Determining the structure of an icosahedral virus crystal by X-ray diffraction follows very much the same course as conventional protein crystallography. The major differences arise from the relatively large sizes of the particles, which significantly affect the data collection process, data processing and management, and later, the refinement of a model. Most of the other differences are due to the high 5 3 2 point group symmetry of icosahedral viruses. This alters dramatically the means by which initial phases are obtained by molecular substitution, extended to higher resolution by electron density averaging and density modification, and the refinement of the structure in the light of high non-crystallographic symmetry. In this review, we attempt to lead the investigator through the various steps involved in solving the structure of a virus crystal. These steps include the purification of viruses, their crystallization, the recording of X-ray diffraction data, and its reduction to structure amplitudes. It further addresses the problems attending phase determination and ultimately the refinement of a model. Finally, we describe the unique properties of virus crystals and the factors that influence their physical and diffraction properties.

Detailed structures are now available for three plant satellite viruses, satellite tobacco necrosis virus (STNV), satellite tobacco mosaic virus (STMV), and satellite panicum mosaic virus (SPMV). It is, therefore, possible to compare the tertiary structure of viral protein subunits, their quaternary interactions, and the interactions of protein subunits with the RNA genome. This analysis indicates that, in spite of common function and preservation of a "jelly-roll" motif in the protein monomer, the three viruses are remarkably different. The differences include the arrangement of secondary structural elements, interactions of adjacent subunits, and the disposition of subunits relative to icosahedral symmetry axes. In each of the three viruses, however, the narrow end of the jelly roll forms fivefold contacts. The fivefold protein interactions are organized about a Ca2+ ion for STNV, an anion for STMV, and, apparently, neither of these for SPMV. Low-resolution neutron diffraction studies using H2O/D2O solvent contrast variation revealed the general location of the RNA genome within the STNV. In the case of SPMV, regions of electron density on the interior of the capsid could be assigned to RNA, although it was not possible to model the nucleic acid. Only for STMV was nucleic acid visible in election density maps, and this was manifested as double-helical RNA segments associated with each coat protein dimer. The observations presented here provide no support for any common evolutionary relationship.

Three new crystal forms of satellite panicum mosaic virus (SPMV) were grown and their structures solved from X-ray diffraction data using molecular replacement techniques. The crystals were grown under conditions of pH and ionic strength that were appreciably different then those used for the original structure determination. In rhombohedral crystals grown at pH 8.5 and low ionic strength PEG 3350 solutions, Fourier syntheses revealed segments, ten amino acid residues long, of amino-terminal polypeptides not previously seen, as well as masses of electron density within concavities on the interior of the capsid, which appeared in the neighborhoods of icosahedral five- and threefold axes. The densities were compatible with secondary structural domains of RNA, and they included a segment of double helical RNA of about four to five base pairs oriented, at least approximately, along the fivefold axes. The distribution of RNA observed for SPMV appears to be distinctly different than the encapsidated nucleic acid conformation previously suggested for another satellite virus, satellite tobacco mosaic virus. This study further shows that analysis of viruses in crystals grown under different chemical conditions may reveal additional information regarding the structure of encapsidated RNA.

Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze–thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-2 and 101-2 radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses.

Xylanase from the bacterial plant pathogen Erwinia chrysanthemi (E.C. 3.2.1.8), expressed in E. coli, has been crystallized for X-ray diffraction analysis both in the presence and the absence of its polymeric substrate 4-O-methyl glucuronoxylan. In all cases it was found that the quality, time of appearance, and reproducibility of both the native and complex crystals were significantly enhanced by heating of the protein to 323 K prior to dispensing the crystallization trials. Crystals of the native protein are ideal for X-ray diffraction analysis, producing Bragg reflections beyond 1.5 A resolution with virtually no degradation with time. The native crystals are in space group P2(1), with a = 39.33, b = 49.46, c = 90.85 A and beta = 101.58 degrees. Other polymorphs have also been obtained and their cell parameters determined. Crystallization of the enzyme in the presence of polymeric substrate yields two distinctly different crystals at different concentrations of the xylan. These are thought to be complexes of the protein with stable products of the enzymatic reaction. A similar result had been obtained previously with pancreatic alpha-amylase and its substrate glycogen.