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

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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.