contrast) occurs where portions of the folded protein chain protrude from the surface. Consequently, surface knobs or projections, termed morphological units, are the main features identified by this method. However, because these surface features are often formed by multiple proteins, their organization does not necessarily correspond to that of the individual proteins that make up the capsid shell. Even when structure is well preserved and a high degree of contrast can be achieved, the minimal size of an object that can be distinguished by classical electron microscopy, its resolution, is limited to 50 to 75 Å. Th is resolution is far too poor to permit molecular interpretation: for example, the diameter of an α-helix in a protein is on the or der of 10 Å. Cryo-electron microscopy (cryo-EM), in which samples are rapidly frozen and examined at very low temperatures in a hydrated, vitrified (noncrystalline, glass-like) state, preserves native structure. Because samples are not stained, this technique allows direct visualization of the contrast inherent to the virus particle, and it also enables higher resolution.
The symmetry of most virus particles facilitates analysis of individual images obtained by cryo-EM for reconstruction of two- or three-dimensional structure (Box 4.1). This approach can be complemented by cryo-electron tomography, in which two-dimensional images are recorded as the vitrified sample is tilted at different angles to the electron beam and subsequently combined into a three-dimensional density map (Fig. 4.3). These methods have been greatly improved since their introduction, to near atomic-level resolution, and are now standard tools of structural biology. Their application has provided unprecedented views of virus particles not amenable to other methods of structural analysis, for example, alphaviruses (Fig. 4.23) and herpesviruses (Fig. 4.27).
The first descriptions of the molecular interactions that dictate the structure of virus particles were obtained by X-ray crystallography (Fig. 4.4) (see the interview with Dr. Michael Rossmann: http://bit.ly/Virology_Rossmann). A plant virus (tobacco mosaic virus) was the first to be crystallized, and the first high-resolution virus structure determined was that of tomato bushy stunt virus. Since this feat was accomplished in 1978, high-resolution structures of increasingly large animal viruses have been determined, placing our understanding of the principles of capsid architecture on a firm foundation.
METHODS
The development of cryo-electron microscopy, a revolution in structural biology
Cryo-EM has now revealed near-atomic resolution of not only symmetric virus particles, some very large, but also asymmetric and dynamic cellular machines built from many components, such as transcription complexes and the spliceosome. The foundations for this revolutionary method of structural biology were laid in the 1970s and 1980s by Jacques Dubochet, Joachim Frank, and Richard Henderson, whose contributions were recognized by the 2017 Nobel Prize in Chemistry.
Henderson was the first to use electron microscopy to investigate the structure of a protein (bacteriorhodopsin in a cell membrane), and obtained a low-resolution model. The development by Frank of algorithms for the sorting of randomly oriented molecules into related groups for averaging improved the resolution of two-dimensional images and facilitated their transformation into three-dimensional structural models (Fig. 4.3). Further increases in resolution were achieved when Dubochet perfected methods for vitrification of samples to produce much sharper images.
Near-atomic resolution, which is now quite routine, was attained with additional refinements, including the use of direct electron detectors (rather than film or CCD cameras) to capture images and increasingly sophisticated data processing software.
The composite image of cryo-EM reconstructions of the enzyme β-galactosidase dramatizes the great improvement in resolution, from the 10 to 20 Å typical a decade ago to, in this case, 2.2 Å (left to right). Courtesy of Sriram Subramanian, National Cancer Institute.
Figure 4.3 Cryo-EM and image reconstruction illustrated with rotavirus.
Concentrated preparations of purified virus particles are prepared for cryo-electron microscopy by rapid freezing on an electron microscope grid so that a glasslike, noncrystalline water layer is produced. This procedure avoids sample damage that can be caused by crystallization of the water or by chemical modification or dehydration during conventional negative-contrast electron microscopy. The sample is maintained at or below −160°C during all subsequent operations. Fields containing sufficient numbers of vitrified virus particles are identified by transmission electron microscopy at low magnification (to minimize sample damage from the electron beam) and photographed at high resolution (top).
These electron micrographs can be treated as two-dimensional projections (Fourier transforms) of the particles. Three-dimensional structures can be reconstructed from such two-dimensional projections by mathematically combining the information included in different views of the particles. For the purpose of reconstruction, the images of different particles are treated as different views of the same structure.
For reconstruction, micrographs are digitized for computer processing. Each particle to be analyzed is then centered inside a box, and its orientation is determined by application of programs that orient the particle on the basis of its icosahedral symmetry. In cryo-electron tomography, images are collected with the sample at different angles to the electron beam and combined computationally to reconstruct a three-dimensional structure. The advantage of this approach is that no assumptions about the symmetry of the structure are required. The parameters that define the orientation of the particle must be determined with a high degree of accuracy, for example, to within 1° for even a low-resolution reconstruction (~40 Å).
Once the orientations of a number of particles sufficient to represent all parts of the asymmetric unit have been determined, a low-resolution three-dimensional reconstruction is calculated from the initial set of two-dimensional projections by using computational methods.
This reconstruction is refined by including data from additional views (particles). The number of views required depends on the size of the particle and the resolution sought. The reconstruction is initially interpreted in terms of the external features of the virus particle. Various computational and computer graphics procedures have been developed to facilitate interpretation of internal features. Courtesy of B.V.V. Prasad, Baylor College of Medicine.
And is it not true that even the small step of a glimpse through the microscope reveals to us images that we should deem fantastic and over-imaginative if we were to see them somewhere accidentally, and lacked the sense to understand them.
Paul Klee, On Modern Art, translated by Paul Findlay (London, United Kingdom, 1948)
