Organized packing of RNA inside viruses as revealed by cryo-electron microscopy and x-ray diffraction analysis

Abstract

The development of modern electron microscopy techniques to visualize the hydrated structures of biological macromolecules has stimulated many new studies, especially with viruses and virus-macromolecule complexes that are too large to study with current x-ray crystallographic methods, Cryo-electron microscopy (cryoEM) in conjunction with three-dimensional reconstruction procedures is capable of revealing both external and internal features of these structures.

Adding the Third Dimension to Virus Life Cycles: Three-Dimensional Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs

“…It has been used widely and successfully in many systems. One of the great advantages of cryo-EM over classical, negative-stain microscopy is that it allows reliable difference imaging (10,17,18,38,156,162,193,240,242,243,247,267,286,297,298,308,325,352,361,362,364). Many difference imaging results have been confirmed by biochemical and immunological means.…”

“…Hence, knowledge of the radial density distribution, for example, from a low-angle X-ray pattern (130), or accurate knowledge of the structure of a component can be used to calculate the appropriate correction to the image data. The availability of X-ray structures for the adenovirus hexon (298), the protein shell of CPMV (10,62), and the HRV (62,233,235) has allowed very accurate difference imaging to be performed after CTF correction.…”

“…One needs to remember that roughly 15% of the readers (and seemingly 97% of referees) will not be able to visualize an image in stereo, so that an alternative presentation should be present. The use of different colors for chemically distinct components (64,145,243,294,352,353), cut-open views (10,132), radially cut surfaces (14,15,20), icosahedrally cut surfaces (40), and polar sections (132) helps to convey the structural details, but they should always be carefully described to allow the reader to distinguish representation from result. Another very useful technique is radial depth cueing (141,264,292), which conveys an immediate, and often quantitative, view of the radial placement of details in a map (145).…”

“…Partially ordered genomes, typically in a layer in close proximity to the inner capsid wall, have been detected with cryoreconstruction methods in many viruses such as the middle and bottom components of cowpea mosaic virus (10), calicivirus (244), FHV (66), native and swollen forms of CCMV (290), rotavirus (247), TYMV (39), and aquareovirus (272). Difference map analysis is often helpful in ascribing genome density (10,39,66,247).…”