RAPID: Structure, Function and Dynamics of SARS Coronavirus-2 Main Protease 3CLpro Determined with Mix-and-Inject Serial XFEL Crystallography

RAPID:使用混合注射串行 XFEL 晶体学测定 SARS 冠状病毒 2 主要蛋白酶 3CLpro 的结构、功能和动力学

基本信息

  • 批准号:
    2030466
  • 负责人:
  • 金额:
    $ 19.96万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2020
  • 资助国家:
    美国
  • 起止时间:
    2020-07-01 至 2023-06-30
  • 项目状态:
    已结题

项目摘要

An award is made to the University of Wisconsin-Milwaukee (UWM) to investigate the molecular mechanism of an essential coronavirus protease reaction in real time. This project addresses fundamental questions how the SARS coronavirus-2 (CoV-2) proliferates. CoV-2 is the source of the worldwide COVID-19 pandemic, which severely impacts public health and national prosperity. The CoV-2 main protease, also called 3CLpro, catalyzes an essential reaction for assembly of CoV-2 infectious particles. If the 3CLpro is blocked, the virus cannot assemble, and its spread is effectively suppressed. The goal is to characterize the 3CLpro’s catalytic cycle at ambient temperatures with X-ray structures. This research takes advantage of opportunities at X-ray Free Electron Lasers (XFELs) such as the Linac Coherent Light Source (LCLS) at Stanford Linear Accelerator Center in Menlo Park, CA. XFELs are the strongest X-ray sources in the world that make it possible to capture molecular reaction intermediates at near atomic resolution within biologically relevant temperatures and time scales. The unique opportunities at XFELs will advance the understanding of 3CLpro catalysis and its function in the proliferation of the virus and contribute to the elimination of the pandemic. This project will involve UWM graduate students and postdoctoral researchers who will be trained in newest data collection and data analysis methods at XFELs.In host cells the SARS-CoV-2’s RNA genome is translated by host ribosomes into a long polypeptide strand that must be cleaved into functional proteins. This is achieved by the CoV-2 3CLpro. If the 3CLpro is inhibited, the newly formed virus particles cannot assemble correctly and become non-infectious. This project structurally characterizes the catalytic cycle of the 3CLpro at XFELs. XFELs are extremely powerful, femtosecond-pulsed X-ray sources, which became available to a wider community a decade ago. At XFELs, crystal structures that are essentially free of radiation damage can be determined at ambient (near physiological) temperatures. Within 3CLpro microcrystals the catalytic cycle will be initiated by diffusion of substrate. Since the crystals are so small, diffusion is not rate-limiting. Microcrystals are mixed with substrate at various time delays before the mixture is injected into the X-ray beam, a method known as ‘mix-and-inject’ serial crystallography (MISC). MISC will be used to follow the 3CLpro enzymatic reaction in real time with X-ray structures of reaction intermediates. The binding of small compounds that inhibit the 3CLpro will be probed at near atomic resolution at relevant temperatures. Results will (i) aid the design and discovery of new inhibitory compounds that affect the function of this essential protease and prevent the formation of infectious viral particles, and (ii) contribute to the development of MISC as an applicable method at XFELs, to be used for the structural characterization of reactions in biologically significant molecules.This RAPID award is made by the Division of Biological Infrastructure (DBI) using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
威斯康星大学密尔沃基分校(UWM)获得了一项奖项,以研究真实的时间内一种重要的冠状病毒蛋白酶反应的分子机制。 该项目解决了SARS冠状病毒-2(CoV-2)如何扩散的基本问题。COV-2是全球COVID-19大流行的源头,严重影响公共卫生和国家繁荣。CoV-2主要蛋白酶,也称为3CLpro,催化CoV-2感染性颗粒组装的必要反应。 如果3CLpro被阻断,病毒就无法组装,其传播也就被有效抑制。目标是用X射线结构表征3CLpro在环境温度下的催化循环。这项研究利用了X射线自由电子激光器(XFEL)的机会,例如位于加利福尼亚州门洛帕克的斯坦福大学直线加速器中心的直线加速器相干光源(LCLS)。XFEL是世界上最强的X射线源,可以在生物相关的温度和时间尺度内以近原子分辨率捕获分子反应中间体。XFEL的独特机会将促进对3CLpro催化作用及其在病毒增殖中的功能的理解,并有助于消除大流行。该项目将涉及UWM研究生和博士后研究人员,他们将在XFEL接受最新数据收集和数据分析方法的培训。在宿主细胞中,SARS-CoV-2的RNA基因组由宿主核糖体翻译成一条长的多肽链,该多肽链必须被切割成功能蛋白。这是由CoV-2 3CLpro实现的。如果3CLpro被抑制,新形成的病毒颗粒就不能正确组装,变得没有感染性。该项目在结构上表征了XFEL中3CLpro的催化循环。XFEL是一种非常强大的飞秒脉冲X射线源,十年前就已经被广泛使用。在XFEL中,基本上没有辐射损伤的晶体结构可以在环境(接近生理)温度下确定。在3CLpro微晶内,催化循环将通过底物的扩散而启动。由于晶体非常小,扩散并不是限速的。在混合物被注入X射线束之前,微晶在不同的时间延迟下与衬底混合,这种方法被称为“混合注入”系列晶体学(MISC)。MISC将用于真实的跟踪3CLpro酶促反应,并具有反应中间体的X射线结构。将在相关温度下以近原子分辨率探测抑制3CLpro的小化合物的结合。结果将(i)有助于设计和发现新的抑制性化合物,这些化合物影响这种必需蛋白酶的功能并防止感染性病毒颗粒的形成,以及(ii)有助于开发MISC作为XFEL的适用方法,用于生物重要分子反应的结构表征。该RAPID奖由生物基础设施部(DBI)颁发。该奖项反映了NSF的法定使命,并通过使用基金会的知识价值和更广泛的影响审查标准进行评估,被认为值得支持。

