Electron Microscopy pp 401-443

Part of the Methods in Molecular Biology book series (MIMB, volume 1117) | Cite as

Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

  • Fei Guo
  • Wen Jiang
Protocol

Abstract

With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

Key words

Cryo-EM Image processing Icosahedral reconstruction Asymmetric reconstruction Near-atomic resolution Virus 

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Fei Guo
    • 1
  • Wen Jiang
    • 1
  1. 1.Department of Biological Sciences, Markey Center for Structural BiologyPurdue UniversityWest LafayetteUSA

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