Protein crystallography facilities for St Andrews

Lead Research Organisation: University of St Andrews
Department Name: Biology


The determination of protein structures using X-ray crystallography has been revolutionized over the last few years. Genetic engineering can produce large amounts of pure protein, and the exploration of the multifarious conditions under which a protein might form crystals has also been revolutionised through the use of robotics, such that 1000 different conditions can now be explored rapidly using only a milligram of pure protein. At St Andrews, we have established the Scottish Structural Proteomics Facility in collaboration with structural biologists, chemists and virologists at other institutions. This facility provides robotics to streamline the production of proteins and crystals for subsequent analysis using X-ray diffraction. We have now reached another bottleneck, namely the ability to effectively screen the crystals to determine their diffraction quality. At the moment, for the small crystals produced by our robotics, we have to travel to an intense X-ray source in Grenoble. These trips need to be planned weeks in advance, and involve a considerable amount of travelling. Our research proposal is requesting funds for enahanced X-ray diffraction equipment in collaboration with arguably the world's foremost supplier of X-ray equipment for single crystal analysis. We are proposing to acquire a high intensity X-ray generator that will produce 1.5x the intensity of our current system. This should allow us to screen far more small crystals in St Andrews. Another means of improving observable diffraction is to dehydrate the protein crystals. The majority of protein crystals have large solvent channels that occupy 30 to 70%, and in some cases up to 90%, of the volume of the crystal. This makes the crystals fragile, as there are few interactions between the protein molecules holding the crystal together. There have been many reports of dehydration improving the ordering of molecules in a crystal with a subsequent improvement in diffraction quality. Such reports have often been hit-and-miss and not very reproducible. We are asking for funds in this proposal to purchase a 'free mounting system', or FMS. This device allows fine control of the state of hydration in a crystal whilst simultaneously observing the changes in the diffraction pattern. This simple methodology will allow us to optimise the diffraction quality of our crystals / in some reports, dehydration has had dramatic effects, in some cases going from no diffraction to the observation of diffraction to a resolution that would enable a protein structure to be determined. Another major advantage of the FMS method, is in freezing crystals. It is routine for protein crystals to be flash-frozen to 100K for data collection. The reason is to limit radiation damage. In order to freeze a crystal, however, a suitable cryoprotectant has to be found, that is something that will stop the water in the crystal forming ice upon freezing, as ice produces its own intense diffraction pattern that can disturb the protein's diffraction pattern. Finding such a cryoprotectant can be difficult, however the FMS system allows simple cryoprotection through the use of a drop of oil being placed in the otherwise 'dry' crystal. Therefore, we can very easily cryoprotect a crystal in its optimum diffracting state. Finally, the process of structure determination requires the determination of 'phases' / this is information lost in the diffraction process that needs to be regained through other means. One method is to detect very small changes in the intensities of the diffraction pattern that arise from absorption of the X-rays by certain atoms at certain X-ray wavelengths. Most proteins contain sulfur atoms which absorb X-rays generated using a chromium anode. We are proposing to develop such a chromium phasing system in-house at St Andrews that will allow us to determine certain protein structures without the need to travel to synchrotrons.

Technical Summary

Structural proteomics has revolutionised the production and crystallisation of proteins aided by robotics. St Andrews is home to the Scottish Structural Proteomics Facility (SSPF), funded by SFC and the BBSRC's SPORT initiative, and provides a pipeline for crystal production. There are currently over 140 proteins on the SSPF target list, with many other proteins being studied through grants held by the applicants in scientific areas such as DNA repair, carbohydrate biosynthesis and transport, and the structural biology of paramyxoviruses. Other researchers from across Scotland are also putting proteins into the pipeline for structural analysis. A current bottleneck is the ability to screen diffraction from small crystals in-house, which currently needs to be undertaken at the synchrotron with the associated planning and delays. This proposal is to develop our in-house X-ray facilities in collaboration with Rigaku/MSC to provide, (1) a high flux rotating anode and CCD detector to enable screening of small crystals, (2) a Proteros free mounting system (FMS) to allow optimisation of diffraction through dehydration, and (3) a chromium anode and associated optics to provide an in-house phasing system. The FMS system has the potential to discover the optimum hydration state giving maximum diffraction, and allows simple cryoprotection using oil. A crystal can then be flash-frozen in its optimal state for further analysis. We see this as having huge potential for extracting the most structural information from our crystals, and to avoid expensive screening at a synchrotron. Most of the proteins we are studying have sufficient sulphurs to theoretically provide an anomalous signal sufficient to phase the protein structure, providing data is collected at the chromium edge. In addition, any calciums, or seleniums would also provide a significant anomalous signal to potentially phase the structure.


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