Epitope dynamics of SARS-CoV-2 S protein by cryo-electron tomography and single particle cryo-EM

通过冷冻电子断层扫描和单粒子冷冻电镜观察 SARS-CoV-2 S 蛋白的表位动态

• 批准号: 10697877
• 负责人: Mario Borgnia
• 金额: $ 37.69万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697877
• 关键词: 2019-nCoV; Acquired Immunodeficiency Syndrome; Antigens; Biophysics; COVID-19; Cell membrane; Cells; Characteristics; Chimeric Proteins; Collaborations; Complex; Coronavirus; Crowding; Cryo-electron tomography; Cryoelectron Microscopy; Data; Data Collection; Development; Dissection; Electron Microscope; Environment; Epithelial; Epitopes; Evaluation; Goals; HIV; Hour; In Situ; Infection; Ions; Length; Ligands; Light; Lipid Bilayers; Machine Learning; Macromolecular Complexes; Maps; Masks; Mediating; Membrane; Methods; Modeling; Modernization; Molecular Conformation; Molecular Structure; National Center for Advancing Translational Sciences; National Institute of Environmental Health Sciences; Pathogenesis; Peptides; Process; Proteins; Publishing; Resolution; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Scanning; Series; Specimen; Structure; Surface; System; Techniques; Universities; Vaccines; Viral; Viral Proteins; Viral Vector; Virion; Virus Diseases; Virus Replication; base; cryogenics; design and construction; fight against; glycosylation; image processing; immunogenicity; improved; instrumentation; interest; macromolecule; mutant; neutralizing antibody; novel; novel strategies; particle; protein expression; protein purification; receptor; reconstruction; temporal measurement; tomography; tool; vaccine development; variants of concern

We have established high-throughput structure determination workflows using single particle analysis cryo-EM (SPA) on variants of the SARS-CoV-2 S protein ectodomain. These workflows allow us to solve the structure of macromolecular complexes of the spike at near atomic resolution with less than 24 hours of combined data collection and image processing. We also developed the first framework for automated evaluation of cryo-EM specimens using machine learning (Bouvette et. Al, eLife 2022). In collaboration with Dr. Robert Petrovich at the Protein Expression and Purification Facility, we established a pipeline to determine the structure of S-protein ectodomain from different variants of concern. Over the past two years we have used this method to solve the structure of the spike in complex with a variety of ligands in several parallel projects. In FY-2022 we published the results of two of these projects (Hong et al. PNAS 2022 and Fu et al. Plos One 2022) resulting from collaborations with the groups of Dr. Mitchell Ho at NCI and Dr. Matthew Hall at NCATs. We have deployed a cryo-electron tomography (cryo-ET) and sub-volume averaging (SVA) pipeline to determine the structure of full length (FL) S-protein and its complexes in the context of the viral envelope. In collaboration with Dr. Alberto Bartesaghi at Duke we are establishing modernized cryo-ET/SVA workflows that make use of the improved quality of DED data and incorporate novel image processing techniques to obtain high-resolution tomographic reconstructions, identify objects of interest in a crowded environment and determine their near-atomic resolution structure (Bouvette et al. Nat. Comm. 2021). In collaboration with Dr. Eric Freed at NCI and Dr. Negin Martin at the NIEHS Viral Vector Core we have established BSL-2 compatible pseudotyped viral systems for expression of wild type and mutant forms of type I fusion proteins from SARS-CoV-2 and HIV. These models will help shed light on aspects of the cellular pathogenesis of AIDS and COVID-19. We are currently using these systems in several collaborative projects aimed to a) characterize epitopes on the surface of the spike, b) map conformational changes along the maturation process, and c) map the interaction of S1/S2 with intracellular receptors and epithelial macromolecules. A model for the mechanism of fusion mediated by these proteins has been proposed based on their structures in the prefusion and post fusion states, and on biophysical data at much lower resolution. The model postulates conformational intermediates which are yet to be confirmed experimentally. Their elucidation will require the development of structural techniques with sufficient temporal resolution to capture intermediate snapshots. In collaboration with Dr. Tony Huang at Duke University, we are developing novel approaches to this problem. In addition to shedding light on the mechanism of fusion, this instrumentation will provide tools for the structural dissection of a wide variety of dynamic processes. Structural characterization of other stages of viral replication will require access to structural determination in situ. We are deploying a cryogenic focused ion beam scanning electron microscope. We will combine this new capability with our high throughput tomographic structure determination pipeline to characterize macromolecular complexes in the context of the cell.

我们已经建立了高通量的结构测定工作流程，使用单粒子分析cryo-EM（SPA）对SARS-CoV-2 S蛋白胞外域的变体。这些工作流程使我们能够在不到24小时的数据收集和图像处理时间内，以近原子分辨率解决尖峰的大分子复合物的结构。我们还开发了第一个框架，用于使用机器学习自动评估冷冻EM标本（Bouvette et. Al，eLife 2022）。与蛋白表达和纯化设施的Robert Petrovich博士合作，我们建立了一个管道来确定不同变体的S蛋白胞外域的结构。在过去的两年中，我们已经使用这种方法来解决的穗在复杂的各种配体在几个并行项目的结构。在2022财年，我们发表了其中两个项目（Hong et al. PNAS 2022和Fu et al. Plos One 2022）的结果，这些项目是与NCI的Mitchell Ho博士和NCAT的Matthew Hall博士合作完成的。 我们已经部署了冷冻电子断层扫描（cryo-ET）和子体积平均（SVA）管道，以确定全长（FL）S-蛋白及其复合物的病毒包膜的背景下的结构。与杜克大学的Alberto Bartesaghi博士合作，我们正在建立现代化的cryo-ET/SVA工作流程，该工作流程利用DED数据的改进质量，并结合新颖的图像处理技术，以获得高分辨率的断层扫描重建，在拥挤的环境中识别感兴趣的对象，并确定其近原子分辨率的结构（Bouvette et al. Nat. Comm. 2021）。与NCI的Eric Freed博士和NIEHS病毒载体核心的Negin Martin博士合作，我们建立了BSL-2兼容的假型病毒系统，用于表达SARS-CoV-2和HIV的I型融合蛋白的野生型和突变型。这些模型将有助于阐明艾滋病和COVID-19的细胞发病机制。我们目前正在几个合作项目中使用这些系统，旨在a）表征刺突表面的表位，B）绘制沿着成熟过程的构象变化，以及c）绘制S1/S2与细胞内受体和上皮大分子的相互作用。 基于它们在融合前和融合后状态的结构，以及在低得多的分辨率下的生物物理数据，已经提出了由这些蛋白质介导的融合机制的模型。该模型假设的构象中间体，尚未得到证实的实验。他们的阐明将需要开发足够的时间分辨率的结构技术，以捕捉中间快照。我们与杜克大学的Tony Huang博士合作，正在开发解决这一问题的新方法。除了阐明融合的机制外，该仪器还将为各种动态过程的结构解剖提供工具。病毒复制的其他阶段的结构表征将需要获得原位结构测定。我们正在部署低温聚焦离子束扫描电子显微镜。我们将联合收割机与我们的高通量层析结构测定管道结合起来，以表征细胞背景下的大分子复合物。