This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In this new, single experiment proposal, beam time is requested to screen crystals of E. coli transhydrogenase (TH) for diffraction and to collect native data. TH is a 206 kDa integral membrane protein and a conformationally coupled protein pump essential for the maintenance of NADPH levels in mitochondria. TH mutations are linked to Type 2 diabetes. Experiments will entail evaluating crystal forms prepared from multiple TH constructs and in the presence of various detergents while screening cryoprotection conditions. Once moderate diffraction is established, native data will be collected for molecular replacement calculations, using known crystal structures of TH soluble domains. If these are unsuccessful, empirical methods of phase determination will be employed.
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