Transcript Downing
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