Determination of virus structure by X-ray diffraction. This method requires crystallization of the sample of interest, such as a virus particle. The conditions under which a supersaturated solution of a protein or virus particles will form crystals suitable for X-ray diffraction, while retaining its native structure, cannot be predicted. Consequently, highly concentrated and purified virus particles are incubated under many different conditions, varying such parameters as concentrations of salt or metal ions, the presence of other polymers, and temperature. This empirical approach has been facilitated by the development of crystallization screening kits and robotic devices to set up crystallization trials. Nevertheless, it can be time-consuming, and success is not guaranteed. A virus crystal is composed of virus particles arranged in a well-ordered three-dimensional lattice. When the crystal is bombarded with a monochromatic X-ray beam traveling through the pinhole, each atom within the virus particle scatters the radiation. Interactions of the scattered rays with one another form a diffraction pattern that is recorded. Each spot contains information about the position and the identity of the atoms in the crystal. The locations and intensities of the spots are stored electronically. Determination of the three-dimensional structure of the virus from the diffraction pattern requires information that is lost in the X-ray diffraction experiment needed for calculating the positions of the atoms. The diffraction pattern collected from the crystal is now most usually interpreted by using the phases from the structure of a related molecule as a starting point, and subsequently applying computer algorithms to calculate the actual values of the phases. This method is known as molecular replacement. Once the phases are known, the intensities and spot positions from the diffraction pattern are used to calculate the locations of the atoms within the crystal.
Not all viruses can be examined directly by X-ray crystallography: some do not form suitable crystals, and the larger viruses lie beyond the power of the current procedures by which X-ray diffraction spots are converted into a structural model. However, their architectures can be determined by using a combination of structural methods. Individual viral proteins can be examined by X-ray crystallography and by multidimensional nuclear magnetic resonance techniques. The latter methods, which allow structural models to be constructed from knowledge of the distances between specific atoms in a polypeptide chain, can be applied to proteins in solution, a significant advantage.
High-resolution structures of individual proteins have been particularly important in deciphering mechanisms of attachment and entry of enveloped viruses. Even more valuable are methods in which high-resolution structures of individual viral proteins are combined with cryo-EM reconstructions of intact virus particles. For example, in difference imaging, the structures of individual proteins are in essence subtracted from the reconstruction of the particle to yield new structural insights (Fig. 4.5). This powerful approach has provided fascinating views of interactions of viral envelope proteins embedded in lipid bilayers and of internal surfaces and components of virus particles.
Atomic-resolution structures of individual proteins or domains can also be modeled into lower-resolution views (currently ~15 Å) obtained by small-angle X-ray scattering. This technique, which is applied to proteins in solution, provides information about the overall size and shape of flexible, asymmetric proteins, and has provided valuable information about viral proteins with multiple functional domains (see Chapter 10). It can also reveal dynamic properties, such as conformational change, a property shared with serial femto-second X-ray crystallography, in which as many as hundreds of thousands of images of small crystals are recorded in a very short time.
Building a Protective Coat
Regardless of their size and architectural sophistication, all virions contain at least one protein coat, the capsid or nucleocapsid, which encases and protects the nucleic acid genome (Table 4.2). As first pointed out by Francis Crick and James Watson in 1956, most virus particles appear to be rod shaped or spherical under the electron microscope. Because the coding capacities of viral genomes known at that time were very limited, these authors proposed that construction of capsids from a small number of subunits would minimize the genetic cost of encoding structural proteins. Such genetic economy dictates that capsids be built from identical copies of a small number of viral proteins with properties that permit regular and repetitive interactions among them. These protein molecules are arranged to provide maximal contact and noncovalent bonding among subunits and structural units. We now know that the capsids of even the largest viruses, with genomes of >1 Mbp, are also built from a small number of proteins. This property indicates that optimization of regular protein-protein interactions is the primary determinant of virus architecture. The repetition of such interactions among a limited number of proteins results in a regular structure, with symmetry that is determined by the spatial patterns of the interactions. The helical or icosahedral symmetry common to many viruses not only satisfies such protein limitations but also has considerable practical value (Box 4.2).
Figure 4.5 Difference mapping illustrated by a 6-Å-resolution reconstruction of adenovirus. (A) Comparison of α-helices of the penton base in the cryo-electron microscopic (cryo-EM) density (gray mesh) and the crystal structure of this protein bound to a fiber peptide (ribbon). The excellent agreement established that α-helices could be reliably discerned in the 6-Å cryo-EM reconstruction. (B) Portion of the cryo-EM difference map corresponding to the surface of one icosahedral face of the capsid. The crystal structures of the penton base (yellow) and the hexons (green, cyan, blue, and magenta at different positions) at appropriate resolution were docked within the cryo-EM density at 6-Å resolution. The cryo-EM density that does not correspond to these structural units (the difference map) is shown in red. At this resolution, the difference map revealed four trimeric structures located between neighboring hexons and three bundles of coiled-coiled α-helices. Both assemblies are now known to be formed by cement protein IX. Adapted from Saban SD et al. 2006. J Virol 80:12049–12059, with permission. Courtesy of Phoebe Stewart, Vanderbilt University Medical Center.
Helical Structures
The nucleocapsids of some enveloped animal viruses, as well as certain plant viruses and bacteriophages, are rod-like or filamentous structures with helical symmetry. Helical symmetry is described by the number of structural units per turn of the helix, the axial rise per unit, and the pitch of the helix (Fig. 4.6A). A characteristic feature of a helical structure is that any volume can be enclosed simply by varying the length of the helix. Such a structure is said to be open. In contrast, capsids with icosahedral symmetry (described below) are closed structures with fixed internal volume.
From a structural point of view, the best-understood helical nucleocapsid is that of tobacco mosaic virus, the very first virus to be identified. The virus particle comprises a single molecule of (+) strand RNA, about 6.4 kb in length, enclosed within a helical protein coat (Fig. 4.6B; see also Fig. 1.9). The coat is built from a single protein with an extended shape. Repetitive interactions among coat protein subunits form disks, which in turn assemble as a long, rod-like, right-handed helix. In the interior of the helix, each coat protein molecule binds three nucleotides of the RNA genome. The coat protein subunits therefore engage in identical interactions with one another and with the genome, allowing the construction of a large, stable structure from multiple copies of a single protein.
The particles of
