Understanding virus structure is key to understanding fundamental processes in the viral life cycle, such as capsid assembly and disassembly, receptor binding, and antibody neutralization. Two important methods used to image virus structure are X-ray crystallography and cryo-electron microscopy (cryo-EM). X-ray crystallography exploits the diffracting power of large (~0.2 mm) three-dimensional (3D) protein crystals irradiated by an X-ray beam. It requires large amounts of sample that have to be induced to form the large ordered arrays, and produces diffraction patterns that later have to be interpreted to yield the structure. Cryo-EM does not require sample crystallization, and so smaller

From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ

amounts of sample can be used. Two-dimensional projections of the biological object in solution are recorded and later combined using image processing procedures to obtain a 3D reconstruction.

X-ray crystallography gives the most detailed picture of viral capsid structure, as it can reach atomic resolution (1). About 45 unique high-resolution viral structures solved by crystallographic techniques are currently available at the Protein Databank (PDB) (2). Crystallographic techniques are increasingly able to deal with very large macromolecular complexes, as shown by the largest virus structures solved so far: the bluetongue virus core (700-Â diameter) (3), and bacteriophage HK97 (660-Â) (4). However, many viruses present particularly challenging problems for crystallography, not only because of their large size and complex capsid composition, but also because they are too difficult to obtain in the amounts required for crystallization, or because the presence of flexible capsid components hinders production of highly ordered crystals.

Individual viral components are generally more amenable to overexpression and crystallization. Their atomic structures give us a very detailed picture of each capsid component but do not provide information on their mutual relation in the virion. On the other hand, nowadays one can routinely obtain 3D virus capsid maps from cryo-EM (5) at medium resolution, i.e., 12-15 Â, and achieving subnanometer resolution is not that uncommon (6-11). Thus, with cryo-EM, a picture of the complete virion is obtained, although the lower resolution makes it a blurry picture compared with crystallography maps.

Multiresolution imaging (Fig. 1) consists in cleverly combining these two sources of 3D information: atomic structure of individual components and medium resolution of whole viral particles. The known crystal structures are fitted to the cryo-EM density, resulting in a 3D model in which the atomic structures are placed in the cryo-EM map with a precision of about 4 Â. The resultant description of the virus is called a quasi-atomic or pseudo-atomic model. This model provides information at higher resolution than the cryo-EM map by itself and offers a more complete picture than the structures of the isolated components. Subsequent analysis of the quasi-atomic model reveals characteristics of the intercapsomere relationships, as well as those between protein shells and other virion components like membranes or DNA (10,12,13). Quasi-atomic models also allow modeling of conformational changes related to capsid maturation (14,15) or assembly (16). They can also be used for determining the phases of X-ray diffraction data for those viruses for which crystals are available (3,4), thus saving the effort needed for isomorphous replacement or Se-methionine derivatization. Difference maps calculated between the quasi-atomic model (containing only those capsid components whose atomic structure is available) and the cryo-EM reconstruction reveal the molecular envelope of those components whose high-resolution structure is unknown (17,18).

Fig. 1. Fundamentals of multiresolution imaging, illustrated with an example from the macroscopic world.

Depending on the cryo-EM map resolution and the biochemical knowledge available, these envelopes can be used to model the missing structures.

In this chapter, procedures for the calculation of a quasi-atomic model, a difference map, and their analysis are described. Both X-ray crystallography and cryo-EM are highly specialized techniques for which there is an extensive bibliography (see, for example, refs. 1,19-21), whose description goes beyond the scope of this chapter. Fortunately for the scientific community, the 3D models obtained by both methods are publicly available in specialized databases. The PDB at (2) contains atomic coordinates for about 20,000 biological macromolecules; the Macromolecular Structure Database (MSD) at (22) has recently started to store 3D electron microscopy (3DEM) maps. Thus, for the purposes of this chapter, it will be assumed that the two pieces of 3D data to be combined (cryo-EM map of a virus capsid and atomic coordinates of a capsid protein) are available for downloading from the databases. As the computational tools used in multiresolution imaging are available through crystallographic, image processing, and visualization software usually running under UNIX operating systems, some knowledge of computing, in particular of the unix environment (23), will also be assumed.

Although the technique described here has hitherto been applied to icosahe-dral capsids, cryo-EM holds the promise of obtaining 3D maps of nonsymmet-ric viruses by way of tomographic techniques (24). Thus, multiresolution imaging is likely to play a significant role in the study of large pleomorphous viruses in the near future.

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