High-throughput, nanoliter-scale macromolecular crystallization at UCSD

描述（由申请人提供）： 通过X射线晶体学确定的生物大分子结构提供了无与伦比的功能见解，并使旨在治疗人类疾病的治疗方法的设计成为可能。蛋白质和其他生物大分子（如有序RNA）的结晶在许多情况下受到可用于结晶试验的纯化样品的量的限制。结晶仍然是一个经验过程，这意味着成功通常取决于尝试的不同条件的数量。因此，必须用有限数量的生物样品筛选大量沉淀物。这个问题的解决方案是结晶过程的机器人自动化，由于机器人建立纳升级液滴的可靠能力，它需要的样品比手动方法少10倍。机器人自动化还能够以高通量的方式尝试大量的条件。该提案旨在为加州大学圣地亚哥分校（UCSD）的NIH支持的研究人员获得集成结晶机器人技术。具体而言，我们建议购买一台用于配制结晶溶液的自动溶液混合机，一台将这些溶液自动分配到结晶托盘中的自动分配器，一台用于设置结晶液滴的机器人，以及一台用于检查和记录结晶试验结果的自动成像系统。该设备将被纳入现有的核心设施，即高分子晶体学设施，该设施目前设有X射线数据收集装置，并有一名经验丰富的工程师管理日常操作。设备将由现有的高分子晶体学设施委员会管理，日常操作将由现有的工程师管理。UCSD在X射线晶体学方面有着悠久的传统，这一承诺一直延续到今天。这一承诺体现在对化学与生物化学系和斯卡格斯药学院管理结晶机器人的人员的财政支持上。对仪器的长期支持将通过作为高分子晶体学设施一部分的用户费来补充。UCSD拥有大量的X射线晶体学家，他们的研究工作将通过定期使用结晶机器人技术而得到显着加强。重要的是，作为核心设施的一部分，该仪器还将使UCSD的研究人员能够将其研究计划转向结构测定，这些研究人员的专业知识不属于X射线晶体学。从X射线晶体学中获得的详细信息可能会使这些研究课题向治疗解决方案发展。简而言之，拟议的仪器是至关重要的结构生物学在加州大学圣地亚哥分校的校园范围内的成功。 公共卫生相关性：通过X射线晶体学确定的生物大分子结构提供了无与伦比的功能见解，并使旨在治疗人类疾病的治疗方法的设计成为可能。该提案旨在获得一个集成的机器人系统的结晶生物大分子在加州大学，圣地亚哥。该设备将使该校区的一大群生物医学科学家能够追求与人类疾病相关的基本问题，并将对蛋白质和从结构测定中获得的有序RNA的基本理解应用于治疗。

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.