The field of structural genomics aims to characterize the structure-function relationships of all proteins by the experimental elucidation of the three dimensional atomic structures of representative members of protein families combined with the accurate computation of models for closely sequence related proteins. The path from gene to structure for eukaryotic proteins is riddled with challenges including the error-prone isolation of gene clones from cDNA libraries, poor yields in protein expression and purification, and time lost between the set-up of crystallization experiments and X-ray diffraction analysis of putative crystals. To address these challenges, we propose the development of integrated instruments, methods and software that will substantially increase the success rate while reducing the time, effort, materials, and cost required for protein structure determination. Among these new developments will be: (1) Rosetta-guided design and gene synthesis of expression optimized DNA sequences encoding proteins that are guided towards more facile purification and improved crystallogenesis; (2) nano-volume microfluidic crystallization devices that embody the major protein crystallization methods allowing extensive crystallization screening of precious protein samples and their complexes; and (3) a tunable in-house MAD X-ray source which will allow in situ room-temperature crystal X-ray diffraction screening and final anomalous dispersion diffraction data collection for rapid structure determination. Starting in year 3, these new technologies will be validated through the planned structure determination and public release of over 550 eukaryotic protein targets belonging to families with high relevance to cancer biology and therapy, including kinases, transcription factors, and metabolic enzymes. The dissemination of the proposed instrumentation, methods and software will allow researchers to pursue protein X-ray crystal structures at or below a total cost of $10,000 per eukaryotic protein structure.
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