Femtosecond nano-crystallography of membrane proteins

膜蛋白的飞秒纳米晶体学

基本信息

  • 批准号:
    8329489
  • 负责人:
  • 金额:
    $ 23.9万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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):本提案的目的是开发用于膜蛋白结构测定的飞秒(fs)晶体学方法,其中x射线结构分析是基于斯坦福大学LCLS使用新型高能fs x射线激光器收集的来自完全水合膜蛋白纳米/微晶体蒸汽的数十万个x射线衍射图。LCLS于2009年秋季开始运行,提供的fs脉冲强度超过第三代同步加速器源12个数量级。膜蛋白在所有活细胞中都非常重要,因为它们催化呼吸、光合作用、运输和细胞通讯等重要功能。人体30%的蛋白质是膜蛋白,60%以上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,对其分子功能的理解受到阻碍;虽然通过x射线晶体学和核磁共振已经确定了6万多种可溶性蛋白质的结构,但迄今为止确定的膜蛋白质结构还不到250种。迄今为止,膜蛋白结构的测定通常涉及一个耗时的过程,需要数年(有时甚至数十年)才能生长出适合x射线结构测定的大而有序的晶体。此外,x射线引起的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/或氧化还原活性辅助因子时。即使在低温条件下,x射线引起的辐射损伤也限制了微晶体的x射线衍射。这一建议是基于2009年12月在LCLS使用fs x射线脉冲收集的膜蛋白光系统I的纳米/微晶体的300万个衍射图,首次证明了fs-纳米晶体学的原理。作为模型系统的光系统I分子量为1,056,000道尔顿,由36个蛋白和381个非共价结合的辅因子组成,是迄今为止已结晶的最复杂的膜蛋白之一。这些实验已经证明了“先衍射后破坏原理”,该原理首次出现在2006年蚀刻在硝酸硅薄膜上的图像上(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之一,这种晶体含有78%的溶剂,只有4个盐桥参与晶体接触。该提案旨在为膜蛋白晶体学开辟一条令人兴奋的新途径,在室温下,使用完全水合的纳米/微晶体在其母液中使用x射线激光脉冲,在几分钟的时间框架内收集成千上万的衍射图案,x射线激光脉冲非常短,以至于x射线诱导的辐射损伤仅在数据收集后开始。这种新方法也有可能在未来通过将光学激光激发与fs x射线数据收集相结合来获得分子激发态的结构。随着该提案进入新的未开发领域,它涉及到从筛选最佳微晶体和定义微晶体生长到高通量数据筛选,数据评估和相位确定的新方法开发的方法开发。

项目成果

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{{ truncateString('PETRA FROMME', 18)}}的其他基金

Center for Membrane Proteins in Infectious Diseases (MPID)
传染病膜蛋白中心 (MPID)
  • 批准号:
    8692880
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Femtosecond nano-crystallography of membrane proteins
膜蛋白的飞秒纳米晶体学
  • 批准号:
    8322064
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Dynamics of membrane proteins unraveled by time-resolved serial crystallography
时间分辨系列晶体学揭示膜蛋白的动力学
  • 批准号:
    10657320
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Femtosecond nano-crystallography of membrane proteins
膜蛋白的飞秒纳米晶体学
  • 批准号:
    8027697
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Femtosecond nano-crystallography of membrane proteins
膜蛋白的飞秒纳米晶体学
  • 批准号:
    9055725
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Center for Membrane Proteins in Infectious Diseases (MPID)
传染病膜蛋白中心 (MPID)
  • 批准号:
    8741167
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Dynamics of membrane proteins unraveled by time-resolved serial crystallography
时间分辨系列晶体学揭示膜蛋白的动力学
  • 批准号:
    9887557
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Center for the Rational Design of Membrane Protein Crystallography
膜蛋白晶体学合理设计中心
  • 批准号:
    8152487
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Dynamics of membrane proteins unraveled by time-resolved serial crystallography
时间分辨系列晶体学揭示膜蛋白的动力学
  • 批准号:
    10334532
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:
Femtosecond nano-crystallography of membrane proteins
膜蛋白的飞秒纳米晶体学
  • 批准号:
    9304242
  • 财政年份:
    2010
  • 资助金额:
    $ 23.9万
  • 项目类别:

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