High-throughput, nanoliter-scale macromolecular crystallization at UCSD

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The structures of biological macromolecules determined by X-ray crystallography provide unparalleled functional insights and make possible the design of therapeutics aimed at treating human illnesses. Crystallization of proteins and other biological macromolecules, such as ordered RNAs, is limited in many cases by the quantity of purified sample available for crystallization trials. Crystallization remains an empirical process, meaning that success generally depends on the number of different conditions tried. Thus, a large number of precipitants must be screened with a limiting quantity of biological sample. The solution to this problem is robotic automation of the crystallization process, which requires ~10-fold less sample than manual means due to the reliable ability of robots to set up nanoliter-scale drops. Robotic automation also enables a vast number of conditions to be tried in high-throughput fashion. This proposal seeks to acquire integrated crystallization robotics for NIH-supported investigators at the University of California, San Diego (UCSD). Specifically, we propose to acquire an automated solution mixing machine for formulating crystallization solutions, an automated dispenser of these solutions into crystallization trays, robotics to set up the crystallization drop, and an automated imaging system for examining and recording the outcome of crystallization trials. The equipment will be incorporated into an existing core facility, the Macromolecular Crystallography Facility, which currently houses an X-ray data collection setup and has an experienced staff engineer who manages daily operations. The equipment will be administered by the existing Macromolecular Crystallography Facility Committee and daily operations will be managed by the existing staff engineer. UCSD has a long tradition of X-ray crystallography, and this commitment continues today. This commitment is evidenced by financial support for personnel to manage the crystallization robotics from the Department of Chemistry & Biochemistry and the Skaggs School of Pharmacy and Pharmaceutical Sciences. The long-term support of the instrumentation will come on a recharge basis through user fees as part of the Macromolecular Crystallography Facility. UCSD has a substantial number of X-ray crystallographers whose research efforts would be markedly enhanced by regular access to crystallization robotics. Significantly, the instrumentation, as part of a core facility, will also enable investigators at UCSD whose expertise is outside of X-ray crystallography to move their research programs towards structure determination. The detailed level of information gained from X-ray crystallography is likely to permit growth of these research topics towards therapeutic solutions. In short, the proposed instrumentation is crucial to the campus-wide success of structural biology at UCSD. PUBLIC HEALTH RELEVANCE: The structures of biological macromolecules determined by X-ray crystallography provide unparalleled functional insights and make possible the design of therapeutics aimed at treating human illnesses. This proposal seeks to obtain an integrated robotic system for the crystallization of biological macromolecules at the University of California, San Diego. This equipment will enable a large group of biomedical scientists at this campus to pursue fundamental questions related to human diseases, and to apply the basic understanding of proteins and ordered RNAs gained from structure determination to therapeutics.