RII Track-4: Strengthening structural biology research with single particle cryo-electron microscopy (cryo-EM)

我们对原子如何在分子中排列的理解在某种程度上依赖于如何将它们可视化。能够可视化分子结构的仪器正在开发中，因此新开发的仪器可能只在少数地方可用。该奖学金将提供一种称为单粒子冷冻电子显微镜（Cryo-EM）的高级显微镜形式的综合培训。一旦在国家大分子成像中心（NCMI）接受培训，PI将使用该设备在原子水平上可视化生物分子结构。这将加强蒙大拿大学综合结构生物学工具的开发。对大分子原子的三维排列的了解使理解关键生物过程的基本机制细节和生物分子之间的相互作用成为可能。多年来，x射线晶体学是测定大分子结构最常用的方法；然而，许多重要的分子，包括膜蛋白，不容易结晶。由于cryo-EM作为结构生物学的下一代技术的最新发展，现在可以分析大型和动态复杂的组件。NCMI是少数几个配备专家的国家低温电镜核心设施之一，提供培训和自动化单粒子数据采集。通过与德克萨斯州休斯顿贝勒大学NCMI的合作访问，PI和一名实习学生将获得cryo-EM的深入知识和操作cryo-EM仪器的实践经验。该奖学金将为PI提供Cryo-EM和相关技术的培训，这些技术将用于他对特定蛋白质结构和功能的分析。特别是，该项目将涉及哺乳动物ric8a及其相关结合化合物的表征，以及沙粒病毒包膜糖蛋白。NCMI的整体低温电镜训练包括样品制备，二维（2-D）数据成像，单粒子对齐和平均，三维地图构建和模型开发。培训的重要部分还将集中在使用最新的软件包进行数据采集、三维重建、结构分析和冷冻电镜结果可视化的实践。这次冷冻电镜培训将加强NCMI和蒙大拿大学的研究人员之间的合作，他们希望将他们的结构生物学研究扩展到包括冷冻电镜技术，特别是对那些表现出构象异质性的样品，使它们不适合x射线晶体学分析。

Non-technical DescriptionOur understanding of how atoms are arranged in molecules relies to some extent to how they can be visualized. Instruments capable of visualization of molecular structures are developing, such that newly developed instruments may be only available at a few locations. This fellowship will enable comprehensive training in an advanced form of microscopy called single-particle Cryo-Electron Microscopy(Cryo-EM). Once trained at the National Center for Macromolecular Imaging (NCMI), the PI will use this equipment to visualize biomolecular structures at the atomic level. This will strengthen the development of comprehensive structural biological tools at the University of Montana. Knowledge of the three-dimensional arrangement of macromolecular atoms makes it possible to understand fundamental mechanistic details of critical biological processes and the interactions between biomolecules. For many years, X-ray crystallography was the most common method for determining macromolecular structure; however, many important molecules, including membrane proteins, do not readily crystallize. Thanks to the recent development of cryo-EM as a next-generation technology for structural biology, analysis of large and dynamic complex assembles is now possible. The NCMI is one of a few national cryo-EM core facilities staffed with experts to provide training and automated single-particle data acquisition. Through collaborative visits with NCMI at Baylor University in Houston, TX, the PI and a student trainee will receive in-depth knowledge of cryo-EM and hands-on experience to operate cryo-EM instrumentation. Technical DescriptionThe fellowship will provide the PI with training on Cryo-EM and associated techniques, which will be used in his work on the analysis of the structure and function of specific proteins. In particular, the project will involve characterization of mammalian Ric-8A and its associated binding compounds, and the arenavirus envelope glycoprotein. The overall cryo-EM training at NCMI includes sample preparation, 2-dimensional (2-D) data imaging, single-particle alignment and averaging, 3-D map construction, and model development. A significant portion of the training will also focus on practicing with the latest software packages developed for data acquisition, 3-D reconstructions, structural analysis and visualization of the cryo-EM results. This cryo-EM training will strengthen collaborations between the NCMI and researchers from the University of Montana who wish to extend their research on structural biology to include cryo-EM technology, especially for samples that exhibit conformational heterogeneity, making them unsuitable for X-ray crystallographic analysis.