Hostname: page-component-76fb5796d-dfsvx Total loading time: 0 Render date: 2024-04-28T04:46:36.943Z Has data issue: false hasContentIssue false
  • English
  • Français

A Negative Stain for Electron Microscopic Tomography

Published online by Cambridge University Press:  27 February 2012

Andrea Fera ,
Jane E. Farrington ,
Joshua Zimmerberg  and
Thomas S. Reese
Andrea Fera*
Affiliation:
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA
Jane E. Farrington
Affiliation:
Laboratory of Cellular Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
Joshua Zimmerberg
Affiliation:
Laboratory of Cellular Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
Thomas S. Reese
Affiliation:
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA
*
Corresponding author. E-mail: feraandr@ninds.nih.gov
Get access

Abstract

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

Keywords

tomographyinfluenza virusnegative stainingback projection methodindividual molecule imagingnative state imaging
Type
Biological and Biomedical Applications
Information
Microscopy and Microanalysis , Volume 18 , Issue 2 , April 2012 , pp. 331 - 335
Copyright
Copyright © Microscopy Society of America 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Booy, F.P., Ruigrok, R.W.H. & van Bruggen, E.F.J. (1985). Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles. J Mol Biol 184, 667–676.CrossRefGoogle ScholarPubMed
Burgess, S.A., Walker, M.L., White, H.D. & Trinick, J. (1997). Flexibility within myosin heads revealed by negative stain and single-particle analysis. J Cell Biol 139, 675–681.CrossRefGoogle ScholarPubMed
Burgess, S.A., Walker, M.L., Sakakibara, H., Oiwa, K. & Knight, P.J. (2004a). The structure of dynein-c by negative stain electron microscopy. J Struct Biol 146, 205–216.CrossRefGoogle ScholarPubMed
Burgess, S.A., Walker, M.L., White, H.D., Thirumurugan, K., Trinick, J. & Knight, P.J. (2004b). Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules. J Struct Biol 147, 247–258.CrossRefGoogle ScholarPubMed
Chen, X., Winters, C.A. & Reese, T.S. (2008). Life inside a thin section: Tomography. J Neurosci 28, 9321–9327.CrossRefGoogle ScholarPubMed
Cohen-Krausz, S. & Trachtenberg, S. (2003). The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: Recovering high-resolution details from a flexible helical assembly. J Mol Biol 331, 1093–1108.CrossRefGoogle ScholarPubMed
Colman, P.M., Varghese, J.N. & Laver, W.G. (1983). Structure of the catalytic and antigenic sites on the influenza virus Neuraminidase. Nature 303, 41–44.CrossRefGoogle ScholarPubMed
Cremlyn, R.J. (1996). An Introduction to Organosulfur Chemistry. Chichester, NY: John Wiley and Sons.Google Scholar
De Carlo, S. & Harris, J.R. (2011). Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron 42, 117–131.CrossRefGoogle ScholarPubMed
Gamblin, S.J., Haire, L.F., Russell, R.J., Stevens, D.J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D.A., Daniels, R.S., Elliot, A., Wiley, D.C. & Skehel, J.J. (2004). The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838–1842.CrossRefGoogle ScholarPubMed
Harris, A., Cardone, G., Winkler, D.C., Heymann, J.B., Brecher, M., White, J.M. & Steven, A.C. (2006). Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Nat Acad Sci USA 103, 19123–19127.CrossRefGoogle ScholarPubMed
Heuser, J.E. & Salpeter, S.R. (1979). Organization of acetylcholine receptors in quickfrozen, deep-etched, and rotary-replicated torpedo postsynaptic membrane. J Cell Biol 82, 150–173.CrossRefGoogle ScholarPubMed
Hosoi, J., Matsuo, T., Ishida, Y. & Harada, Y. (1981). Process of irradiation damage to negatively stained helical structure observed by low dose method. J Electron Microsc 30, 1–7.Google ScholarPubMed
Kolodziej, S.J., Hudmon, A., Waxham, M.N. & Stoops, J.K. (2000). Three-dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase II-a and truncated CaM kinase II-a reveal a unique organization for its structural core and functional domains. J Biol Chem 275, 14354–14359.CrossRefGoogle Scholar
Kolodziej, S.J., Penczek, P.A. & Stoops, J.K. (1997). Utility of Butvar support film and methylamine tungstate stain in three-dimensional electron microscopy: Agreement between stain and frozen-hydrated reconstructions. J Struct Biol 120(2), 158–167.CrossRefGoogle ScholarPubMed
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 71–76.CrossRefGoogle ScholarPubMed
Mast, J. & Demeestere, L. (2009). Electron tomography of negatively stained complex viruses: Application in their diagnosis. Diagn Pathol 4, 5.CrossRefGoogle ScholarPubMed
Mastronarde, D.N. (1997). Dual-axis tomography: An approach with alignment methods that preserve resolution. J Struct Biol 120, 343–352.CrossRefGoogle ScholarPubMed
McIntosh, R., Nicastro, D. & Mastronarde, D. (2005). New views of cells in 3D: An introduction to electron tomography. Trends Cell Biol 15, 43–51.CrossRefGoogle ScholarPubMed
Ruigrok, R.W.H., Andree, P.J., Hooft Van Huysduynen, R.A.M. & Mellema, J.E. (1984). Characterization of three highly purified influenza virus strains by electron microscopy. J Gen Virol 65, 799–802.CrossRefGoogle ScholarPubMed
Ruigrok, R.W.H., Krijgsman, P.C.J., de Ronde-Verloop, F.M. & de Jong, J.C. (1985). Natural heterogeneity of shape, infectivity and protein composition in an influenza A (H3N2) virus preparation. Virus Res 3, 69–76.CrossRefGoogle Scholar
Ruigrok, R.W.H., Wrigley, N.G., Calder, L.J., Cusack, S., Wharton, S.A., Brown, E.B. & Skehel, J.J. (1986). Electron microscopy of the low pH structure of influenza virus haemagglutinin. EMBO J 5, 41–49.CrossRefGoogle Scholar
Stroud, R.M. (1983). Acetylcholine receptor structure. Neurosci Comm 1, 124–138.Google Scholar
Unwin, P.N. (1974). Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. II. The influence of electron irradiation of the stain distribution. J Mol Biol 87, 657–670.CrossRefGoogle ScholarPubMed
Varghese, J.N., Laver, W.G. & Colman, P.M. (1983). Structure of the influenza virus glycoprotein neuraminidase at 2.9 Å resolution. Nature 303, 35–40.CrossRefGoogle ScholarPubMed
Wabl, M.R. (1974). Electron microscopic localization of two proteins on the surface of the 50 S ribosomal subunit of Escherichia coli using specific antibody markers. J Mol Biol 84, 241–247.CrossRefGoogle ScholarPubMed
Wilson, I.A., Skehel, J.J. & Wiley, D.C. (1981). Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3Å resolution. Nature 289, 366–373.CrossRefGoogle Scholar
Xu, X., Zhu, X., Dwek, R.A., Stevens, J. & Wilson, I.A. (2008). Structural characterization of the 1918 influenza virus H1N1 neuraminidase. J Virol 82, 10493–10501.CrossRefGoogle ScholarPubMed
Supplementary material: PDF

Fera supplementary material

Figure 1.pdf

Download Fera supplementary material(PDF)
PDF 106.2 KB