With the advent of improved methods for phasing, the rate-limiting step to determine the structure of a macromolecular using X-ray crystallography is most often crystallization. The goal of crystallization is to find the best combination of parameters to produce crystals of sufficient quality so they can be used to generate high resolution diffraction data. Such a search involves testing an almost limitless number of parameters, including protein concentration, type of buffer and its pH, nature and concentration of precipitant, temperature, and use of additives or ligands. Hence, """"""""crystallization space"""""""" is immense and if searched by low-throughput methods where crystal screens are pipetted manually, the process is severely limited by the amount of biological material that can be purified. The application of dispensing robots can dramatically change this situation because these can pipette protein in nanoliter volumes and therefore allow a much greater sampling of conditions with less biological material. Trays can also be set up considerably faster than manual methods. Although MUSC has a developing and thriving program in X-ray crystallography, including shared resources for protein production, our ability to produce crystal structures is severely hampered by the lack of crystallization robotics. This application aims to address that deficiency requesting the Phoenix RE nanoliter dispensing robot from Rigaku Inc. This will not only enhance the productivity of existing crystallography projects at MUSC, but will also stimulate new activity involving a wider group of investigators who aspire to use X-ray crystallography and have been unable to do so for lack of crystals. The broad benefits will be (a) the ability to tackle more challenging and potentially rewarding projects, including membrane proteins, (b) enhancement of the research environment at MUSC due to a greater emphasis on collaborative mechanistic studies of important biomedical problems that can be published in the higher impact journals, (c) increased competitiveness for NIH funding, (d) improved training environment for graduate students and post-docs and, (e) further development of X-ray crystallography and of structural biology in general through recruitment of new faculty.
This proposal requests funds to purchase crystallization robotics to enhance the X-ray crystallography facility that supports biomedical research at the Medical University of South Carolina. X-ray crystallography is a method of determining the structural details of macromolecules whose biological functions may be significant in human health and disease. Precise knowledge of the structure and function of such macromolecules contributes to better understanding of normal and abnormal biological processes and development of improved therapies to treat or cure major illnesses such as cancer, infectious diseases, inflammatory diseases and Alzheimer's disease.
