Establishing a cryo-EM pipeline for protein structure elucidation

建立用于蛋白质结构阐明的冷冻电镜管道

• 批准号: RTI-2023-00484
• 负责人: Baenziger, John
• 金额: $ 10.93万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744311
• 关键词: Establishing; cryo; EM; pipeline; protein

Equipment Requested: We are requesting support to establish a cryo-electron microscopy (cryo-EM) pipeline for protein structure elucidation. uOttawa is lacking a central component of this pipeline, an automated freeze-plunger for the preparation of vitrified protein grids. This equipment is not available in Roger Guindon Hall, the laboratory space of the participating co-applicants. The absence of such equipment is not only having a detrimental effect on the progress of uOttawa NSERC-funded research programs but is placing these programs at risk. A freeze-plunger is urgently needed to facilitate the structural characterization of biological macromolecules and for the training of our HQP in state-of-the-art structural biology methods. Research Activities to Support: Recent advancements have made it possible for scientists to visualize biological molecules at atomic resolution by single particle cryo-EM. Cryo-EM allows researchers to elucidate the structures of macromolecules, such as membrane proteins and large protein complexes, that are extremely difficult to crystallize and that are thus not amenable to traditional x-ray crystallographic approaches. The four co-applicants on this grant each have NSERC-funded research programs that will exploit this transformative technology to address fundamental questions regarding the structure and function of membrane proteins or protein complexes. Each of these labs is poised to make major breakthroughs in our understanding of protein structure-function relationships but in each case the inability to prepare cryo-EM grids at uOttawa is preventing or limiting the application of cryo-EM to their chosen targets. This application will support research focused on pentameric ligand-gated ion channels, lipid transporters, G-protein coupled hormone sensors, histone proteins, bacteriophages, and lipid metabolism enzymes, many of which are not stable enough for the long-distance transportation to remote cryo-EM facilities. There is thus an urgent need for the freeze-plunger for the preparation of vitrified protein sample grids at uOttawa. Expected Outcomes: The cryo-EM freeze plunger will allow researchers to elucidate structures of membrane proteins or protein complexes that previously were inaccessible by crystallographic methods. The in-house freeze plunger will allow uOttawa researchers to screen vitrification conditions and, once appropriate conditions are obtained, transport the prepared grids in liquid nitrogen to single-particle data collection facilities anywhere in the world. The freeze-plunger will be used in both graduate and undergraduate courses, which will expose our HQP to this state-of-the-art technology and thus equip them with the necessary research skills for career development and biotech jobs in Canada. The establishment of a cryo-EM pipeline will be transformative for several NSERC-funded research programs at uOttawa and will strengthen the training of over 100 HQP.

所需设备：我们正在请求支持，以建立用于蛋白质结构解析的冷冻电子显微镜（cryo-EM）管道。uOttawa缺乏这条管道的核心组件，即用于制备玻璃化蛋白质网格的自动冷冻柱塞。该设备在参与共同申请人的实验室空间Roger Guindon Hall中不可用。缺乏这种设备不仅对uOttawa NSERC资助的研究项目的进展产生不利影响，而且使这些项目处于危险之中。一个冷冻柱塞是迫切需要的，以促进生物大分子的结构表征和培训我们的HQP在国家的最先进的结构生物学方法。 支持的研究活动：最近的进展使科学家能够通过单粒子冷冻EM以原子分辨率可视化生物分子。Cryo-EM使研究人员能够阐明大分子的结构，如膜蛋白和大蛋白复合物，这些大分子非常难以结晶，因此不适合传统的X射线晶体学方法。这项资助的四个共同申请人都有NSERC资助的研究项目，这些项目将利用这种变革性技术来解决有关膜蛋白或蛋白质复合物的结构和功能的基本问题。这些实验室中的每一个都准备在我们对蛋白质结构与功能关系的理解方面取得重大突破，但在每种情况下，uOttawa无法制备冷冻EM网格都阻碍或限制了冷冻EM在其选定目标上的应用。该应用程序将支持重点研究五聚体配体门控离子通道，脂质转运蛋白，G蛋白偶联激素传感器，组蛋白，噬菌体和脂质代谢酶，其中许多都不够稳定，无法长距离运输到远程cryo-EM设施。因此，迫切需要在uOttawa制备玻璃化蛋白质样品网格的冷冻柱塞。预期结果：cryo-EM冷冻柱塞将使研究人员能够阐明以前无法通过晶体学方法获得的膜蛋白或蛋白质复合物的结构。内部冷冻柱塞将允许uOttawa研究人员筛选玻璃化条件，一旦获得适当的条件，将液氮中准备好的网格运输到世界任何地方的单粒子数据收集设施。冷冻柱塞将在研究生和本科生课程中使用，这将使我们的HQP接触到这种最先进的技术，从而为他们的职业发展和加拿大生物技术工作提供必要的研究技能。低温EM管道的建立将对uOttawa的几个NSERC资助的研究项目产生变革性影响，并将加强对100多名HQP的培训。