Skip to main content
SpringerLink
Log in

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

  • Chapter
  • First Online:
Structure and Physics of Viruses

Part of the book series: Subcellular Biochemistry ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    Also especially recommended for further reading are references [3, 4, 11, 23, 25, 26, 27, 35] listed above.

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

References and Further Reading

  1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2007) Molecular biology of the cell. Garland Science, New York

    Google Scholar 

  2. Nogales E, Grigorieff N (2001) Molecular machines: putting the pieces together. J Cell Biol 152:F1–F10

    Article  PubMed  CAS  Google Scholar 

  3. Steven A, Belnap D (2005) Electron microscopy and image processing: an essential tool for structural analysis of macromolecules. Curr Protoc Protein Sci Chapter 17:Unit 17 12

    Google Scholar 

  4. Klug A (2010) From virus structure to chromatin: X-ray diffraction to three-dimensional electron microscopy. Annu Rev Biochem 79:1–35

    Article  PubMed  CAS  Google Scholar 

  5. Harrison SC (2004) Whither structural biology? Nat Struct Mol Biol 11:12–15

    Article  PubMed  CAS  Google Scholar 

  6. Baumeister W, Steven AC (2000) Macromolecular electron microscopy in the era of structural genomics. Trends Biochem Sci 25:624–631

    Article  PubMed  CAS  Google Scholar 

  7. Steven AC, Baumeister W (2008) The future is hybrid. J Struct Biol 163:186–195

    Article  PubMed  CAS  Google Scholar 

  8. Lasker K, Phillips JL, Russel D, Velazquez-Muriel J, Schneidman-Duhovny D, Tjioe E, Webb B, Schlessinger A, Sali A (2010) Integrative structure modeling of macromolecular assemblies from proteomics data. Mol Cell Proteomics 9:1689–1702

    Article  PubMed  CAS  Google Scholar 

  9. Goldsmith CS, Miller SE (2009) Modern uses of electron microscopy for detection of viruses. Clin Microbiol Rev 22:552–563

    Article  PubMed  Google Scholar 

  10. Bozzola JJ, Russell LD (1999) Electron microscopy. Principles and techniques for biologist. Jones and Bartlett Publishers, Boston

    Google Scholar 

  11. Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228

    Article  PubMed  CAS  Google Scholar 

  12. Amos LA, Henderson R, Unwin PN (1982) Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol 39:183–231

    Article  PubMed  CAS  Google Scholar 

  13. Henderson R (2004) Realizing the potential of electron cryo-microscopy. Q Rev Biophys 37:3–13

    Article  PubMed  CAS  Google Scholar 

  14. Harris JR (1997) Negative staining and cryoelectron microscopy: the thin film techniques. BIOS Scientific Publishers Ltd., Oxford

    Google Scholar 

  15. Wild P (2008) Electron microscopy of viruses and virus-cell interactions. Methods Cell Biol 88:497–524

    Article  PubMed  CAS  Google Scholar 

  16. Leiman PG, Kanamaru S, Mesyanzhinov VV, Arisaka F, Rossmann MG (2003) Structure and morphogenesis of bacteriophage T4. Cell Mol Life Sci 60:2356–2370

    Article  PubMed  CAS  Google Scholar 

  17. Cavalier A, Spehner D, Humbel BM (eds) (2008) Handbook of cryo-preparation methods for electron microscopy. CRC Press, London

    Google Scholar 

  18. Baker TS, Olson NH, Fuller SD (1999) Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. Microbiol Mol Biol Rev 63:862–922

    PubMed  CAS  Google Scholar 

  19. De Carlo S, Stark H (2010) Cryonegative staining of macromolecular assemblies. Methods Enzymol 481:127–145

    Article  PubMed  Google Scholar 

  20. Crowther RA (2008) The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets. Philos Trans R Soc Lond B Biol Sci 363:2441–2451

    Article  PubMed  CAS  Google Scholar 

  21. De Rosier DJ, Klug A (1968) Reconstruction of three dimensional structures from electron micrographs. Nature 217:130–134

    Article  PubMed  Google Scholar 

  22. Moody MF (1990) Image analysis of electron micrographs. In: Hawkes PW, Valdré U (eds) Biophysical electron microscopy. Basis concepts and modern techniques. Academic Press, London, pp 145–288

    Google Scholar 

  23. Rochat RH, Chiu W (2012) Cryo-electron microscopy and tomography of virus particles. In: Egelman EH (ed) Comprehensive biophysics, biophysical techniques for structural characterization of macromolecules, vol 1. Academic Press, Oxford, pp 311–340

