Cryo‐Electron Tomography for Structural Characterization of Macromolecular Complexes

Julia Cope1, John Heumann1, Andreas Hoenger1

1 Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.13
DOI:  10.1002/0471140864.ps1713s65
Online Posting Date:  August, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Cryo‐electron tomography (cryo‐ET) is an emerging 3‐D reconstruction technology that combines the principles of tomographic 3‐D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo‐ET is particularly suited to investigating cell‐biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging‐based 3‐D reconstruction procedures. This unit aims to make cryo‐ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo‐ET. Protocols describe specimen preparation, data recording and 3‐D data reconstruction for cryo‐ET, with a special focus on macromolecular complexes. A step‐by‐step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt‐series acquisition for cryo‐ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo‐tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal‐to‐noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions. Curr. Protoc. Protein Sci. 65:17.13.1‐17.13.31. © 2011 by John Wiley & Sons, Inc.

Keywords: cryo‐electron microscopy (cryo‐EM); cryo‐electron tomography (cryo‐ET); vitrification of macromolecules; tilt‐series acquisition; sub‐volume averaging

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Plunge Freezing Isolated Macromolecules Using a Homemade Plunge Freezer
  • Alternate Protocol 1: Plunge Freezing Isolated Macromolecules Using a Commercial Plunge Freezer
  • Alternate Protocol 2: High‐Pressure Freezing and Cryo‐Sectioning for Cryo‐Electron Tomography
  • Basic Protocol 2: Setting up the Microscope for Cryo‐Electron Tomography
  • Basic Protocol 3: Tilt Series Data Collection
  • Basic Protocol 4: Tomogram Reconstruction
  • Basic Protocol 5: Averaging Subvolumes of Identical Structures from Tomograms
  • Support Protocol 1: Applying Symmetry with Subvolume Averaging
  • Support Protocol 2: Estimating Resolution in Subvolume Averages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Plunge Freezing Isolated Macromolecules Using a Homemade Plunge Freezer

  Materials
  • Liquid nitrogen
  • Gaseous ethane or ethane/propane mixture with tubing and pipet tip
  • Purified protein/macromolecule sample or thin cells
  • Suitable dilution buffer (if needed)
  • 5‐ or 10‐nm colloidal gold (see recipe)
  • Plunge freezing device (Fig. )
  • Glow discharger (e.g., Emitech K100X, Quorum Technologies)/plasma cleaner (e.g., Gatan Solarus 950, Gatan)
  • Holey‐carbon grids, 200‐mesh (e.g., C‐flat or Quantifoil, EMS)
  • Forceps (straight, fine tip)
  • Filter paper cut into strips (Whatman no. 1 or similar)
  • Grid storage boxes (EMS)
  • 50‐ml conical tubes or other container to store grid boxes in liquid nitrogen

Alternate Protocol 1: Plunge Freezing Isolated Macromolecules Using a Commercial Plunge Freezer

  Materials
  • Liquid nitrogen
  • Samples in liquid nitrogen
  • Cryo‐holder (Fig. A)
  • Vacuum station
  • Dissecting microscope
  • Transfer station (Fig. A)
  • Funnel with wire mesh filter (≤0.5‐mm)
  • Styrofoam box
  • Ladle (Fig. C)
  • Grid box lid opener (Fig. D)
  • Forceps
  • Electron microscope with cryo‐capabilities (e.g., Tecnai F20, FEI Company)

Alternate Protocol 2: High‐Pressure Freezing and Cryo‐Sectioning for Cryo‐Electron Tomography

  Materials
  • Grids
  • Electron microscope equipped with a computer‐controlled goniometer and a high‐sensitivity charge‐coupled device, CCD, camera
  • Automated tilt series acquisition software (e.g., SerialEM, http://bio3d.colorado.edu/SerialEM/)

Basic Protocol 2: Setting up the Microscope for Cryo‐Electron Tomography

  Materials
  • Computer workstation (Linux, Macintosh, or Windows with Cygwin)
  • Tomogram reconstruction software (e.g., eTomo, part of the IMOD software package, http://bio3d.colorado.edu/imod/)

