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

单粒子低温电子显微镜(Cryo-EM)是研究细胞中蛋白质和其他分子结构的有力工具。冷冻-EM技术的最新进展促进了捕捉蛋白质、蛋白质复合体、抗体和病毒的精细图像的能力，这一技术被恰如其分地称为“分辨率革命”。在密歇根大学和纽约国家冷冻-EM访问和培训中心(NCCAT)的低温电磁专家的支持下，PI将接受培训，并获得使用单粒子低温-EM方法回答围绕基因调控机制的关键科学问题的新专业知识。这种单粒子低温电子显微镜的训练将使PI能够获得高分辨率的蛋白质-DNA结构，而由于尺寸和其他限制，目前无法通过X射线结晶学或核磁共振获得这些结构。低温EM已经成为现代生物研究的重要组成部分，迫切需要培养一支高技能的劳动力来满足日益增长的需求。通过成为这一领域的专家，PI将重振佛蒙特州大学(UVM)现有的Cryo-EM设施，并为其他UVM教员、实验室人员、博士后、研究生和本科生建立培训。该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)实验的能力，以阐明大型蛋白质和寡聚复合体的分子结构。具体地说，这个项目将研究染色质读取域，如溴域，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模板过程。利用Cryo-EM收集细胞内形成的天然复合体的结构数据，对于进一步了解表观遗传信号如何调控关键的细胞过程是必不可少的。为了获得这方面的专业知识，国际电工学会制定了一项培训计划，向密歇根大学和国家低温电磁获取与培训中心(NCCAT)的专家学习单粒子低温电磁技术。通过这些伙伴关系，将建立数据处理和培训工作流程，以确定染色质管制复合体的结构。这一合作努力还将被用于开发新的教育和培训资源，以促进在地方和国家层面上获得低温EM技术。该项目由生物科学局分子和细胞生物科学部的分子生物物理学计划和既定的刺激竞争研究计划(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智力优势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.