Data collection virus

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the use of white beam (modified) Laue methods preliminary work on this is in progress (Bloomer and Helliwell (1985), unpublished at the SRS and Rossmann et al. at Cornell unpublished (1986)).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. 

It took the short time of one year or so to solve the structure of rhinovirus which causes the common cold. This relied on two major advances in methods. The first was the use of synchrotron radiation in data collection. Nearly a million reflections were collected on the protein crystallography facility at the Cornell Synchrotron source in a matter of days. This conveyed a speed advantage over data collection on a conventional source and also ameliorated an otherwise impossible problem of radiation damage when long exposure times were used. The far greater rate of radiation damage in the X-ray beam in relation to plant viruses is symptomatic of an inherently less stable protein capsid and the absence of quasi-symmetry. The capsid consists of 60 copies each of four proteins and the virus with about 30 % RNA has a total molecular weight of about 8.5 million. 

An immediate 1000-fold dilution of blood resulted in stabilization of the clinical samples for up to 48 h. A final clinical protocol was established which included the 1000-fold dilution of blood into DM EM medium plus antibiotics, immediate cold shipment, and assay within 24 h of collection. Using the EPD infectivity assay, the limit of detection for infectious virus in the original blood sample was approximately 1000 IFU mL . Clinical data collected from patients receiving Ad5FGF-4 showed increasing detectable infectious virus depen-... 

Photographic film has been and is used for a variety of macromolecular data collection. It is being replaced by use of other detectors. However, its use for very dense diffraction patterns from viruses and/or Laue patterns is likely to continue. 

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

A promising application of multipole wigglers (at 0.5 A) or undulators (i.e. a harmonic at —0.3 A) is to solve the problem of data collection from very radiation sensitive crystals. For example, in virus data collection several hundred crystals are needed for structure determination. Of course, once the basic structure is known, a greatly reduced amount of data and far fewer crystals are needed in drug binding (difference Fourier) studies. 

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. 

The combination of all the advantages of SR is especially needed in virus and ribosome crystallography where the unit cells are very large. Data collection from very large unit cell constant crystals benefits from... 

The extreme radiation sensitivity of rhinovirus crystals can be contrasted with poliovirus. Poliovirus was solved using data collected on a conventional X-ray source (Hogle, Chow and Filman 1985) as were various plant viruses much earlier (see Harrison (1978) for a review). 

In the early days of virus crystallography at the synchrotron, the largest lattice constant dealt with was that of cow pea mosaic virus (Usha et al 1984). This virus crystallised in space group P6j22 (or its enantiomorph) with a=451 A and c=1038A. Data collected at LURE (figure 10.18) were analysed and the results showed that quality data... 

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 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. 

The synchrotron should not be seen as a replacement for facilities in the home laboratory but as a means for meeting technically challenging data collection problems. Of course, in the absence of any home X-ray facilities, the central facility can be used but this is not terribly efficient because of the necessity of long-term scheduling of many users. Hence, characterisation of samples (e.g. of heavy atom derivatives) should be done at home unless the project is entirely reliant on the SR source in this category are many virus studies and ribosome crystallography as well as small crystal projects. 

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