Femtosecond nano-crystallography of membrane proteins

膜蛋白的飞秒纳米晶体学

• 批准号: 8322064
• 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/8322064
• 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; research study

Summary 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.

总结 本提案的目的是发展飞秒（fs）晶体学方法， 膜蛋白的结构测定，其中X射线结构分析是基于 在数十万个X射线衍射图上， 膜蛋白的纳米/微晶，使用新的高能fs X射线收集 在斯坦福大学的LCLS工作。LCLS于2009年秋季开始运营， 飞秒脉冲的强度超过第三代同步辐射源的12个数量级 大小 膜蛋白在所有活细胞中都是极其重要的，因为它们催化重要的 像呼吸作用、光合作用、运输和细胞通讯。30%的 人类蛋白质是膜蛋白，超过60%的药物靶向于 膜蛋白尽管它们极其重要，但对它们的理解 分子功能受到结构信息缺乏的阻碍;而超过 6万种可溶性蛋白质的结构已被X射线晶体学解析， 根据核磁共振谱，到目前为止已经确定了不到250种不同的膜蛋白结构。 迄今为止，膜蛋白结构的确定通常涉及一个时间- 消费过程中，它花了几年（有时甚至几十年）的增长大，以及- 适合于X射线结构测定的有序晶体。此外，X射线诱导的 辐射损伤是许多膜蛋白晶体的主要问题， 当它们含有金属和/或氧化还原活性辅因子时。X射线引起的辐射 即使在低温下，损伤也限制了微晶的X射线衍射 条件这个建议是基于第一次证明原则的fs- 通过在纳米/纳米晶体上收集300万个衍射图案， 2009年12月在LCLS，使用 fs X射线脉冲。作为模型系统的光系统I具有一个分子 分子量为1，056，000道尔顿，由36种蛋白质和381种辅助因子组成， 共价结合，使光系统I成为最复杂的膜蛋白之一 迄今为止已经被结晶化了。这些实验已经证明， “破坏前的衍射原理”，首次出现在2006年的一个图像蚀刻成一个 硝酸硅薄膜（Chapman 2006，Nature Physics），可以直接扩展到 迄今为止存在的最脆弱的蛋白质晶体，其中含有78%的溶剂和只有4 盐桥参与晶体接触。该提案旨在开启一个令人兴奋的新 膜蛋白晶体学的途径，其中数十万个 衍射图案可以在几分钟的时间范围内使用完全水合的纳米/ 微晶在其母液中，在室温下，用X射线激光脉冲， 短到X射线引起的辐射损伤仅在数据收集之后开始。新 方法也有可能获得分子的激发态结构， 将光学激光激发与飞秒X射线数据采集相结合。为 该提案打破了新的未探索的领域，它涉及方法的发展， 从筛选最好的微晶和确定微晶的生长， 用于高通量数据筛选、数据评估和分阶段的新方法开发 保持战略定力