Growth of EM Method for Determining Structures of Macromolecular Assemblies

What is cryo EM? EM = (Transmission) Electron Microscopy Cryo EM = technique where biological samples are preserved in vitreous ice and imaged by EM at cryogenic temperatures. EM reconstruction = 3D maps are generated by averaging over many EM images.

From Sample to Structure Slide from Wah Chiu, Baylor

Reconstruction Method Sample Types Type Examples Reconstruction Method Typical resolution Single particles Icosahedral viruses, GroEL, ribosome Single particle reconstruction 20-4 Å Filaments Flagella, filamentous viruses, actin, tubular crystals Helical reconstruction 15-3 Å 2D crystals Catalase, aquaporin, tubulin 2D electron crystallography 10-2 Å Ensembles HIV capsids Electron Tomography 40-20 Å

Single Particles MW lower limit: ~200 kDa Icosahedral virus Ribosome Image sources: EMDB, Joachim Frank, Sacha De Carlo

Filaments Acetylcholine receptor tubular crystal Tobacco Mosaic Virus gingi.uchicago.edu/achr.html Actin-myosin complex ami.scripps.edu

2D Crystals Aquaporin 2D crystals, electron diffraction Lipid-protein interactions Gonen et al. 2005. Nature 438:633-8.

Ensembles Reconstruction of HIV capsids by cryoEM tomography Benjamin et al. 2005. J. Mol Biol. 346:577-588.

specimen preparation SUPPORT GRID 3.05 mm 400 divisons/inch 1 division/66 microns Copper or other metal Manufactured “holey carbon grid” Holes typically 2 microns diameter 2spi.com/catalog

Negative Stain vs. Vitreous Ice Specimen in Stain Cryogenic Specimen vitreous ice layer uranyl acetate High contrast image No special temperature control Essentially no radiation damage Particle distorted Image = stain “shell” around the particle Low resolution method: 20-15 Å Great choice for initial sample screening Low contrast image Sample maintained at cryogenic temperature (85 K) High radiation damage Particle undistorted Image is of the actual particle Higher resolution obtained: 15-4 Å Best choice for reconstruction

Imaging--TEM microscope Sample Goes here Swap out to view diffraction Swap out screen to record image Film/CCD camera cryoem.berkeley.edu/~nieder

Cryo EM Experiment Characteristics Images must be taken with low electron doses to prevent radiation damage Averaging over thousands of individual particles and/or “asymmetric units” Image: http://ami.scripps.edu

Data Collection and Initial Image Processing Collect image set (20-100 images, vary focus) Pick Particles (4000-100,000) Perform contrast-transfer-function (CTF) correction for each image Center, align, classify, make “class averages”

Particle Selection Raw image Auto-Select particles Particle Composite For 1 raw image

CTF Correction

Class Averages

Recovering 3D from 2D figure from Joachim Frank

Reconstruction Cycle cryoem.berkeley.edu/~nieder Final map

Map Quality figure from Joachim Frank

Methods to interpret cryo EM map volumes: Structure Analysis Methods to interpret cryo EM map volumes: “segmentation” -- identifying different parts of the map “fitting” --placing atomic coordinates into the map, e.g., from X-ray structures very new: normal mode refinement to improve fit

Segmentation herpes simplex capsid heterotrimer Epsilon 15 bacteriophage Images from Wah Chiu

Fitting Rossmann, M. G. et al 2004. Curr Opin Struct Biol 14:171-80. Kostyuchenko, V. A. et al 2005 Nat Struct Mol Biol 12:810-3.

Soon…a single archive site for maps and models Archiving wwpdb.org Coordinates www.ebi.ac.uk/msd/emdep Maps Soon…a single archive site for maps and models

References The Definitive Textbook: Frank, J. 2006. Three-dimensional electron microscopy of macromolecular assemblies : visualization of biological molecules in their native state. 2nd ed. New York : Oxford University Press. Reviews: Chiu, W., M. L. Baker, and S. C. Almo. 2006. Structural biology of cellular machines. Trends Cell Biol 16:144-50. Nickell, S., C. Kofler, A. P. Leis, and W. Baumeister. 2006. A visual approach to proteomics. Nat Rev Mol Cell Biol 7:225-30.

Resources Research-Resource Centers for Molecular Microscopy: www.ncrr.nih.gov/ncrrprog/btdir/Microsco.asp EM software tools: en.wikipedia.org/wiki/Software_tools_for_molecular_microscopy Visualization: www.cgl.ucsf.edu/chimera/ EMDB map database: www.ebi.ac.uk/msd-srv/emsearch Icosahedral Viruses: viperdb.scripps.edu

Cool Movies based on cryoEM data Microtubules: http://cryoem.berkeley.edu/animations.shtml T4 virus: www.nsf.gov/news/news_summ.jsp?cntn_id=100420&org=MCB&from=news Ribosome: www.wadsworth.org/databank/electron Actomyosin/kinesin: www.scripps.edu/cb/milligan/index.html T4 virus