Structural Dynamics at LCLS

LCLS 结构动力学

• 批准号: 10089007
• 负责人: Sebastien Boutet
• 金额: $ 159.46万
• 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/10089007
• 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 该提案的目标是在SLAC国家中心建立一个生物医学技术研究资源（BTRR） 加速器实验室，涉及一系列相互关联的技术研究和开发（TR&D） 旨在增强和发展SLAC直线加速器相干光源独特能力的项目 （LCLS）用于生物医学应用。BTRR将使结构生物学实验， 难以或不可能在同步加速器（SR）或电子显微镜（cryoEM）设施中进行， 这些能力对更广泛的结构生物学社区的可用性。启用的实验将 促进在各种各样的主题，包括神经传递，信号， 转导、细胞代谢、转录和病毒感染。拟议的TR& D与以下内容紧密结合： 九个驱动生物医学项目（DBPs）的研究主题。这些研究主题集中在 开发可视化大的复合物和膜蛋白，如GPCR，并提供准确的 金属酶的活性部位结构，如核糖核苷酸还原酶和细胞色素c氧化酶，和 复杂的大分子机器，如RNA聚合酶-II。最后，所有DBPs的共同研究领域 涉及时间分辨（TR）研究，包括研究腺嘌呤参与的动态过程 核糖开关信号，N.淋病MtrF，抗生素与β-内酰胺酶结合， 检查CypA变体的相互作用特异性。 所有DBP都依赖于高效的数据收集方法，这是成功的大分子生物学所必需的。 晶体学（MC）实验在X射线自由电子激光。X射线自由电子激光脉冲的高峰亮度破坏了 晶体体积暴露，带来了MC SR以前未知的样品更新挑战 社区因此，在整个实验过程中必须不断补充样品。的一部分 TR& D，快速将晶体和样品溶液输送到X射线束的进样器将得到优化 并在LCLS实验期间自动化，沿着数据分析，以衡量实验成功并优化 使用有限的样本和射束时间。时间分辨研究取决于混合注射器、激光器的改进 激活和补充光谱方法。X射线自由电子激光束时间是稀缺的， 样品和复杂的实验设置前束时间是至关重要的，以确保实验成功， 特别是用于敏感金属酶中间体的复杂时间分辨测量。实验 设计和测试，样品生产，样品表征（包括光谱分析）和晶体 质量筛选支持在实验室，在斯坦福大学同步辐射光源（SSRL）和 在LCLS的筛选光束时间。整合并加强SSRL和LCLS的现有项目， BTRR将为生物医学界提供支持、专业知识和培训。