Center for Structural Biology of HIV RNA

核心3.成像 摘要 CRNA成像核心的目的是为CRNA研究人员提供访问尖端成像的途径 在光学显微镜和低温电子显微镜(Cryo-EM)方面的方法和专业知识。核心意志 与CRNA调查人员合作，使用高分辨率光学显微镜定位和跟踪HIV-1 RNA- 蛋白质复合体的细胞背景，并使用单粒子冷冻电子显微镜(Cryo-EM)来 确定RNA、RNA-蛋白质复合体和HIV相关蛋白质复合体的结构。核心将起作用 通过集成活细胞和超分辨率光来弥合这两种方法之间的分辨率差距 在冷冻电子断层扫描(CRYO-ET)快速改进的方法的推动下，显微技术的发展得到了进一步的发展。这个 总的目标是能够确定HIV RNA-蛋白质复合体的3D结构，包括在体外和在 蜂窝环境。核心实验室由两名PI共同领导。密歇根大学的这一部分专注于 《冷冻-EM》由Pi Melanie Ohi执导。威斯康星大学的实验室由派·内森·谢勒领导，将 提供光学显微镜方法方面的资源和专业知识。密歇根大学核心实验室将为 作为材料和协议的储存库。核心团队将专注于三个目标，包括利用单一 用粒子冷冻-EM方法确定RNA、RNA-蛋白质复合体和HIV-1相关蛋白质的结构 开发有效的“样品到结构”管道，用于有效地分析样品，这将利用 核心的图像分辨率从~50？到亚3？的能力；并正在转向使用相关 光学和电子显微镜(Clem)和冷冻-ET检测和确定细胞内的结构。为了支持…… 作为其培训使命，核心还将为CRNA学员制定创新的教育计划，寻求 成为这些方法和技术的独立实践者。

Core 3. Imaging Summary The purpose of the CRNA Imaging Core is to provide CRNA investigators with access to cutting edge imaging approaches and expertise in both light microscopy and cryo-electron microscopy (cryo-EM). The Core will collaborate with CRNA investigators to use high-resolution light microscopy to localize and track HIV-1 RNA- protein complexes in their cellular context and to use single particle cryo-electron microscopy (cryo-EM) to determine structures of RNAs, RNA-protein complexes, and HIV-related protein complexes. The Core will work to bridge the resolution gap between the two approaches by integrating live cell and superresolution light microscopy approaches, facilitated by the rapidly improving methods in cryo-electron tomography (cryo-ET). The overall goal is to be able to determine 3D structures of HIV RNA-protein complexes both in vitro and in their cellular context. The Core lab is co-led by two PIs. The component at the University of Michigan is focused on cryo-EM and is directed by PI Melanie Ohi. The lab at the University of Wisconsin, run by PI Nathan Sherer, will provide resources and expertise in light microscopy approaches. The University of Michigan Core lab will serve as a repository for materials and protocols. The Core team will focus on three aims that include utilizing single particle cryo-EM approaches to determine structures of RNAs, RNA-protein complexes and HIV-1-related protein complexes; developing effective “sample-to-structure” pipelines for efficient analysis of samples that will leverage the Core’s ability to image at resolutions ranging from ~50 Å to sub-3 Å; and moving towards using correlative light and electron microscopy (CLEM) and cryo-ET to detect and determine structures inside cells. In support of its training mission, the Core will also develop innovative educational programs for CRNA trainees seeking to become independent practitioners of these methods and techniques.