Structural Dynamics at LCLS

LCLS 结构动力学

• 批准号: 10379223
• 负责人: Sebastien Boutet
• 金额: $ 202.29万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10379223
• 关键词: Active Sites; Address; Adenine; Antibiotics; Area; Automation; Automobile Driving; Back; Beds; Binding; Biological; Biomedical Research; Biomedical Technology; Collaborations; Communities; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; Data Analyses; Data Collection; Development; Drug Design; Educational workshop; Electron Microscopy; Ensure; Enzymatic Biochemistry; Experimental Designs; Foundations; Future; G-Protein-Coupled Receptors; Generations; Genetic Transcription; Goals; Grant; Health; Health Services Research; Human; Image; Internet; Laboratories; Lasers; Length; Life; Light; Macromolecular Complexes; Measurement; Membrane Proteins; Metabolism; Methodological Studies; Methodology; Methods; Names; Neisseria gonorrhoeae; Physiologic pulse; Process; Production; Property; RNA Polymerase II; Regulation; Research; Resource Development; Resources; Ribonucleotide Reductase; Roentgen Rays; Sampling; Science; Scientist; Services; Signal Transduction; Source; Specificity; Structure; Synchrotrons; Technology; Testing; Time; Training; Variant; Virus Diseases; Work; base; beta-Lactamase; community engagement; cytochrome c oxidase; design; driving force; experimental study; improved; innovative technologies; instrumentation; metalloenzyme; movie; neurotransmission; new technology; novel strategies; programs; screening; structural biology; success; synchrotron radiation; technology research and development; tool; x-ray free-electron laser

ABSTRACT: OVERALL The goal of this proposal is to form a Biomedical Technology Research Resource (BTRR) at SLAC National Accelerator Laboratory that involves a set of interrelated Technology Research and Development (TR&D) projects aimed at enhancing and developing the unique capabilities of the SLAC Linac Coherent Light Source (LCLS) for biomedical applications. The BTRR will enable structural biology experiments that are extremely difficult or impossible to perform at synchrotron (SR) or electron microscopy (cryoEM) facilities and will increase the availability of these capabilities to the broader structural biology community. The enabled experiments will facilitate paradigm-shifting advances on a wide variety of topics, including neurotransmission, signal transduction, cellular metabolism, transcription and viral infection. The proposed TR&Ds are tightly coupled with the research themes of the nine Driving Biomedical Projects (DBPs). These research themes focus on developments to visualize large complexes and membrane proteins, such as GPCRs and that provide accurate active site structures of metalloenzymes, such as ribonucleotide reductase and cytochrome c oxidase, and complex macromolecular machines, such as RNA polymerase-II. Finally, a common research area of all DBPs involve time-resolved (TR) studies that include research to follow dynamic processes involved in adenine riboswitch signaling, the transport mechanism of N. gonorrhoeae MtrF, antibiotic binding to β-lactamase and examination of interaction specificity of CypA variants. All DBPs hinge on highly efficient data collection methods, which are required for successful macromolecular crystallography (MC) experiments at X-ray FELs. The high peak brightness of an X-ray FEL pulse destroys the crystal volume exposed, bringing about sample refreshment challenges previously unknown to the MC SR community. As a result, the sample must be continually replenished throughout the experiment. As part of the TR&Ds, sample injectors that rapidly deliver crystals and sample solutions to the X-ray beam will be optimized and automated during LCLS experiments along with data analysis to gauge experimental success and optimize use of limited sample and beam time. Time resolved studies hinge on improvements to mixing injectors, laser activation and complementary spectroscopic methods. X-ray FEL beam time is scarce so careful characterization of samples and complex experimental setups prior to beam time is critical to ensure experimental success, in particular for complex time resolved measurements of sensitive metalloenzymes intermediates. Experimental design and testing, sample production, sample characterization (including spectroscopic analysis) and crystal quality screening are supported in the laboratory, at the Stanford Synchrotron Radiation Lightsource (SSRL) and during screening beam time at LCLS. Integrating with, and enhancing the existing programs at SSRL and LCLS, the BTRR will provide support, expertise and training to the biomedical community.

摘要：总体 该提案的目的是在SLAC National建立生物医学技术研究资源（BTRR） 涉及一组相互关联的技术研发（TR＆D）的加速器实验室（TR＆D） 旨在增强和开发SLAC LINAC连贯光源的独特功能的项目 （LCL）用于生物医学应用。 BTRR将实现非常极端的结构生物学实验 在同步加速器（SR）或电子显微镜（Cryoem）设施上难以或不可能执行，并且会增加 这些能力可用于更广泛的结构生物学社区。启用实验将 促进各种主题的范式移动进步，包括神经传递，信号 转导，细胞代谢，转录和病毒感染。建议的TR＆D与 九个驾驶生物医学项目（DBP）的研究主题。这些研究主题着重于 可视化大型复合物和膜蛋白（例如GPCR）的开发项目，并提供准确的 金属酶的活性位点结构，例如核糖核苷酸还原酶和细胞色素C氧化酶，以及 复杂的大分子机器，例如RNA聚合酶-II。最后，所有DBP的共同研究领域 涉及时间分辨（TR）研究，其中包括遵循腺嘌呤涉及的动态过程的研究 核糖开关信号传导，淋病猪笼草的转运机制，抗生素与β-内酰胺酶的结合和 检查CYPA变体的相互作用特异性。 所有DBP在高效的数据收集方法上都取决于成功的大分子分子所必需的 X射线长石的晶体学（MC）实验。 X射线FEL脉冲的高峰亮度破坏了 暴露的晶体体积，带来了MC SR以前未知的样品茶点挑战 社区。结果，必须在整个实验中连续复制样品。作为一部分 TR＆D，将快速输送晶体和样品溶液向X射线束提供的样品喷射器将被优化 并在LCLS实验中自动化以及数据分析以衡量实验成功并优化 使用有限的样品和光束时间。时间解决研究取决于改进混合喷油器，激光 激活和完整的光谱法。 X射线FEL光束时间很少，因此仔细的表征 在光束时间之前的样品和复杂的实验设置对于确保实验成功至关重要 特别针对复杂的时间解析敏感的金属酶中间体的测量。实验 设计和测试，样品生产，样品表征（包括光谱分析）和晶体 实验室，斯坦福同步辐射光线（SSRL）和 在LCLS的筛选时间期间。与SSRL和LCLS的现有程序集成并增强， BTRR将为生物医学界提供支持，专业知识和培训。