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Sample Preparation Induced Artifacts in Cryo-Electron Tomographs

Published online by Cambridge University Press:  08 October 2012

Pavel Plevka ,
Anthony J. Battisti ,
Dennis C. Winkler ,
Kaspars Tars ,
Heather A. Holdaway ,
Carol M. Bator  and
Michael G. Rossmann
Pavel Plevka
Affiliation:
Department of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA
Anthony J. Battisti
Affiliation:
Department of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA
Dennis C. Winkler
Affiliation:
Laboratory of Structural Biology Research, National Institute for Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Kaspars Tars
Affiliation:
Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, Riga, LV 1067, Latvia
Heather A. Holdaway
Affiliation:
Department of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA
Carol M. Bator
Affiliation:
Department of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA
Michael G. Rossmann*
Affiliation:
Department of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA
*
*Corresponding author. E-mail: mr@purdue.edu
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Abstract

We investigated the effects of sample preparation and of the exposure to an electron beam on particles in cryo-electron tomographs. Various virus particles with icosahedral symmetry were examined, allowing a comparison of symmetrically related components that should be identical in structure but might be affected differently by these imaging artifacts. Comparison of tomographic reconstructions with previously determined structures established by an independent method showed that neither freezing nor electron beam exposure produced a significant amount of shrinkage along the z axis (thickness). However, we observed damage to regions of the particles located close to the surface of the vitreous ice.

Keywords

cryo-EMmicroscopyice-vacuum interfaceartifactsvirustomography
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1043 - 1048
Copyright
Copyright © Microscopy Society of America 2012

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Footnotes

†

Present address: Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH 44106, USA

𠈁

Present address: Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA

References

Berriman, J. & Leonard, K.R. (1986). Methods for specimen thickness determination in electron microscopy. II. Changes in thickness with dose. Ultramicroscopy 19, 349–366.Google Scholar
Braunfeld, M.B., Koster, A.J., Sedat, J.W. & Agard, D.A. (1994). Cryo automated electron tomography: Towards high-resolution reconstructions of plastic-embedded structures. J Microsc-Oxford 174, 75–84.CrossRefGoogle ScholarPubMed
Cheng, R.H., Kuhn, R.J., Olson, N.H., Rossmann, M.G., Choi, H.K., Smith, T.J. & Baker, T.S. (1995). Nucleocapsid and glycoprotein organization in an enveloped virus. Cell 80, 621–630.Google Scholar
Egerton, R.F., Li, P. & Malac, M. (2004). Radiation damage in the TEM and SEM. Micron 35, 399–409.CrossRefGoogle ScholarPubMed
Jésior, J.C. & Wade, R.H. (1987). Electron-irradiation-inducing flattening of negatively stained 2D protein crystals. Ultramicroscopy 21, 313–319.Google Scholar
Kleywegt, G.J. & Jones, T.A. (1994). Halloween … masks and bones. In From First Map to Final Model, Bailey, S., Hubbard, R. & Waller, D. (Eds.), pp. 59–66. Daresbury, Warrington, UK: SERC Daresbury Laboratory.Google Scholar
Kleywegt, G.J. & Read, R.J. (1997). Not your average density. Structure 5, 1557–1569.Google Scholar
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.Google Scholar
Ludtke, S.J., Baldwin, P.R. & Chiu, W. (1999). EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128, 82–97.Google Scholar
Luther, P.K., Lawrence, M.C. & Crowther, R.A. (1988). A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 24, 7–18.Google Scholar
Mastronarde, D.N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36–51.Google Scholar
Pletnev, S.V., Zhang, W., Mukhopadhyay, S., Fisher, B.R., Hernandez, R., Brown, D.T., Baker, T.S., Rossmann, M.G. & Kuhn, R.J. (2001). Locations of carbohydrate sites on Sindbis virus glycoproteins show that E1 forms an icosahedral scaffold. Cell 105, 127–136.CrossRefGoogle ScholarPubMed
Rossmann, M.G. & Blow, D.M. (1962). The detection of sub-units within the crystallographic asymmetric unit. Acta Crystallogr 15, 24–31.Google Scholar
Trachtenberg, S., Pinnick, B. & Kessel, M. (2000). The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Halofax volcanii: Cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes. J Struct Biol 130, 10–26.CrossRefGoogle ScholarPubMed
Valegård, K., Liljas, L., Fridborg, K. & Unge, T. (1990). The three-dimensional structure of the bacterial virus MS2. Nature 345, 36–41.CrossRefGoogle ScholarPubMed
Valegård, K., Unge, T., Montelius, I., Strandberg, B. & Fiers, W. (1986). Purification, crystallization and preliminary X-ray data of the bacteriophage MS2. J Mol Biol 190, 587–591.Google Scholar
Wright, E.R., Iancu, C.V., Tivol, W.F. & Jensen, G.J. (2006). Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K. J Struct Biol 153, 241–252.Google Scholar
Zhang, W., Mukhopadhyay, S., Pletnev, S.V., Baker, T.S., Kuhn, R.J. & Rossmann, M.G. (2002). Placement of the structural proteins in Sindbis virus. J Virol 76, 11645–11658.Google Scholar
Zhang, Y., Corver, J., Chipman, P.R., Zhang, W., Pletnev, S.V., Sedlak, D., Baker, T.S., Strauss, J.H., Kuhn, R.J. & Rossmann, M.G. (2003). Structures of immature flavivirus particles. EMBO J 22, 2604–2613.Google Scholar