Viruses crystallization

Examples. 2D SAXS/WAXS experiments on highly anisotropic polymer materials during melting and crystallization can be used to visualize and understand the evolution of nanostructure [56,57], Transformations of biopolymers in solution, e.g., virus crystallization can be studied in situ [58], It is possible to study solidification mechanisms of spider silk [59], or the self-assembly of micelles on a time-scale of milliseconds [60],... 

At present, the lower size limit for virus crystals suitable for room temperature data collection at a... 

Certain factors are likely to influence future analyses of more complex viruses. Crystal stability is governed by packing interactions and, as can be seen from Fig. 16.4, is, to a first approximation, inversely proportional to the square of the virus radius, presumably underl)dng the problems with crystal stability for analyses such as that of PRDl. Even assuming that well-ordered, stable crystals can be formed, technical considerations will place an upper limit on the unit cell size from which useful data can be collected. Nevertheless, with some improvements in beam and detector technology, we expect that data collection from cells up to 2000 A should be feasible for even a primitive unit cell. 

Further reductions in exposure time and hence radiation damage in virus crystallography may accrue from the use of white beam (modified) Laue methods preliminary work on this is in progress (Bloomer and Helliwell (1985), unpublished at the SRS and Rossmann et al. at Cornell unpublished (1986)). Data collection on some virus crystals is virtually impossible in the home laboratory. 

Hajdu, J., and Johnson, L. N. Progress with Laue diffraction studies on proteins and virus crystals. Biochemistry 29, 1669-1678 (1990). 

Diffraction on small crystals with large unit cells (e.g. virus crystals or proteins) or small polymer crystals (e.g. Polyethylene). 

Several plant viruses have been studied using X-ray crystallography and conventional X-ray sources. These are, in no particular order, TBSV (tomato bushy stunt virus), SBMV (southern bean mosaic virus) and STN V (satellite tobacco necrosis virus) - all spherical viruses - and TMV (tobacco mosaic virus) - a cylindrical virus. These virus crystals diffract relatively well and are reasonably stable to radiation. 

One of the first virus crystal data collection runs took place on DCI-1 (Usha et al 1984, see section 10.5.1). 

Ten years ago it would have seemed inconceivable that the structure of viruses would be solved using data collected at short wavelengths like 0.9 A. After all in the home laboratory MoKa (0.71 A) is reserved solely for unit cells up to =20A and CuKa (1.54 A) for macromolecules. Yet 0.9 A data collection on today s bending magnets and wigglers is commonplace. It is not unreasonable to consider data collection from radiation sensitive samples like virus crystals, in future, using an undulator harmonic at 0.33 A with an IP placed 0.5-1.0m from the crystal. 

Calcium binding to tomato bushy stunt virus has been studied using the synchrotron Laue method (Hajdu et al 1989). The non-crystallo-graphic symmetry of the virus crystal aided the clarity of the final map in showing up the calcium sites. A novel spatial deconvolution algorithm was used to obtain intensity estimates of the individual diffraction spots (Shrive, Clifton, Hajdu and Greenhough 1990). 

These preliminary studies culminated in a wealth of activity at SR sources devoted to virus crystal data collection both for structure solving and also drug binding-studies. Table 10.10 gathers the various references to this type of work up to 1990. We now give further details on the rhinovirus work which was done primarily on CHESS after the initial work at Hamburg and Daresbury. This case study will then be followed by a description of the FMDV work done at Daresbury using the SRS. 

The containment and security handling of the FMDV virus crystals was a particularly important aspect of this project. Transport of the virus crystals to the synchrotron from the south of England was approved months in advance by the Chief Veterinary Officer in the United Kingdom. Each trip could only be 24 hr in duration and a Safety Officer had to accompany the scientists on their trips. Perhaps the most important aspect of safety and security was that FMDV is unstable below pH 7. Hence, citric acid swabs were placed on the oscillation camera under the glass capillary holding an FMDV crystal hence, in case of accidental breakage of a capillary the FMDV crystal would be neutralised by the citric acid (actually the breakage of a capillary never did happen). The lability of the virus to acid was explained qualitatively once the struc-... 

The exploitation of this radiation, particularly the brilliance and use of short A s, has made virus crystal data collection routine from difficult samples although it is at present necessary to use hundreds of crystals in the gathering of just one data set. Maybe the use of ultra-short wavelength beams ( =0.33 A) from a harmonic of an undulator could be harnessed to improve the lifetime of one such sample sufficiently to give a complete data set. Much larger macromolecular assemblies are currently under study, such as the ribosome, which possess little or no symmetry (unlike viruses) and are therefore more difficult to solve. 

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