Basic Protocol 3: Tilt Series Data Collection

  Materials
  • Tomogram modeling software (e.g., 3dmod, part of the IMOD package; http://bio3d.colorado.edu/imod/)
  • Subvolume averaging software (e.g., PEET; http://bio3d.colorado.edu/PEET/)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Aebi, U. and Pollard, T.D. 1987. A glow discharge unit to render electron microscope grids and other surfaces hydrophilic. J. Electron. Microsc. Tech. 7:29‐33.
   Al‐Amoudi, A., Diez, D.C., Betts, M.J., and Frangakis, A.S. 2007. The molecular architecture of cadherins in native epidermal desmosomes. Nature 450:832‐837.
   Amat, F., Moussavi, F., Comolli, L.R., Elidan, G., Downing, K.H., and Horowitz, M. 2008. Markov random field based automatic image alignment for electron tomography. J. Struct. Biol. 161:260‐275.
   Bartesaghi, A., Sprechmann, P., Liu, J., Randall, G., Sapiro, G., and Subramaniam, S. 2008. Classification and 3D averaging with missing wedge correction in biological electron tomography. J. Struct. Biol. 162:436‐450.
   Beck, M., Forster, F., Ecke, M., Plitzko, J.M., Melchior, F., Gerisch, G., Baumeister, W., and Medalia, O. 2004. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306:1387‐1390.
   Bouchet‐Marquis, C. and Fakan, S. 2009. Cryoelectron microscopy of vitreous sections: A step further towards the native state. Methods Mol. Biol. 464:425‐439.
   Bouchet‐Marquis, C. and Hoenger, A. 2011. Cryo‐electron tomography on vitrified sections: A critical analysis of benefits and limitations for structural cell biology. Micron 42:152‐162.
   Bouchet‐Marquis, C., Zuber, B., Glynn, A.M., Eltsov, M., Grabenbauer, M., Goldie, K.N., Thomas, D., Frangakis, A.S., Dubochet, J., and Chretien, D. 2007. Visualization of cell microtubules in their native state. Biol. Cell 99:45‐53.
   Bouchet‐Marquis, C., Starkuviene, V., and Grabenbauer, M. 2008. Golgi apparatus studied in vitreous sections. J. Microsc. 230:308‐316.
   Braunfeld, M.B., Koster, A.J., Sedat, J.W., and Agard, D.A. 1994. Cryo automated electron tomography: Towards high‐resolution reconstructions of plastic‐embedded structures. J. Microsc. 174:75‐84.
   Castaño Díez, D., Scheffer, M., Al‐Amoudi, A., and Frangakis, A.S. 2010. Alignator: A GPU powered software package for robust fiducial‐less alignment of cryo tilt‐series. J. Struct. Biol. 170:117‐126.
   Cope, J., Gilbert, S., Rayment, I., Mastronarde, D., and Hoenger, A. 2010. Cryo‐electron tomography of microtubule‐kinesin motor complexes. J. Struct. Biol. 170:257‐265.
   Danev, R., Kanamaru, S., Marko, M., and Nagayama, K. 2010. Zernike phase contrast cryo‐electron tomography. J. Struct. Biol. 171:174‐181.
   De Rosier, D.J. and Klug, A. 1968. Reconstruction of three dimensional structures from electron micrographs. Nature 217:130‐134.
   Dubochet, J. 2007. The physics of rapid cooling and its implications for cryoimmobilization of cells. Methods Cell Biol. 79:7‐21.
   Dubochet, J., Booy, F.P., Freeman, R., Jones, A.V., and Walter, C.A. 1981. Low temperature electron microscopy. Annu. Rev. Biophys. Bioeng. 10:133‐149.
   Dubochet, J., Adrian, M., Chang, J.J., Homo, J.C., Lepault, J., Mcdowall, A.W., and Schultz, P. 1988. Cryo‐electron microscopy of vitrified specimens. Q. Rev. Biophys. 21:129‐228.
   Dubochet, J., Zuber, B., Eltsov, M., Bouchet‐Marquis, C., Al‐Amoudi, A., and Livolant, F. 2007. How to “read” a vitreous section. Methods Cell Biol. 79:385‐406.
