Tools for High-Throughput Protein Crystallization and Structure Determination

高通量蛋白质结晶和结构测定工具

• 批准号: 8294602
• 负责人: Robert E. Thorne
• 金额: $ 11.58万
• 依托单位: MITEGEN, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-23 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294602
• 关键词: Acceleration; Affect; Applied Research; Area; Asia; Biotechnology; Calibration; Chemicals; Collaborations; Commit; Complex; Crystallization; Crystallography; Data Collection; Development; Drops; Europe; Evaluation; Film; Genes; Government; Growth; Harvest; Image; Image Analysis; Imaging technology; In Situ; Injection of therapeutic agent; Kinetics; Knowledge; Life; Liquid substance; Marketing; Mass Spectrum Analysis; Measurement; Membrane Proteins; Methods; Molds; Molecular; Molecular Medicine; Molecular Structure; Nucleic Acids; Optics; Organized by Structure Protein; Outcome; Pattern; Performance; Persons; Pharmacologic Substance; Phase; Positioning Attribute; Process; Production; Productivity; Property; Protein Structure Initiative; Proteins; Resources; Retrieval; Roentgen Rays; Sampling; Screening procedure; Series; Shapes; Shipping; Ships; Small Business Technology Transfer Research; Source; Speed; Spottings; Structure; Surface; Synchrotrons; Technology; Testing; United States National Institutes of Health; Universities; Virus; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; commercialization; cost; design; drug discovery; improved; insight; meetings; new technology; novel strategies; programs; protein expression; protein structure; prototype; response; structural genomics; success; three dimensional structure; tool

DESCRIPTION (provided by applicant): Our understanding of the molecular mechanisms of life is increasingly based upon our knowledge of the three dimensional structure of proteins, nucleic acids, viruses and biomolecular complexes. Structure provides insight into function (or malfunction), and provides a starting point for modern drug discovery and molecular medicine. Biomolecular structures are most often determined using X-ray crystallography of crystallized biomolecules. Over the last decade, high-throughput methods have been introduced that have automated many aspects of protein expression, purification, crystallization and crystallography, and that have lowered the cost per structure determined. However, the growth and harvesting of protein crystals remains a major bottleneck in the pipeline from gene to three-dimensional molecular structure and from structure to pharmaceutical therapy. This Phase II STTR proposal is focused on developing and commercializing improved methods for conventional and high-throughput crystallization and for crystal harvesting and X-ray data collection. An examination at Cornell University of how liquid contact lines interact with surfaces has led to a simple technology for precisely defining the positions of dispensed liquid drops and firmly holding them to those positions, regardless of their chemical composition. This technology forms the basis for a new approach to protein crystallization plates that eliminates the liquid-confining wells of conventional plates. These new plates promise to provide precise control over drop position and shape, resulting in more reproducible crystallization kinetics and simplified image analysis. They will allow hanging and sitting drop growth of soluble and membrane proteins using a single plate, and in situ optical, UV and X-ray analysis with low background. They will meet a critical need for plates optimized for easy X-ray examination of screening and crystallization outcomes and also allow in situ structure determination. They will be compatible with all existing drop dispensing and plate handling hardware, lowering barriers to market entry. This project will continue the scientific and commercial development of these plates and explore other biomedical applications of drop pinning technology. In Phase I we successfully developed and commercialized several new tools for crystal retrieval and X-ray data collection. We will continue this development in Phase II, focusing on improved tools for microcrystallography, for automated sample mounting, and for X-ray beam alignment and energy measurement. Together, these technologies should have significant impact on the productivity of high- throughput structural genomics and drug discovery efforts.

描述（由申请人提供）：我们对生命分子机制的理解越来越多地基于我们对蛋白质、核酸、病毒和生物分子复合物的三维结构的了解。结构提供了对功能（或故障）的洞察，并为现代药物发现和分子医学提供了起点。生物分子结构最常使用结晶生物分子的 X 射线晶体学来确定。在过去的十年中，高通量方法的引入使蛋白质表达、纯化、结晶和晶体学的许多方面实现了自动化，并且降低了每个确定结构的成本。然而，蛋白质晶体的生长和收获仍然是从基因到三维分子结构、从结构到药物治疗的主要瓶颈。 该第二阶段 STTR 提案的重点是开发和商业化传统和高通量结晶以及晶体收获和 X 射线数据收集的改进方法。康奈尔大学对液体接触线如何与表面相互作用的研究产生了一种简单的技术，可以精确定义分配的液滴的位置并将其牢固地保持在这些位置，无论其化学成分如何。该技术构成了蛋白质结晶板新方法的基础，该方法消除了传统板的液体限制孔。这些新板有望提供对液滴位置和形状的精确控制，从而实现更可重复的结晶动力学并简化图像分析。它们将允许使用单板进行可溶性和膜蛋白的悬滴和坐滴生长，并进行低背景的原位光学、紫外线和 X 射线分析。它们将满足对经过优化的板的关键需求，以方便对筛选和结晶结果进行 X 射线检查，并且还可以进行原位结构测定。它们将与所有现有的滴注和板处理硬件兼容，从而降低市场进入壁垒。该项目将继续这些板的科学和商业开发，并探索滴固定技术的其他生物医学应用。 在第一阶段，我们成功开发并商业化了几种用于晶体检索和 X 射线数据收集的新工具。我们将在第二阶段继续这一开发，重点是改进微晶体学、自动样品安装以及 X 射线束对准和能量测量工具。总之，这些技术应对高通量结构基因组学和药物发现工作的生产力产生重大影响。