Femtosecond nano-crystallography of membrane proteins

膜蛋白的飞秒纳米晶体学

• 批准号: 8149876
• 负责人: PETRA FROMME
• 金额: $ 29.7万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8149876
• 关键词: Binding; Biological Models; Cell Communication; Cells; Collection; Complex; Crystallography; Data; Data Collection; Data Set; Development; Drug Delivery Systems; Evaluation; Film; Future; Generations; Growth; Human; Image; Individual; Lasers; Life; Membrane Proteins; Metals; Methods; Molecular; Molecular Weight; Mothers; Nature; Nitrates; Optics; Oxidation-Reduction; Pattern; Phase; Photosynthesis; Photosystem I; Physics; Physiologic pulse; Powder Diffraction; Process; Proteins; Radiation; Radiation induced damage; Research Activity; Respiration; Roentgen Rays; Screening procedure; Silicon; Sodium Chloride; Solvents; Source; Steam; Stream; Structure; Synchrotrons; Techniques; Temperature; Time; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; cofactor; cryogenics; dalton; distilled alcoholic beverage; falls; indexing; method development; nano; nanocrystal; novel; operation; protein structure; public health relevance; research study

DESCRIPTION (provided by applicant): The aim of this proposal is to develop the method of femtosecond (fs) crystallography for the structure determination of membrane proteins, where X-ray structure analysis is based on hundreds of thousands of X-ray diffraction patterns from a steam of fully hydrated nano/ microcrystals of membrane proteins, collected using the new high energy fs X-ray laser at LCLS in Stanford. The LCLS started its operation in the fall of 2009 and provides fs-pulses of an intensity that exceeds third-generation synchrotron sources by 12 orders of magnitude. Membrane proteins are of extreme importance in all living cells as they catalyze vital functions like respiration, photosynthesis, transport, and cell communication. 30% of all human proteins are membrane proteins and more than 60% of all drugs are targeted to membrane proteins. Despite their extreme importance, the understanding of their molecular function is hampered by the lack of structure information; while more than 60,000 structures of soluble proteins have been solved by X-ray crystallography and NMR, less than 250 different membrane protein structures have so far been determined. The determination of membrane protein structures solved to date often involved a time- consuming process where it took years (or sometimes even decades) to grow large, well- ordered crystals suitable for X-ray structure determination. Furthermore, X-ray-induced radiation damage is a major problem for many membrane protein crystals, especially when they contain metals and/or redox active cofactors. The X-ray-induced radiation damage imposes a limitation for X-ray diffraction on microcrystals, even under cryogenic conditions. This proposal is based on the first proof of principle for fs- nanocrystallography by the collection of 3 million diffraction patterns on nano/ microcrystals of the membrane protein Photosystem I in December 2009 at LCLS, using fs X-ray pulses. Photosystem I, which served as the model system, has a molecular weight of 1,056,000 Daltons and consists of 36 proteins and 381 cofactors that are non- covalently bound, making Photosystem I one of the most complex membrane proteins that has been crystallized to date. These experiments have already proven that the "diffraction before destroy principle," first shown in 2006 for an image etched into a silicon-nitrate film, (Chapman 2006, Nature Physics), can be directly extended to one of the most fragile protein crystals that exists to date, which contain 78% solvent and only 4 salt bridges involved in crystal contact. This proposal aims to open an exciting new avenue for membrane protein crystallography, where hundreds of thousands of diffraction patterns can be collected in a time frame of minutes using fully hydrated nano/ microcrystals in their mother liquor, at room temperature, with X-ray laser pulses that are so short that X-ray-induced radiation damage only starts after data collection. The new method has also the potential to obtain structures of excited states of the molecules by combining optical laser excitation with fs X-ray data collection in the future. As the proposal breaks into new unexplored grounds, it involves method developments ranging from the screening for the best microcrystals and the defined growth of microcrystals to new method developments for high throughput data screening, data evaluation and phase determination. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to develop a new method for the structure determination of membrane proteins. This uses ultra-short femtosecond X-ray pulses, provided by the first hard-X-ray laser (the "LCLS" at Stanford) to collect X-ray diffraction data from a continuous stream of fully-hydrated membrane protein nanocrystals. The pulses are so brief that they terminate before radiation damage processes can begin.

描述(申请人提供)：这项提议的目的是开发用于确定膜蛋白结构的飞秒(Fs)结晶学方法，其中X射线结构分析基于来自完全水化的膜蛋白纳米/微晶蒸汽的数十万个X射线衍射图案，这些图案是使用斯坦福大学LCLS的新的高能FS X射线激光收集的。LCLS于2009年秋季开始运行，提供的飞秒脉冲强度比第三代同步加速器源高出12个数量级。膜蛋白在所有活细胞中都是极其重要的，因为它们催化呼吸、光合作用、运输和细胞通讯等重要功能。30%的人类蛋白质是膜蛋白，超过60%的药物是针对膜蛋白的。尽管它们非常重要，但由于缺乏结构信息，人们对它们的分子功能的了解受到阻碍；虽然通过X射线结晶学和核磁共振技术已经解决了60,000多个可溶性蛋白质的结构，但到目前为止，确定的不同的膜蛋白质结构还不到250个。到目前为止，膜蛋白质结构的测定通常涉及一个耗时的过程，需要数年(有时甚至几十年)才能生长出适合于X射线结构测定的大而有序的晶体。此外，X射线辐射损伤是许多膜蛋白晶体的主要问题，特别是当它们含有金属和/或氧化还原活性辅因子时。即使在低温条件下，X射线引起的辐射损伤也对微晶的X射线衍射造成了限制。这一建议的基础是利用飞秒X射线脉冲，于2009年12月在LCLS收集了膜蛋白光系统I的纳米/微晶上的300万个衍射图，这是飞秒纳米晶体学原理的第一次证明。作为模型系统的光系统I具有1056,000道尔顿的相对分子质量，由36个蛋白质和381个非共价结合的辅因子组成，使光系统I成为迄今已结晶的最复杂的膜蛋白之一。这些实验已经证明，2006年首次展示蚀刻到硝酸硅薄膜上的图像的“破坏前衍射原理”(Chapman 2006，自然物理)，可以直接扩展到迄今存在的最脆弱的蛋白质晶体之一，它含有78%的溶剂，只涉及晶体接触的4个盐桥。这一提议旨在为膜蛋白质结晶学开辟一条令人兴奋的新途径，在几分钟的时间框架内，使用室温下其母液中完全水合的纳米/微晶，以及如此短的X射线激光脉冲，可以在几分钟内收集数十万个衍射图，以至于X射线辐射损伤只有在数据收集之后才开始。这种新的方法也有可能在未来通过将光学激光激发与飞秒X射线数据收集相结合来获得分子的激发态结构。随着该提案进入新的未开发领域，它涉及的方法开发范围从筛选最佳微晶体和定义微晶体的生长，到高通量数据筛选、数据评估和相态确定的新方法开发。 与公众健康相关：这项提议的目的是开发一种新的膜蛋白结构测定方法。它使用由第一台硬X射线激光器(斯坦福大学的LCLS)提供的超短飞秒X射线脉冲，从连续的完全水合的膜蛋白纳米晶体中收集X射线衍射数据。这些脉冲如此短暂，以至于它们在辐射损害过程开始之前就终止了。