Understanding the molecular mechanisms of biological systems requires both functional and structural information. Structures of biomolecules are typically determined by one of several methods, including x-ray crystallography, NMR, or EM. Because crystallography can afford some of the highest-resolution views of a particular system, and is not limited by the particle size, the use of this method is widespread, and has contributed the greatest numbers of structures to the field . An innate challenge to x-ray crystallographic investigations is the requirement for high-quality crystals. Crystallization relies on the identification of chemical conditions that can support growth, which conditions are unique to a given protein, protein complex, or protein-nucleic acid complex. Identifying crystallization conditions involves screening a target against hundreds or thousands of different chemistries, typically using 24 or 96-well trays. Ideally, trays are incubated in a vibration-free environment to aid crystal formation, and are incubated at varied temperatures (usually 4?C and 18?C) to broaden the depth of the search space. Trays are viewed by light microscopy at regular intervals to search for the presence of crystals, which often are small and/or irregular. The conditions under which these initial """"""""hits"""""""" are obtained are then cross-correlated to look for trends, and optimized to improve crystal quality for diffraction studies. The Rigaku Minstrel imaging system requested in this proposal offers benefits that will transform how crystallization experiments are conducted at UC Berkeley. The instrument incubates crystal trays at a given temperature and automatically takes digital images of each drop on a scheduled basis using both visible and UV light. Scheduled screening allows for systematic coverage of crystallization conditions over time, greatly increasing the likelihood of finding successful hits. UV illumination can uncover crystals hiding within heavy precipitates, allowing users to distinguish micro-crystals from granular precipitates. UV visualization further discriminates between salt and protein or nucleic acid crystals, ensuring that precious x-ray beam time at synchrotron sources is used efficiently on biological samples. Trays can be incubated at 4?C, a regime inaccessible to us due to a paucity of cold-room space, thereby opening up an entirely new realm of low-temperature conditions for screening. In addition, the Minstrel system will enable users to more rapidly and thoroughly cover crystallization space, speeding structural investigations into a diverse number of highly significant biological systems.
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