltered
filtered

34. 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

35. 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