Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy

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Nine different near-atomic resolution structures of icosahedral viruses, determined by electron cryo-microscopy and published between early 2008 and late 2010, fulfil predictions made 15 years ago that single-particle cryo-EM techniques could visualize molecular detail at 3–4 Å resolution. This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures, which advance our understanding of virus assembly and cell entry.

Introduction

Most cellular activities are outcomes of interactions among many components, including proteins, nucleic acids and lipids. Electron cryo-microscopy of isolated macromolecular complexes (‘single particle cryo-EM’) can now visualize icosahedral viruses at near-atomic resolution (Table 1, [1••, 2••, 3••, 4••, 5••, 6••, 7•, 8••, 9••]), and it should soon achieve similar resolution with less symmetrical particles. In a seminal review 15 years ago, Henderson predicted the success of single-particle techniques in visualizing detail at 3–4 Å resolution [10], and Glaeser extended the analysis a few years later [11]. A major step toward this goal was visualization of the hepatitis B icosahedral capsid at subnanometer resolution [12, 13], allowing for the first time the identification of a protein fold using single-particle cryo-EM. Reconstructions at subnanometer resolution of particles with lower symmetry followed, and it is now possible to obtain reconstructions of ribosomes at about 5 Å resolution [14].

The leading role of icosahedral viruses in achieving near-atomic resolution is due primarily to: (i) their high symmetry, which effectively increases the size of the data set 60-fold, or more if quasi-equivalent subunits can be averaged; (ii) their large molecular mass, producing strong image contrast and hence more accurate alignments and reconstructions; and (iii) their rigidity and uniformity, ensuring near-perfect superposition of structural features in three-dimensional (3D) reconstructions. In this review, we consider technological improvements made over the last decade that have enabled the recent successes, explain some of the remaining challenges and discuss some of the resulting functional conclusions about the organization of nonenveloped virions.

Section snippets

Radiation damage and beam-induced sample movement

Inelastic scattering of high-energy electrons by the sample leads to radiolysis of macromolecules and of the embedding medium, usually vitrified water. The extent of radiation damage depends on the electron dose, sample temperature and beam energy, thus limiting the useful dose for imaging to 5–10 e2 at liquid nitrogen temperature when using 100 kV electrons [10, 15]. The doses used for the near-atomic resolution reconstructions listed in Table 1 range between 15 and 30 e2, which is a bit

Double-stranded DNA (dsDNA) bacteriophage

The head-protein subunits of a large class of dsDNA bacteriophages have a folded structure first recognized in crystallographic analyses of the HK97 phage head and its assembly precursors [58]. Unlike the largely α-helical HepB capsid protein, which could be traced correctly at about 7 Å resolution [12, 13], the phage-head subunit is largely β-sheet, for which strands are resolved only if the resolution extends beyond 5 Å. One of the first group of higher-resolution cryo-EM reconstructions showed

Conclusions

The first image reconstructions of icosahedral viruses, derived from images of negatively stained particles, were published 40 years ago [48]. Reconstructions of icosahedral viruses at near-atomic resolution published during the past two years show that in favorable cases, atomic models can be derived from cryo-EM images [1••, 2••, 3••, 4••, 5••, 6••, 7•, 8••, 9••], yielding valuable new information about virus assembly. Advances in EM instrumentation, in computational algorithms, and in

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgments

The authors are grateful to David DeRosier and Alexis Rohou for comments and careful reading of the manuscript. Supported by NIH Grant P01 GM-62580. The authors are Investigators in the Howard Hughes Medical Institute.

References (73)

  • A. Stewart et al.

    Noise bias in the refinement of structures derived from single particles

    Ultramicroscopy

    (2004)
  • H. Liu et al.

    Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions

    J Struct Biol

    (2008)
  • J. Navaza

    On the three-dimensional reconstruction of icosahedral particles

    J Struct Biol

    (2003)
  • S.H.W. Scheres et al.

    Fast maximum likelihood refinement of electron microscopy images

    Bioinformatics

    (2005)
  • F.J. Sigworth

    A maximum-likelihood approach to single-particle image refinement

    J Struct Biol

    (1998)
  • A. Aldroubi et al.

    Magnification mismatches between micrographs: corrective procedures and implications for structural analysis

    Ultramicroscopy

    (1992)
  • F. Thon

    Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung

    Zeitschrift für Naturforschung

    (1966)
  • F. Zemlin et al.

    Coma-free alignment of high resolution electron microscopes with the aid of optical diffractograms

    Ultramicroscopy

    (1978)
  • D.J. Smith et al.

    The importance of beam alignment and crystal tilt in high resolution electron microscopy

    Ultramicroscopy

    (1983)
  • R. Henderson et al.

    Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution

    Ultramicroscopy

    (1986)
  • N. Grigorieff

    FREALIGN: high-resolution refinement of single particle structures

    J Struct Biol

    (2007)
  • M. Wolf et al.

    Ewald sphere correction for single-particle electron microscopy

    Ultramicroscopy

    (2006)
  • R.A. Crowther et al.

    Three dimensional reconstructions of spherical viruses by fourier synthesis from electron micrographs

    Nature

    (1970)
  • L.F. Estrozi et al.

    Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way

    J Struct Biol

    (2010)
  • Y. Liang et al.

    IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database

    J Struct Biol

    (2002)
  • S.J. Ludtke et al.

    EMAN: semiautomated software for high-resolution single-particle reconstructions

    J Struct Biol

    (1999)
  • P.A. Penczek

    Fundamentals of three-dimensional reconstruction from projections

    Methods Enzymol

    (2010)
  • M.F. Schmid et al.

    Scaling structure factor amplitudes in electron cryomicroscopy using X-ray solution scattering

    J Struct Biol

    (1999)
  • G. Harauz et al.

    Exact filters for general geometry three dimensional reconstruction

    Optik

    (1986)
  • N. Grigorieff

    Resolution measurement in structures derived from single particles

    Acta Crystallogr D Biol Crystallogr

    (2000)
  • B. McClain et al.

    X-ray crystal structure of the rotavirus inner capsid particle at 3.8 A resolution

    J Mol Biol

    (2010)
  • S.D. Trask et al.

    Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins

    J Virol

    (2006)
  • K.M. Reinisch et al.

    Structure of the reovirus core at 3.6 A resolution

    Nature

    (2000)
  • J.M. Grimes et al.

    The atomic structure of the bluetongue virus core

    Nature

    (1998)
  • K.A. Dryden et al.

    Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction

    J Cell Biol

    (1993)
  • X.S. Chen et al.

    Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16

    Mol Cell

    (2000)
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