   Frangakis, A.S. and Hegerl, R. 2001. Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion. J. Struct. Biol. 135:239‐250.
   Frank, J. 2006. Electron Tomography: Methods for Three‐Dimensional Visualization of Structures in the Cell, Second Edition. Springer, New York.
   Gilbert, P. 1972a. Iterative methods for the three‐dimensional reconstruction of an object from projections. J. Theor. Biol. 36:105‐117.
   Gilbert, P.F. 1972b. The reconstruction of a three‐dimensional structure from projections and its application to electron microscopy. II. Direct methods. Proc. R. Soc. Lond. B. Biol. Sci. 182:89‐102.
   Gonen, T., Cheng, Y., Sliz, P., Hiroaki, Y., Fujiyoshi, Y., Harrison, S. C., and Walz, T. 2005. Lipid‐protein interactions in double‐layered two‐dimensional AQP0 crystals. Nature 438:633‐638.
   Harauz, G. and van Heel, M. 1986. Exact filters for general geometry three dimensional reconstruction. Optik 73:146‐156.
   Herman, G.T., Lent, A., and Rowland, S.W. 1973. ART: Mathematics and applications. A report on the mathematical foundations and on the applicability to real data of the algebraic reconstruction techniques. J. Theor. Biol. 42:1‐32.
   Heymann, J.B., Cardone, G., Winkler, D.C., and Steven, A.C. 2008. Computational resources for cryo‐electron tomography in Bsoft. J. Struct. Biol. 161:232‐242.
   Hoog, J.L., Schwartz, C., Noon, A.T., O'toole, E.T., Mastronarde, D.N., Mcintosh, J.R., and Antony, C. 2007. Organization of interphase microtubules in fission yeast analyzed by electron tomography. Dev. Cell 12:349‐361.
   Iancu, C.V., Tivol, W.F., Schooler, J.B., Dias, D.P., Henderson, G.P., Murphy, G.E., Wright, E.R., Li, Z., Yu, Z., Briegel, A., Gan, L., He, Y., and Jensen, G.J. 2006. Electron cryotomography sample preparation using the Vitrobot. Nat. Protoc. 1:2813‐2819.
   Jin, L., Milazzo, A.C., Kleinfelder, S., Li, S., Leblanc, P., Duttweiler, F., Bouwer, J.C., Peltier, S.T., Ellisman, M.H., and Xuong, N.H. 2008. Applications of direct detection device in transmission electron microscopy. J. Struct. Biol. 161:352‐358.
   Kremer, J.R., Mastronarde, D.N., and Mcintosh, J.R. 1996. Computer visualization of three‐dimensional image data using IMOD. J. Struct. Biol. 116:71‐76.
   Ladinsky, M.S. 2010. Micromanipulator‐assisted vitreous cryosectioning and sample preparation by high‐pressure freezing. Methods Enzymol. 481:165‐194.
   Lučić , V., Forster, F., and Baumeister, W. 2005. Structural studies by electron tomography: From cells to molecules. Annu. Rev. Biochem. 74:833‐865.
   Luther, P.K., Lawrence, M.C., and Crowther, R.A. 1988. A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 24:7‐18.
   Marsh, B.J., Mastronarde, D.N., Buttle, K.F., Howell, K.E., and Mcintosh, J.R. 2001. Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT‐T15, visualized by high resolution electron tomography. Proc. Natl. Acad. Sci. U.S.A. 98:2399‐2406.
   Mastronarde, D.N. 2005. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152:36‐51.
   Medalia, O., Weber, I., Frangakis, A.S., Nicastro, D., Gerisch, G., and Baumeister, W. 2002. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science 298:1209‐1213.
   Messaoudii, C., Boudier, T., Sanchez Sorzano, C.O., and Marco, S. 2007. TomoJ: Tomography software for three‐dimensional reconstruction in transmission electron microscopy. BMC Bioinformatics 8:288‐296.