项目成果

期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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Marius Schmidt其他文献

Sensitivity analysis of a source partitioning method for H2O and CO2 fluxes based on high frequency eddy covariance data: Findings from field data and large eddy simulations
基于高频涡度协方差数据的 H2O 和 CO2 通量源划分方法的灵敏度分析:现场数据和大涡模拟的结果
  • DOI:
  • 发表时间:
    2019
  • 期刊:
  • 影响因子:
    6.2
  • 作者:
    A. Klosterhalfen;A. Moene;Marius Schmidt;Todd M. Scanlon;H. Vereecken;Alexander Graf
  • 通讯作者:
    Alexander Graf
New software for the singular value decomposition of time-resolved crystallographic data
用于时间分辨晶体数据奇异值分解的新软件
  • DOI:
  • 发表时间:
    2009
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Yi Zhao;Marius Schmidt
  • 通讯作者:
    Marius Schmidt
Time-Resolved Macromolecular Crystallography at Modern X-Ray Sources.
现代 X 射线源的时间分辨大分子晶体学。
  • DOI:
    10.1007/978-1-4939-7000-1_11
  • 发表时间:
    2017
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Marius Schmidt
  • 通讯作者:
    Marius Schmidt
Time-Resolved Crystallography at X-ray Free Electron Lasers and Synchrotron Light Sources
X 射线自由电子激光器和同步加速器光源的时间分辨晶体学
  • DOI:
  • 发表时间:
    2015
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Marius Schmidt
  • 通讯作者:
    Marius Schmidt
Time-Resolved Serial Femtosecond Crystallography at the European X-ray Free Electron Laser
欧洲 X 射线自由电子激光器的时间分辨串行飞秒晶体学

Marius Schmidt的其他文献

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{{ truncateString('Marius Schmidt', 18)}}的其他基金

CAREER: Experiencing the 5th Dimension, cis/trans and Z/E Isomerizations in Biomolecules
职业:体验生物分子的第五维、顺式/反式和 Z/E 异构化
  • 批准号:
    0952643
  • 财政年份:
    2010
  • 资助金额:
    $ 19.96万
  • 项目类别:
    Continuing Grant

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