    Google Scholar 

  24. Chang J, Liu X, Rochat RH, Baker ML, Chiu W (2012) Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography. Adv Exp Med Biol 726:49–90

    Article  PubMed  CAS  Google Scholar 

  25. Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21:265–273

    Article  PubMed  CAS  Google Scholar 

  26. Steven AC, Heymann JB, Cheng N, Trus BL, Conway JF (2005) Virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity. Curr Opin Struct Biol 15:227–236

    Article  PubMed  CAS  Google Scholar 

  27. Luque D, Gonzalez JM, Garriga D, Ghabrial SA, Havens WM, Trus B, Verdaguer N, Carrascosa JL, Castón JR (2010) The T=1 capsid protein of penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication. J Virol 84:7256–7266

    Article  PubMed  CAS  Google Scholar 

  28. Johnson JE (2008) Multi-disciplinary studies of viruses: the role of structure in shaping the questions and answers. J Struct Biol 163:246–253

    Article  PubMed  CAS  Google Scholar 

  29. Russel D, Lasker K, Phillips J, Schneidman-Duhovny D, Velazquez-Muriel JA, Sali A (2009) The structural dynamics of macromolecular processes. Curr Opin Cell Biol 21:97–108

    Article  PubMed  CAS  Google Scholar 

  30. Tang L, Johnson JE (2002) Structural biology of viruses by the combination of electron cryomicroscopy and X-ray crystallography. Biochemistry 41:11517–11524

    Article  PubMed  CAS  Google Scholar 

  31. Topf M, Lasker K, Webb B, Wolfson H, Chiu W, Sali A (2008) Protein structure fitting and refinement guided by cryo-EM density. Structure 16:295–307

    Article  PubMed  CAS  Google Scholar 

  32. Rossmann MG, Morais MC, Leiman PG, Zhang W (2005) Combining X-ray crystallography and electron microscopy. Structure 13:355–362

    Article  PubMed  CAS  Google Scholar 

  33. Topf M, Sali A (2005) Combining electron microscopy and comparative protein structure modeling. Curr Opin Struct Biol 15:578–585

    Article  PubMed  CAS  Google Scholar 

  34. Luque D, Saugar I, Rodriguez JF, Verdaguer N, Garriga D, Martin CS, Velazquez-Muriel JA, Trus BL, Carrascosa JL, Castón JR (2007) Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch. J Virol 81:6869–6878

    Article  PubMed  CAS  Google Scholar 

  35. Zhou ZH (2011) Atomic resolution cryo electron microscopy of macromolecular complexes. Adv Protein Chem Struct Biol 82:1–35

    Article  PubMed  CAS  Google Scholar 

  36. Zhou ZH (2008) Towards atomic resolution structural determination by single-particle cryo-electron microscopy. Curr Opin Struct Biol 18:218–228

    Article  PubMed  CAS  Google Scholar 

  37. Hryc CF, Chen DH, Chiu W (2011) Near-atomic-resolution cryo-EM for molecular virology. Curr Opin Virol 1:110–117

    Article  PubMed  CAS  Google Scholar 

  38. Yu X, Jin L, Zhou ZH (2008) 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453:415–419

    Article  PubMed  CAS  Google Scholar 

  39. Zhang X, Settembre E, Xu C, Dormitzer PR, Bellamy R, Harrison SC, Grigorieff N (2008) Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proc Natl Acad Sci U S A 105:1867–1872

    Article  PubMed  CAS  Google Scholar 

  40. Briggs JA, Huiskonen JT, Fernando KV, Gilbert RJ, Scotti P, Butcher SJ, Fuller SD (2005) Classification and three-dimensional reconstruction of unevenly distributed or symmetry mismatched features of icosahedral particles. J Struct Biol 150:332–339

    Article  PubMed  Google Scholar 

  41. Kostyuchenko VA, Chipman PR, Leiman PG, Arisaka F, Mesyanzhinov VV, Rossmann MG (2005) The tail structure of bacteriophage T4 and its mechanism of contraction. Nat Struct Mol Biol 12:810–813

    Article  PubMed  CAS  Google Scholar 

  42. Aksyuk AA, Rossmann MG (2011) Bacteriophage assembly. Viruses 3:172–203

    Article  PubMed  CAS  Google Scholar 

  43. Stubbs G, Kendall A (2012) Helical viruses. Adv Exp Med Biol 726:631–658

    Article  PubMed  CAS  Google Scholar 

  44. Ge P, Tsao J, Schein S, Green TJ, Luo M, Zhou ZH (2010) Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327:689–693

