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ELECTRON CRYSTALLOGRAPHY: Its role in proteomics, Present and future

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.

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ELECTRON CRYSTALLOGRAPHY: Its role in proteomics, Present and future

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

  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. 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. 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

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