PROJECT SUMMARY Structural biology is a field with a goal to delineate the physical architecture of biological macromolecules; a primary structural method employed in this endeavor is macromolecular X- ray crystallography (MX). MX methods account for nearly 90% of the greater than 165,000 structures deposited to the Protein Data Bank, but a primary challenge to successful MX structure determination is finding conditions in which a macromolecule will crystallize. To address this obstacle, an arsenal of chemical cocktail screens and high-throughput screening methods, coupled with specialized imaging, have been brought to bear on the problem of determining the conditions in which a macromolecular target will form a crystal that is of sufficient quality to be amenable to X-ray diffraction structure determination. This proposal focuses on continuing and extending the capabilities of a central resource providing critical high-throughput crystallization screening to the scientific community, the High-Throughput Crystallization Screening Center (HTX) at Hauptman-Woodward Medical Research Institute. The HTX Center has been in operation for two decades, providing a unique national crystallization resource specifically geared to help overcome the obstacle of coaxing a macromolecule to crystallize. The HTX Center provides a high-throughput screening format designed to minimize sample requirements via use of small volume liquid-handling robotics, a unique experimental set-up, and state-of-the-art imaging for screening. Notably, the technologies, instruments, and expertise available at the HTX Center are not widely available. This proposal focuses on extending the capabilities of the HTX Center by developing an expanded repertoire of experimental screening options and improved image analysis and processing, while performing instrumentation upgrades to maintain the HTX Center as a premier resource for crystallization screening for the scientific user community. We propose developing and implementing the necessary computational infrastructure and software to accommodate new screening plate definitions and optimization of successful crystallization hits, and improved user interfaces to maximize the information accessibility from screening experiment outcomes. A major goal of this proposal is to increase access to the state-of-the-art crystallization screening instrumentation and expertise to researchers from a wide array of laboratories in academic, non-profit and government institutes, with an objective to ensure access that enables a broad range of biomedically important research.