Structure determination of icosahedral viruses by cryo-electron microscopy and image analysis
Abstract
The structures of the Cabb-B and CM1841 strains of cauliflower mosaic virus (CaMV) have been solved to about 3 nm resolution from images of unstained, frozen-hydrated samples by cryo-electron microscopy (cryoEM) and image analysis. CaMV, with a maximum diameter of 53.8 nm, is composed of three concentric layers (I-III) of solvent-excluded density surrounding a large, solvent-filled cavity ($\sim$27 nm dia.). The outermost layer (I) contains 72 capsomeric morphological units. This is the first T = 7 virus known to obey the rules of stoichiometry proposed for isometric viruses by Caspar and Klug (1962, Cold Spring Harb. Symp. Quant. Biol. 27, 1-24). The double-stranded DNA genome is distributed in layers II and III along with a portion of the viral protein. The CaMV reconstructions confirm a structural model of neutron diffraction (Kruse et al., 1987, Virology 159, 166-168) and a replication-assembly hypothesis (Hull et al., 1987, J. Cell Sci. Suppl. 7, 213-229). However, since the interpretation of cryoEM images of biological macromolecules is strongly influenced by the effect of phase contrast transfer function (CTF) of the microscope, it is important to restore the unequally weighted phase-contrast for the range of spatial frequencies included in the analysis. Correct compensation for the CTF is difficult because of ambiguities in measuring the exact level of defocus and the relative contributions of amplitude contrast, beam coherence, and inelastic scattering. The availability of atomic resolution data for cowpea mosaic virus (CPMV) and bacteriophage $\phi$X174 allows one to empirically correct the cryoEM images. A protocol involving using correlation and least-squares algorithms was developed to compensate for the CTF effect with the scattering amplitudes calculated from X-ray crystallographic results. The CTF-compensated CPMV model was further used to investigate both internal and external features in the virus with the aim of obtaining a more accurate representation of the density distribution. The existence of different CPMV particles, separated on cesium chloride gradient, was used to investigate RNA structure inside CPMV. The RNA was found to be primarily confined between adjacent icosahedral three-fold axes, and on the five-fold axes beneath the capsid inner surface (r = 8-10 nm). This suggests that the capsids may strongly influence a common packing arrangement of the separate RNA molecules. In addition, two electrophoretic forms of CPMV capsids, produced by proteolysis in vivo, were also examined by SDS/PAGE, anion-exchange chromatography, and negative-stain electron microscopy. CryoEM and image analysis of these forms further showed that the compact pentamers in the fast-electrophoretic form of the capsid probably resulted from the C-terminus cleavage in the small protein of CPMV.
Degree
Ph.D.
Advisors
Bracker, Purdue University.
Subject Area
Biophysics|Molecular biology|Microbiology
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