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Conventional Electron Microscopy, Cryo-Electron Microscopy and Cryo-Electron Tomography of Viruses

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Part of the Subcellular Biochemistry book series (SCBI, volume 68)

Abstract

Electron microscopy (EM) techniques have been crucial for understanding the structure of biological specimens such as cells, tissues and macromolecular assemblies. Viruses and related viral assemblies are ideal targets for structural studies that help to define essential biological functions. Whereas conventional EM methods use chemical fixation, dehydration, and staining of the specimens, cryo-electron microscopy (cryo-EM) preserves the native hydrated state. Combined with image processing and three-dimensional reconstruction techniques, cryo-EM provides 3D maps of these macromolecular complexes from projection images, at subnanometer to near-atomic resolutions. Cryo-EM is also a major technique in structural biology for dynamic studies of functional complexes, which are often unstable, flexible, scarce or transient in their native environments. As a tool, cryo-EM complements high-resolution techniques such as X-ray diffraction and NMR spectroscopy; these synergistic hybrid approaches provide important new information. Three-dimensional cryo-electron tomography goes further, and allows the study of viruses not only in their physiological state, but also in their natural environment in the cell, thereby bridging structural studies at the molecular and cellular levels.

Keywords

Capsid Cryo-electron microscopy Cryo-electron tomography Electron microscopy Fourier transform Image processing Resolution Three-dimensional reconstruction Viral macromolecular assembly Virus 

Abbreviations

2D

Two-dimensional, two dimensions

3D

Three-dimensional, three dimensions

3DR

Three-dimensional reconstruction

CCD

Charge-coupled device

cryo-EM

Cryo-electron microscopy

cryo-ET

Cryo-electron tomography

CTF

Contrast transfer function

EM

Electron microscopy, electron microscope

FEG

Field emission gun

FSC

Fourier shell correlation

FT

Fourier transform

SEM

Scanning electron microscopy

SSE

Secondary structure element

TEM

Transmission electron microscopy

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Notes

Acknowledgements

I thank Daniel Luque for stimulating discussions, José L. Carrascosa and José M. Valpuesta for continuous support, comments and careful reading of the manuscript, Alasdair C. Steven and Benes L. Trus for advice and encouragement and Catherine Mark for editorial help. I am indebted to current and former members of my group (Irene Saugar, Daniel Luque, Nerea Irigoyen, Elena Pascual, Josué Gómez-Blanco, Mariana Castrillo, Ana Correia and Carlos Pérez) and other colleagues for their hard work, skills and enthusiasm that made work possible and enjoyable. This work was supported by grant BFU2011-25902 from the Spanish Ministry of Science and Innovation.

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Further Reading1

  1. Agbandje-McKenna M, McKenna R (eds) (2011) Structural virology. RSC Publishing, CambridgeGoogle Scholar
  2. Jensen GJ (ed) (2010) Cryo-EM, Part A. Sample preparation and data collection. Methods in Enzymology, vol 481; Cryo-EM, part B. 3-D Reconstruction. Methods in Enzymology, vol 482; Cryo-EM, part C. Analysis, interpretation and case studies. Methods in Enzymology, vol 483. Academic PressGoogle Scholar
  3. Rossmann MG, Rao VB (eds) (2012) Viral molecular machines. Adv Exp Med Biol, vol 726. Springer, New YorkGoogle Scholar

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© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Department of Macromolecular StructureCentro Nacional de Biotecnología (CSIC)MadridSpain

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