Technology Development for Biological Imaging with XFELs

XFEL 生物成像技术开发

• 批准号: 10654727
• 负责人: MATTHIAS FRANK
• 金额: $ 52.57万
• 依托单位: LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654727
• 关键词: Address; Area; Behavior; Benchmarking; Biological; Cell physiology; Cellular Membrane; Consumption; Cryoelectron Microscopy; Crystallization; Crystallography; Data Set; Development; Drug Targeting; Electrodes; Electronics; Electrons; Encapsulated; Environment; European; Film; Freezing; Generations; Health; Human; Hydration status; Image; Investigation; Knowledge; Laboratories; Light; Lipids; Liquid substance; Location; Measurement; Medical; Membrane; Membrane Potentials; Membrane Proteins; Methodology; Methods; Molecular Conformation; Physiologic pulse; Polymers; Preparation; Protein Dynamics; Proteins; Protons; Public Health; Radiation Dose Unit; Radiation induced damage; Reaction; Reproducibility; Research; Resolution; Roentgen Rays; Sampling; Scanning; Science; Signal Transduction; Silicon; Source; Speed; Structure; Styrenes; Techniques; Technology; Temperature; Thinness; Time; Transmembrane Domain; Vacuum; Visualization; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Medical Imaging; biological development; biosecurity; cryogenics; data acquisition; density; design; electric field; flexibility; graphene; improved; maleic acid; manufacture; millisecond; mimetics; nano-objects; nanolipoprotein particles; nanoparticle; new technology; next generation; novel; novel strategies; protein complex; protein function; protein structure; scaffold; spatiotemporal; structural biology; structural imaging; technology development; tool; voltage gated channel; x-ray free-electron laser

Project Summary / Abstract Determining the structure and conformational dynamics of large protein complexes as well as other biological nanoparticles at room temperature with near atomic resolution has the potential to greatly impact structural biology and our knowledge of biomolecular function and interactions. A major bottleneck in structural biology is that while many critical cellular functions are performed by membrane proteins, they have proven intractable to structure determination by traditional x-ray crystallography, in which x-ray radiation damage is mitigated by spreading the radiation dose over many molecules in a crystal. Consequently, most membrane protein structures remain unknown to date. While cryo-electron microscopy (cryo-EM) has been successful in obtaining high-resolution structural information from large biomolecules and nanoparticles, it requires freezing of the sample to mitigate electron-induced radiation damage and cryogenic measurement makes it impossible to visualize fast conformational changes. X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow us to break this nexus between resolution and radiation damage by utilizing the “diffraction-before-destruction” principle and promise imaging at unprecedented spatio-temporal resolution. Over the last decade since the opening of the world's first XFEL, the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, protein structure determination at room temperature to near-atomic resolution by serial- femtosecond nanocrystallography (SFX) has been demonstrated. However, several challenges and limitations remain that need to be addressed to fully utilize the capabilities of these new light sources and the upcoming next generation XFELs for structural biology. The overall objective of this proposal is to enable new science by addressing several of the current technological and methodological challenges in x-ray diffractive imaging of biological samples with XFELs, in particular in the areas of sample preparation for membrane proteins that, generally, suffer from low abundance and/or are hard to crystallize, sample introduction technologies enabling high data acquisition rates, and novel approaches to time-resolved structure determination of membrane proteins. This work will also drastically reduce sample consumption and will increase the diversity of membrane protein and other biological nano- objects that can be studied with XFELs. The proposed work also aims to develop new capabilities for time- resolved structural studies at XFELs to include cross-membrane potential triggered membrane protein dynamics, enabling investigation of a broader range of biomolecular and cellular reactions and the associated structural change over a large range of times scales from microseconds to milliseconds. If successful, this work would greatly aid our experimental capabilities to study and understand function of protein complexes and biological nanoparticles in a wide range of fields including human health and biosecurity.

