Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy
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 e−/Å2 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 e−/Å2, 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.
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