    Article  PubMed  CAS  Google Scholar 

  45. Ge P, Zhou ZH (2011) Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches. Proc Natl Acad Sci U S A 108:9637–9642

    Article  PubMed  CAS  Google Scholar 

  46. Egelman EH (2007) Single-particle reconstruction from EM images of helical filaments. Curr Opin Struct Biol 17:556–561

    Article  PubMed  CAS  Google Scholar 

  47. Grunewald K, Medalia O, Gross A, Steven AC, Baumeister W (2003) Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding. Biophys Chem 100:577–591

    Article  PubMed  CAS  Google Scholar 

  48. Lucic V, Forster F, Baumeister W (2005) Structural studies by electron tomography: from cells to molecules. Annu Rev Biochem 74:833–865

    Article  PubMed  CAS  Google Scholar 

  49. Cope J, Heumann J, Hoenger A (2011) Cryo-electron tomography for structural characterization of macromolecular complexes. Curr Protoc Protein Sci Chapter 17:Unit17 13

    Google Scholar 

  50. Subramaniam S, Bartesaghi A, Liu J, Bennett AE, Sougrat R (2007) Electron tomography of viruses. Curr Opin Struct Biol 17:596–602

    Article  PubMed  CAS  Google Scholar 

  51. Grunewald K, Cyrklaff M (2006) Structure of complex viruses and virus-infected cells by electron cryo tomography. Curr Opin Microbiol 9:437–442

    Article  PubMed  Google Scholar 

  52. Grunewald K, Desai P, Winkler DC, Heymann JB, Belnap DM, Baumeister W, Steven AC (2003) Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302:1396–1398

    Article  PubMed  Google Scholar 

  53. Butan C, Winkler DC, Heymann JB, Craven RC, Steven AC (2008) RSV capsid polymorphism correlates with polymerization efficiency and envelope glycoprotein content: implications that nucleation controls morphogenesis. J Mol Biol 376:1168–1181

    Article  PubMed  CAS  Google Scholar 

  54. Harris A, Cardone G, Winkler DC, Heymann JB, Brecher M, White JM, Steven AC (2006) Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Natl Acad Sci U S A 103:19123–19127

    Article  PubMed  CAS  Google Scholar 

  55. Cyrklaff M, Risco C, Fernandez JJ, Jimenez MV, Esteban M, Baumeister W, Carrascosa JL (2005) Cryo-electron tomography of vaccinia virus. Proc Natl Acad Sci U S A 102:2772–2777

    Article  PubMed  CAS  Google Scholar 

  56. Ibiricu I, Huiskonen JT, Dohner K, Bradke F, Sodeik B, Grunewald K (2011) Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons. PLoS Pathog 7:e1002406

    Article  PubMed  CAS  Google Scholar 

  57. Fu CY, Johnson JE (2011) Viral life cycles captured in three-dimensions with electron microscopy tomography. Curr Opin Virol 1:125–133

    Article  PubMed  CAS  Google Scholar 

  58. Iwasaki K, Omura T (2010) Electron tomography of the supramolecular structure of virus-infected cells. Curr Opin Struct Biol 20:632–639

    Article  PubMed  CAS  Google Scholar 

  59. Crowther RA, Amos LA, Finch JT, De Rosier DJ, Klug A (1970) Three dimensional reconstructions of spherical viruses by fourier synthesis from electron micrographs. Nature 226:421–425

    Article  PubMed  CAS  Google Scholar 

Further Reading

  • Agbandje-McKenna M, McKenna R (eds) (2011) Structural virology. RSC Publishing, Cambridge

    Google Scholar 

  • 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 Press

    Google Scholar 

  • Rossmann MG, Rao VB (eds) (2012) Viral molecular machines. Adv Exp Med Biol, vol 726. Springer, New York

    Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Macromolecular Structure, Centro Nacional de Biotecnología (CSIC), c/Darwin 3, Campus de Cantoblanco, 28049, Madrid, Spain

    José R. Castón

Authors
  1. José R. Castón
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José R. Castón .

Editor information

Editors and Affiliations

  1. "Severo Ochoa" (CSIC_UAM), And Dept. of Molecular Biology, Centro de Biología Molecular, Cantoblanco, Madrid, 28049, Madrid, Spain

    Mauricio G. Mateu

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media Dordrecht

About this chapter

Cite this chapter

Castón, J.R. (2013). Conventional Electron Microscopy, Cryo-Electron Microscopy and Cryo-Electron Tomography of Viruses. In: Mateu, M. (eds) Structure and Physics of Viruses. Subcellular Biochemistry, vol 68. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-6552-8_3

Download citation

Publish with us

Policies and ethics

Search

Navigation