项目总结/摘要 确定大型蛋白质复合物以及其他复合物的结构和构象动力学 在室温下具有近原子分辨率的生物纳米颗粒具有极大影响 结构生物学和我们对生物分子功能和相互作用的知识。的一大瓶颈 结构生物学是，虽然许多关键的细胞功能是由膜蛋白，他们有 已经证明难以通过传统的X射线晶体学确定结构，其中X射线辐射 通过将辐射剂量分散在晶体中的许多分子上来减轻损伤。因此，大多数 膜蛋白结构至今仍是未知的。虽然冷冻电子显微镜（cryo-EM）已经被 成功地从大生物分子和纳米颗粒中获得高分辨率的结构信息， 需要冷冻样品以减轻电子诱导的辐射损伤和低温测量 使得我们无法观察到快速的构象变化。 X射线自由电子激光器（XFEL）可以产生超短和超亮的X射线脉冲，使我们能够 利用“破坏前衍射”打破分辨率和辐射损伤之间的这种联系 原理和承诺成像在前所未有的时空分辨率。在过去的十年里， 世界上第一个XFEL，直线加速器相干光源（LCLS）在SLAC国家加速器上开放 实验室，蛋白质结构测定在室温下，近原子分辨率的系列- 已经证明了飞秒纳米照相术（SFX）。然而，一些挑战和限制 仍然需要解决的是，充分利用这些新光源的能力和即将到来的 结构生物学的下一代XFEL。 该提案的总体目标是通过解决当前的几个问题， 利用XFEL对生物样品进行X射线衍射成像的技术和方法学挑战， 特别是在膜蛋白的样品制备领域， 和/或难以结晶，能够实现高数据采集速率的样品引入技术， 膜蛋白的时间分辨结构测定方法。这项工作也将大大 减少样品消耗，增加膜蛋白等生物纳米的多样性， 可以用XFEL研究的物体。拟议的工作还旨在开发新的时间能力- 在XFEL中解决了结构研究，以包括跨膜电位触发的膜蛋白 动力学，使研究更广泛的生物分子和细胞反应和相关的 结构变化的时间跨度很大，从微秒到毫秒。如果成功，这项工作 这将极大地帮助我们研究和理解蛋白质复合物的功能， 生物纳米颗粒在包括人类健康和生物安全在内的广泛领域中发挥着重要作用。

项目成果

Biophysical Characterization of Membrane Proteins Embedded in Nanodiscs Using Fluorescence Correlation Spectroscopy.

• DOI: 10.3390/membranes12040392
• 发表时间: 2022-03-31
• 期刊: Membranes
• 影响因子: 4.2

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

• DOI: 10.1038/s41467-018-04116-9
• 发表时间: 2018-05-09
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Seuring，Carolin;Ayyer，Kartik;Chapman，Henry N.
• 通讯作者: Chapman，Henry N.

Structural enzymology using X-ray free electron lasers.

• DOI: 10.1063/1.4972069
• 发表时间: 2017-07
• 期刊: Structural dynamics (Melville, N.Y.)
• 作者: Kupitz C;Olmos JL Jr;Holl M;Tremblay L;Pande K;Pandey S;Oberthür D;Hunter M;Liang M;Aquila A;Tenboer J;Calvey G;Katz A;Chen Y;Wiedorn MO;Knoska J;Meents A;Majriani V;Norwood T;Poudyal I;Grant T;Miller MD;Xu W;Tolstikova A;Morgan A;Metz M;Martin-Garcia JM;Zook JD;Roy-Chowdhury S;Coe J;Nagaratnam N;Meza D;Fromme R;Basu S;Frank M;White T;Barty A;Bajt S;Yefanov O;Chapman HN;Zatsepin N;Nelson G;Weierstall U;Spence J;Schwander P;Pollack L;Fromme P;Ourmazd A;Phillips GN Jr;Schmidt M
• 通讯作者: Schmidt M

Analysis of XFEL serial diffraction data from individual crystalline fibrils.

• DOI: 10.1107/s2052252517014324
• 发表时间: 2017-11-01
• 期刊: IUCrJ
• 影响因子: 3.9
• 作者: Wojtas DH;Ayyer K;Liang M;Mossou E;Romoli F;Seuring C;Beyerlein KR;Bean RJ;Morgan AJ;Oberthuer D;Fleckenstein H;Heymann M;Gati C;Yefanov O;Barthelmess M;Ornithopoulou E;Galli L;Xavier PL;Ling WL;Frank M;Yoon CH;White TA;Bajt S;Mitraki A;Boutet S;Aquila A;Barty A;Forsyth VT;Chapman HN;Millane RP
• 通讯作者: Millane RP

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins.

用于生产哺乳动物受体的无细胞共翻译方法：使用纳米脂蛋白扩展无细胞表达工具箱。

• DOI: 10.3389/fphar.2019.00744
• 发表时间: 2019
• 期刊: Frontiers in pharmacology
• 影响因子: 5.6
• 作者: Shelby,MeganL;He,Wei;Dang,AmandaT;Kuhl,TonyaL;Coleman,MatthewA
• 通讯作者: Coleman,MatthewA