   Milazzo, A.C., Moldovan, G., Lanman, J., Jin, L., Bouwer, J.C., Klienfelder, S., Peltier, S.T., Ellisman, M.H., Kirkland, A.I., and Xuong, N.H. 2010. Characterization of a direct detection device imaging camera for transmission electron microscopy. Ultramicroscopy 110:744‐747.
   Murata, K., Liu, X., Danev, R., Jakana, J., Schmid, M.F., King, J., Nagayama, K., and Chiu, W. 2010. Zernike phase contrast cryo‐electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure 18:903‐912.
   Nicastro, D., Frangakis, A.S., Typke, D., and Baumeister, W. 2000. Cryo‐electron tomography of neurospora mitochondria. J. Struct. Biol. 129:48‐56.
   Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M.E., and Mcintosh, J.R. 2006. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313:944‐948.
   Nickell, S., Forster, F., Linaroudis, A., Net, W.D., Beck, F., Hegerl, R., Baumeister, W., and Plitzko, J.M. 2005. TOM software toolbox: Acquisition and analysis for electron tomography. J. Struct. Biol. 149:227‐234.
   O'Toole, E.T., Giddings, T.H., Mcintosh, J.R., and Dutcher, S.K. 2003. Three‐dimensional organization of basal bodies from wild‐type and delta‐tubulin deletion strains of Chlamydomonas reinhardtii. Mol. Biol. Cell 14:2999‐3012.
   Penczek, P.A. 2002. Three‐dimensional spectral signal‐to‐noise ratio for a class of reconstruction algorithms. J. Struct. Biol. 138:34‐46.
   Penczek, P.A. 2010. Fundamentals of three‐dimensional reconstruction from projections. Methods. Enzymol. 482:1‐33.
   Sachse, C., Chen, J.Z., Coureux, P.D., Stroupe, M.E., Fandrich, M., and Grigorieff, N. 2007. High‐resolution electron microscopy of helical specimens: A fresh look at tobacco mosaic virus. J. Mol. Biol. 371:812‐835.
   Saxton, W.O., Baumeister, W., and Hahn, M. 1984. Three‐dimensional reconstruction of imperfect two‐dimensional crystals. Ultramicroscopy 13:57‐70.
   Tivol, W.F., Briegel, A., and Jensen, G.J. 2008. An improved cryogen for plunge freezing. Microsc. Microanal. 14:375‐379.
   Vanhecke, D., Asano, S., Kochovski, Z., Fernandez‐Busnadiego, R., Schrod, N., Baumeister, W., and Lučić, V. 2010. Cryo‐electron tomography: Methodology, developments and biological applications. J. Microsc. 242:221‐227.
   van Heel, M. and Schatz, M. 2005. Fourier shell correlation threshold criteria. J. Struct. Biol. 151:250‐262.
   Winkler, H. and Taylor, K.A. 2006. Accurate marker‐free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy 106:240‐254.
   Xiong, Q., Morphew, M.K., Schwartz, C.L., Hoenger, A.H., and Mastronarde, D.N. 2009. CTF determination and correction for low dose tomographic tilt series. J. Struct. Biol. 168:378‐387.
   Zanetti, G., Riches, J.D., Fuller, S.D., and Briggs, J.A. 2009. Contrast transfer function correction applied to cryo‐electron tomography and sub‐tomogram averaging. J. Struct. Biol. 168:305‐312.
   Zhang, X., Jin, L., Fang, Q., Hui, W.H., and Zhou, Z.H. 2010. 3.3 A cryo‐EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell 141:472‐482.
   Zheng, S.Q., Keszthelyi, B., Branlund, E., Lyle, J.M., Braunfeld, M.B., Sedat, J.W., and Agard, D.A. 2007. UCSF tomography: An integrated software suite for real‐time electron microscopic tomographic data collection, alignment, and reconstruction. J. Struct. Biol. 157:138‐147.
Key Reference
  Vanhecke et al., 2010. See above.
  A current review discussing sample preparation, data acquisition, data processing and interpretation, and some recent examples of biological applications using cryo‐ET.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library