Femtosecond nano-crystallography of membrane proteins

膜蛋白的飞秒纳米晶体学

• 批准号: 8027697
• 负责人: PETRA FROMME
• 金额: $ 30万
• 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/8027697
• 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

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（由申请人提供）：该提案的目的是开发膜蛋白的结构测定的飞秒（FS）晶体学方法，其中X射线结构分析基于数十万X射线衍射模式，该模式是从完全水合的Nano/ Microcrystals使用新能量的lcl forn forn forn flas funford forn fors x-fors x-ray x-ray x-ray的全水合纳米/微晶的蒸汽中的。 LCLS于2009年秋季开始运行，并提供了超过第三代同步源的强度的FS脉冲，乘以12个数量级。膜蛋白在所有活细胞中都非常重要，因为它们催化了呼吸，光合作用，运输和细胞通信等重要功能。所有人类蛋白中有30％是膜蛋白，所有药物中有60％旨在针对膜蛋白。尽管缺乏结构信息，但他们对分子功能的理解受到阻碍。尽管已经通过X射线晶体学和NMR溶解了60,000多个可溶性蛋白结构，但迄今已确定了少于250种不同的膜蛋白结构。迄今为止解决的膜蛋白结构的确定通常涉及一个耗时的过程，在该过程中，花费了数年（甚至几十年）才能生长出适合X射线结构确定的大型，有序的晶体。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，尤其是当它们含有金属和/或氧化还原活性辅助因子时。 X射线诱导的辐射损伤甚至在低温条件下也对微晶体施加了X射线衍射的限制。该建议基于FS-纳米晶体图的第一个原理证明，通过使用FS X射线脉冲在2009年12月在LCLS收集了膜蛋白光系统I I的纳米/微晶上的300万个衍射模式。光系统I（作为模型系统）的分子量为1,056,000个达尔顿人，由36种蛋白质和381个辅助因子组成，这些蛋白质与非共价结合，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，“破坏原理之前的衍射”是2006年首次显示的图像，用于将其蚀刻到二氧化硅膜（Chapman 2006，自然物理学）中，可以直接扩展到迄今为止存在的最脆弱的蛋白质晶体之一，其中包含78％的溶剂和4个盐桥，并且在水晶接触中只有4个盐桥。该提案旨在为膜蛋白质晶体学开辟一个令人兴奋的新途径，其中数十万个衍射模式可以在几分钟的时间范围内使用全部水合的纳米/微晶在其母液中，在室温下，X射线激光脉冲仅在X射线诱导的辐射损失后才开始收集数据。新方法还具有通过将光激光激发与FS X射线数据收集相结合的分子激发态结构的潜力。随着该提案分为新的未开发的理由，它涉及方法的开发，从筛选最佳微晶以及微晶的定义增长到用于高吞吐量数据筛选，数据评估和相位确定的新方法开发。 公共卫生相关性：该提案的目的是开发一种新方法来确定膜蛋白的结构。这使用了超短的飞秒X射线脉冲，该脉冲由第一个硬X射线激光器（斯坦福大学的“ LCLS”）提供，以从连续的全含膜蛋白纳米晶体的连续流中收集X射线衍射数据。脉冲是如此的简短，以至于在辐射损伤过程开始之前终止。