Structural Dynamics at LCLS

摘要：总体 这项提议的目标是在SLAC国家实验室建立一个生物医学技术研究资源(BTRR 加速器实验室，涉及一整套相互关联的技术研发(tr&d) 旨在加强和发展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能够非常 很难或不可能在同步加速器(SR)或电子显微镜(CryoEM)设施中执行，并将增加 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 促进各种主题的范式转换进展，包括神经传递、信号 转导、细胞代谢、转录和病毒感染。建议的研发及发展署与 九个驱动力生物医学项目(DBP)的研究主题。这些研究主题集中在 可视化大的复合体和膜蛋白的发展，如GPCRs和提供准确 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； 复杂的大分子机器，如RNA聚合酶-II。最后，所有DBP的一个共同研究领域 参与时间分辨(Tr)研究，包括跟踪腺嘌呤相关的动态过程的研究 核糖开关信号、淋球菌mtrF的转运机制、抗生素与β-内酰胺酶的结合以及 CypA变异体相互作用特异性的检测。 所有的DBP都依赖于高效的数据收集方法，这是成功的大分子所必需的 X射线自由电子能谱(MC)实验。X射线自由电子激光脉冲的高峰值亮度破坏了 晶体体积暴露，带来了MC SR以前未知的样品刷新挑战 社区。因此，在整个实验过程中，必须不断补充样本。作为该计划的一部分 将对快速将晶体和样品溶液输送到X射线束的样品注射器进行优化 并在LCLS实验期间实现自动化，同时进行数据分析以评估实验成功并进行优化 使用有限的样品和光束时间。时间分辨研究取决于对混合喷射器、激光器的改进 活化法和互补法。X射线自由电子激光的时间很少，所以要仔细描述 在束流时间之前对样品和复杂的实验装置进行分析是确保实验成功的关键 特别适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、样品制作、样品表征(包括光谱分析)和晶体 在实验室、斯坦福同步辐射光源(SSRL)和 在LCLS的筛选光束时间内。整合并增强SSRL和LCLS的现有计划， BTRR将为生物医学界提供支持、专业知识和培训。

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.