A guide to the crystallographic analysis of icosahedral viruses

Abstract

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.

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“…256−258 To prepare and purify the viruses from the extracellular/ tissue culture media, purification kits are readily accessible from the commercial sources. 259 During the preparation steps, to maintain the icosahedral symmetry of the virus, the handling procedures should be performed as gentle as possible. Following the purification stage, crystallization is required to bring the aqueous protein solution to supersaturation.…”

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“…[200][201][202] Kits are freely available commercially for the preparation and purication of viruses from the extracellular matrix. 203 Handling should be performed as gently as possible to uphold the icosahedral symmetry of the virus. Consequently, crystallization is mandatory to achieve the supersaturation of the aqueous protein solution.…”

“…Batch crystallization, dialysis, liquid-liquid diffusion, and vapor diffusion are the major four steps involved in growing the crystals of virus samples. 203 Furthermore, the crystallographic data is collected at 100 K, i.e., cryogenic temperature, to prevent secondary radiation damage to the virus specimen and to improve the resolution. However, growing high-quality virus crystal via X-ray crystallography is quite challenging and the currently used methods are slow and centered on trial and errors.…”

“…Batch crystallization, dialysis, liquid–liquid diffusion, and vapor diffusion are the major four steps involved in growing the crystals of virus samples. 203 Furthermore, the crystallographic data is collected at 100 K, i.e. , cryogenic temperature, to prevent secondary radiation damage to the virus specimen and to improve the resolution.…”

The solvent component of macromolecular crystals

“…The various density-modification and phase-improvement methods have been reviewed, for example, in Podjarny et al (1996), Rupp (2009) and Cowtan (2010) and in additional references provided below. The role and application of density modification and density averaging in the ab initio phasing of virus crystal structures has been reviewed separately by McPherson & Larson (2015).…”