MCA: Application of Cryo-Electron Microscopy to Determine the Structure of Epigenetic Regulatory Complexes

单粒子冷冻电子显微镜（Cryo-EM）是研究细胞中蛋白质和其他分子结构的强大工具。捕捉蛋白质、蛋白质复合物、抗体和病毒的精美细节图像的能力已经被冷冻EM技术的最新进展所促进，被恰当地称为“分辨率革命”。“在密歇根大学和纽约国家冷冻EM获取和培训中心（NCCAT）的冷冻EM专家的支持下，PI将接受培训，并获得使用单粒子冷冻EM方法回答基因调控机制相关关键科学问题的新专业知识。 这种单粒子cryoEM培训将使PI能够以高分辨率获得蛋白质-DNA结构，而由于尺寸和其他限制，目前无法通过X射线晶体学或NMR获得这些结构。 Cryo-EM已成为现代生物研究的重要组成部分，迫切需要培养高技能的劳动力，以满足不断增长的需求。通过成为这一领域的专家，PI将振兴佛蒙特大学（UVM）现有的Cryo-EM设施，并为其他UVM教师、实验室人员、博士后、研究生、本项目的主要目标是获得领导和进行单粒子冷冻电子显微镜（Cryo-EM）的能力。阐明大蛋白质和低聚复合物的分子结构的实验。具体来说，该项目将研究染色质阅读器结构域，如布罗莫结构域，如何与组蛋白和核小体上的翻译后修饰相互作用，以调节必要的DNA模板化过程。利用Cryo-EM收集细胞内形成的天然复合物的结构数据，对于进一步了解表观遗传信号如何调节关键细胞过程至关重要。为了获得这种专业知识，PI制定了一项培训计划，从密歇根大学和国家冷冻EM访问和培训中心（NCCAT）的专家那里学习单粒子冷冻EM技术。通过这些伙伴关系，将建立数据处理和培训工作流程，以确定染色质调控复合物的结构。这一合作努力还将用于开发新的教育和培训资源，以促进在地方和国家一级获得冷冻EM技术。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学项目和刺激竞争性研究的既定项目（EPSCoR）共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Single particle Cryo-electron microscopy (Cryo-EM) is a powerful tool for studying the structure of proteins and other molecules in our cells. The ability to capture exquisitely detailed images of proteins, protein complexes, antibodies, and viruses has been facilitated by recent advancements in Cryo-EM technology, aptly termed the "resolution revolution." With the support of Cryo-EM experts at the University of Michigan and the National Center for Cryo-EM Access and Training (NCCAT) in New York, the PI will receive training and gain new expertise in using single-particle Cryo-EM methods to answer key scientific questions surrounding gene regulatory mechanisms. This training in single particle cryoEM will allow the PI to obtain protein-DNA structures at high resolution that are not currently available through X-ray crystallography or NMR due to size and other limitations. Cryo-EM has become an essential component of modern biological research, and there is a great need to train a highly skilled workforce to meet the growing demand. By becoming an expert in this area, the PI will revitalize an existing University of Vermont (UVM) Cryo-EM facility and set up training for other UVM faculty, lab personnel, postdocs, graduate, and undergraduate students.The primary objective of this project is to acquire the ability to lead and conduct single particle Cryo-electron microscopy (Cryo-EM) experiments to elucidate the molecular structures of large proteins and oligomeric complexes. Specifically, this project will investigate how chromatin reader domains, such as the bromodomain, interact with post-translational modifications on histone proteins and the nucleosome to regulate essential DNA templated processes. Utilizing Cryo-EM to collect structural data on the native complexes formed within the cell will be essential to further current knowledge of how epigenetic signaling regulates critical cellular processes. To obtain this expertise, the PI has developed a training plan to learn single-particle Cryo-EM techniques from experts at the University of Michigan and at the National Center for Cryo-EM Access and Training (NCCAT). Through these partnerships data processing and training workflows will be established to determine the structures of chromatin regulatory complexes. This collaborative effort will also be utilized to develop new educational and training resources that will facilitate access to Cryo-EM technology at the local and national levels.This project is jointly funded by the Molecular Biophysics Program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Established Program to Stimulate Competitive Research (EPSCoR).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.