RAPID: IIBR: Instrumentation: Time-resolved studies of the SARS-CoV-2 endonuclease NP15

RAPID：IIBR：仪器：SARS-CoV-2 核酸内切酶 NP15 的时间分辨研究

• 批准号: 2031343
• 负责人: Petra Fromme
• 金额: $ 20万
• 依托单位: Arizona State University
• 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: 2020-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2031343&HistoricalAwards=false
• 关键词: RAPID; IIBR; Instrumentation; Time; resolved

The coronavirus disease 2019 (COVID-19) crisis has produced an urgent need to understand the structure and dynamics of the proteins of SARS-CoV-2 (CoV-2), the coronavirus that is the cause of this severe infectious disease. Research supported by this award will unravel the catalytic mechanisms of one of the most interesting and unique CoV-2 enzymes, the RNA endoribonuclease, NendoU, which is only found in the highly virulent coronaviruses. NendoU plays a key role in limiting the sensing of viral RNA by host sensors and is thus a very important target to elucidate the mechanisms of viral pathogenesis at the molecular level. The researchers will use a combination of advanced techniques and new innovations in sample preparation to make “molecular movies” that reveal the mechanism of action for the NendoU protein to advance our understanding of viral biology and may aid in the identification of potential therapeutic compounds. Results from these studies will be shared through on-line seminars, at scientific meetings, in public data repositories, and through publications in peer-reviewed journals.Static structures of the NendoU from SARS, MERS and most recently, also from CoV-2 have been determined by standard crystallography under cryogenic conditions, but no structures have been captured with the substrate RNA bound nor have any time-resolved studies been performed on the enzyme. This COVID-2019 RAPID award focuses on time resolved studies using a combination of time-resolved femtosecond crystallography (TR-SFX) and time resolved cryo-electron microscopy (TR-cryo-EM) to capture a molecular movie of the catalytic reaction of NendoU. After the establishment of micro/nano-crystallization conditions for NendoU, mix-and-inject TR-SFX studies will be performed at an x-ray free electron laser (XFEL) to capture snapshots of key reaction steps, including substrate binding as well as the catalytic reaction. Secondly, the mix-and-inject technology developed for Tr-SFX, will be adapted for TR-cryo-EM, which complement the TR-SFX study by unravelling the later time points in catalysis, including product release and the steady state. These time-resolved studies of NendoU will unravel the structure of the transition state and may provide a basis for development of novel drugs for severe coronavirus infections or investigations of the role of NendoU in suppression of the immune response. This RAPID award to Arizona State University is made by the Division of Biological Infrastructure 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.

2019年冠状病毒病（COVID-19）危机迫切需要了解SARS-CoV-2（CoV-2）蛋白质的结构和动力学，这种冠状病毒是这种严重传染病的原因。该奖项支持的研究将揭示最有趣和独特的CoV-2酶之一，RNA内切核糖核酸酶NendoU的催化机制，该酶仅在高毒力冠状病毒中发现。NendoU在限制宿主传感器对病毒RNA的传感中起关键作用，因此是在分子水平上阐明病毒致病机制的非常重要的靶标。 研究人员将使用先进技术和样品制备中的新创新相结合来制作“分子电影”，揭示NendoU蛋白的作用机制，以促进我们对病毒生物学的理解，并可能有助于识别潜在的治疗化合物。 这些研究的结果将通过在线研讨会、科学会议、公共数据库和同行评审期刊上的出版物分享。来自SARS、MERS和最近来自CoV-2的NendoU的静态结构已经在低温条件下通过标准晶体学确定，但是没有捕获到与底物RNA结合的结构，也没有对酶进行任何时间分辨的研究。这个COVID-2019 RAPID奖项专注于时间分辨研究，使用时间分辨飞秒晶体学（TR-SFX）和时间分辨冷冻电子显微镜（TR-cryo-EM）的组合来捕获NendoU催化反应的分子电影。在建立NendoU的微/纳米结晶条件后，将在X射线自由电子激光器（XFEL）下进行混合和注入TR-SFX研究，以捕获关键反应步骤的快照，包括底物结合以及催化反应。其次，为Tr-SFX开发的混合和注射技术将适用于TR-cryo-EM，其通过解开催化中的后期时间点（包括产品释放和稳态）来补充TR-SFX研究。NendoU的这些时间分辨研究将揭示过渡态的结构，并可能为开发治疗严重冠状病毒感染的新药或研究NendoU在抑制免疫反应中的作用提供基础。亚利桑那州立大学的RAPID奖由生物基础设施部使用冠状病毒援助，救济和经济安全（CARES）法案的资金颁发。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

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

• DOI: 10.1016/j.str.2022.12.009
• 发表时间: 2023-02-02
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Jernigan, Rebecca J.;Logeswaran, Dhenugen;Doppler, Diandra;Nagaratnam, Nirupa;Sonker, Mukul;Yang, Jay-How;Ketawala, Gihan;Martin-Garcia, Jose M.;Shelby, Megan L.;Grant, Thomas D.;Mariani, Valerio;Tolstikova, Alexandra;Sheikh, Michelle Z.;Yung, Mimi Cho;Coleman, Matthew A.;Zaare, Sahba;Kaschner, Emily K.;Rabbani, Mohammad Towshif;Nazari, Reza;Zacks, Michele A.;Hayes, Brandon;Sierra, Raymond G.;Hunter, Mark S.;Lisova, Stella;Batyuk, Alexander;Kupitz, Christopher;Boutet, Sebastien;Hansen, Debra T.;Kirian, Richard A.;Schmidt, Marius;Fromme, Raimund;Frank, Matthias;Ros, Alexandra;Chen, Julian J. -L.;Botha, Sabine;Fromme, Petra
• 通讯作者: Fromme, Petra