Protein X-ray Crystallographic Infrastructure for the Edinburgh Centre for Translational and Chemical Biology

The information that is being generated from DNA sequencing is now having a major impact on the sorts of scientific questions being asked in the fields of chemistry biology and medicine. Recent examples are the discoveries linking differences in gene sequences to a number of diseases including certain types of cancers, diabetes, bipolar disorder and rheumatoid arthritis. These differences in gene sequence are translated into differences in protein structure and it is these small differences in protein structure (and therefore function) that result in differences in metabolism or signalling in the cell which are the root causes of the various disease states. Protein X-ray crystallography is a very efficient method that can be used to determine the 3D structure of these proteins and in Edinburgh there is an urgent need to upgrade our equipment to generate more results in this area. A significant amount of basic research in medicine, biology and chemistry is being driven by the new information from gene sequences. A common strand in this research is the expression and purification of the proteins derived from these key genes. At Edinburgh University we have founded a new Centre for Translational and Chemical Biology with central resources to facilitate the efficient production and purification of proteins. Over 50 independent research groups working on a wide range of medically and biologically important problems have pooled resources to speed up the process of producing and characterising proteins. Obtaining an accurate picture of the detailed structure of the protein (or protein complex) of interest provides a wealth of biologically important information that leads amongst other things, to the more efficient design of drugs. Thus, a clear picture of the molecular structure is an important research goal for every protein-based project. In order to examine the increased numbers of proteins generated at Edinburgh, we need another X-ray generator. This need is particularly urgent as one of our two generators has now failed irreparably. The data collection equipment (the image plate system, a kind of reusable X-ray photographic plate) is however still fully functioning and could be used on another X-ray source. We would like to buy a generator that can produce a more powerful beam so that we can collect better data from smaller crystals.

We request a Microfocus rotating anode X-Ray generator and associated optics system. New protein crystals: Increasingly we are working with smaller crystals and find that they show little or no diffraction on our current rotating anode source, however if we screen these at synchrotron sources we can frequently obtain useful diffraction. Our MAR detector systems are capable of recording weaker diffraction, however the current source (Bruker-Nonius FR591 with multilayer optics) does not provide the necessary flux at the sample to give recordable diffraction patterns. In order to make the best use of synchrotron visits we need an extensive screening effort to optimise the crystallisation and freezing conditions. Protein/Ligand complexes. We have a large group involved in protein ligand binding simulation and have also pioneered variable concentration soaking methods as a way of studying binding modes and kinetics. Extensive crystal engineering involving multiple soaking in/out of ligands inevitably damages crystals and reduces the recordable diffraction for a given flux. In real terms this means that a crystal system which is initially amenable to data collection on our current equipment eventually becomes usable only at synchrotron sources. In choosing the requested equipment we have also considered sealed tube microfocus systems from Oxford diffraction and Xenocs and while these bring savings in running costs we feel that they do not produce the increase in performance necessary for our projects. We have also considered the possibility of an upgrade to a CCD detector however since we have a reliable high performance detector available for this installation (A Mar Research MAR345) we do not believe the extra cost and increased running costs can be justified. The purchase of a microfocus rotating anode represents a cost effective method to increase the screening and data collection capability within the Centre for Translational and Chemical Biology.