HIGH RESOLUTION 3-D RECONSTRUCTION OF VIRUSES USING SINGLE PARTICLE ELECTRON CR

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 病毒感染一直是并且仍然是人类健康的主要威胁之一。病毒是蛋白质和核酸的大型组合体，依赖宿主的感染来完成其生命周期并维持其繁殖。许多病毒由高度对称的二十面体蛋白质外壳组成，其中包裹着遗传物质。病毒必须发生多个过程，包括衣壳组装、基因组包装、成熟和病毒-宿主相互作用，才能成功完成其生命周期并对宿主造成严重的、通常是致命的损害。病毒颗粒的高分辨率 3D 结构（如果可用）将为理解这些过程以及制定有效的预防和治疗策略提供重要的见解。冷冻电子显微镜（cryo-EM）是一种用于大型大分子复合物和病毒结构测定的新兴技术。近年来，冷冻电镜技术取得了重大进展，将病毒结构解析到亚纳米分辨率 (6-10 ) 已成为常规，最近更接近原子分辨率 (~4 )。然而，冷冻电镜图像的图像处理和 3D 重建对计算资源要求很高。在这个提案中，我们申请计算资源来解决细菌病毒T7/T3在组装和DNA包装过程的多个阶段的结构，这是一个由NIH资助的活跃的R01项目。 T7 和 T3 是密切同源物，是 Podoviridae 家族中的双链 DNA 噬菌体，具有短尾并感染大肠杆菌。在本项目中，我们的目标是使用冷冻电镜和 3-D 重建技术来表征 T7/T3 衣壳组装、成熟和基因组包装的机制。对于噬菌体生命周期的四种主要状态，衣壳、衣壳 II、dsDNA 部分包装颗粒 (ipDNA-衣壳) 和最终的感染性颗粒，我们将纯化颗粒，使用冷冻电镜对颗粒进行成像，并通过图像处理和 3D 重建来解析高分辨率结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Viral infections have been and remain one of the major threats to human health. Viruses are large assemblies of proteins and nucleic acids that rely on infection of hosts to complete their life cycle and sustain their propagation. Many viruses consist of a highly symmetric icosahedral protein shell that encapsulates the genetic materials. Multiple processes, including capsid assembly, genome packaging, maturation and virus-host interaction, must occur for a virus to successfully complete its life cycle and cause severe, often fatal, damage to the host. High resolution 3-D structure of the virus particles, if available, will provide important insights to understanding of these processes and the development of effective prevention and treatment strategies. Cryo-electron microscopy (cryo-EM) is an emerging technique for structural determination of large macromolecular complexes and viruses. In recent years, there are significant progresses in cryo-EM and it is now becoming routine to solve the structure of a virus to sub-nanometer resolutions (6-10 ), and more recently to near atomic resolution (~4 ). However, the image processing and 3-D reconstruction of cryo-EM images are computational resource demanding. In this proposal, we apply for the computational resources to solve the structures of bacterial virus T7/T3 at their many stages of assembly and DNA packaging process, which is an active NIH-funded R01 project. T7 and T3, which are close homologues, are dsDNA phages in the Podoviridae family that have a short tail and infect E. coli.. In this project, we aim to characterize the mechanisms of T7/T3 capsid assembly, maturation and genome packaging using cryo-EM and 3-D reconstruction techniques. For each of the four major states of the phage life cycle, procapsid, capsid II, dsDNA partially packaged particles (ipDNA-capsid), and the final infectious particles, we will purify the particles, image the particles using cryo-EM, and solve the high resolution structure by image processing and 3-D reconstruction.