Epitope dynamics and functional analysis of viral envelope glycoprotein by cryo-electron tomography and single particle cryo-EM

我们已经建立了高通量的结构确定工作流程，使用单粒子分析冷冻-EM(SPA)对SARS-CoV-2 S蛋白胞外结构域的变体进行分析。这些工作流程使我们能够在接近原子分辨率的情况下，以不到24小时的数据收集和图像处理组合来解决SPEK的大分子络合物的结构。我们还开发了第一个使用机器学习对低温EM样本进行自动评估的框架(Bouvette et.Al，eLife 2022)。在蛋白质表达和纯化设施的罗伯特·彼得罗维奇博士的合作下，我们建立了一条管道，从不同的关注变体中确定S蛋白胞外结构域的结构。在过去的三年里，我们已经在几个平行的项目中使用这种方法来解决各种配体络合物中的尖峰结构。在2022财年，我们公布了其中两个项目的结果(Hong et al.PNAS 2022和Fu等人。PLOS One 2022)，这是与NCI的Mitchell Ho博士和NCATS的Matthew Hall博士合作的结果。 我们已经部署了冷冻电子断层扫描和亚体积平均管道来确定病毒被膜全长(FL)S蛋白及其复合体的结构。与杜克大学的Alberto Bartesaghi博士合作，我们正在建立现代化的冷冻/SVA工作流程，利用改进的DED数据质量并结合新的图像处理技术来获得高分辨率断层扫描重建，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结构(Bouvette等人)。纳特。通讯。2021年)。在与NCI的Eric Freed博士和NIEHS病毒载体核心的Negin Martin博士的合作下，我们建立了BSL-2兼容的伪型病毒系统，用于表达SARS-CoV-2和HIV的I型融合蛋白的野生型和突变形式。这些模型将有助于阐明艾滋病和新冠肺炎的细胞发病机制。我们目前正在几个合作项目中使用这些系统，旨在a)表征棘突表面的表位，b)映射成熟过程中的构象变化，以及c)映射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的结构，以及低分辨率的生物物理数据，提出了一个由这些蛋白质介导的融合机制的模型。该模型假设了构象中间体，这些构象中间体尚未得到实验证实。它们的阐明将需要开发具有足够时间分辨率的结构技术来捕获中间快照。与杜克大学的Tony Huang博士合作，我们正在开发解决这个问题的新方法。除了揭示核聚变的机制外，这种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特征将需要获得原位结构测定。我们正在部署一台低温聚焦离子束扫描电子显微镜。我们将把这一新能力与我们的高通量断层扫描结构确定流水线相结合，以在细胞环境中表征大分子复合体。

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 three 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.