Establishing a cryo-EM pipeline for protein structure elucidation

请求设备：我们请求支持建立冷冻电子显微镜（cryo-EM）管道，用于蛋白质结构解析。渥太华缺乏这条管道的核心组成部分，用于制备玻璃化蛋白质网格的自动冷冻柱塞。Roger Guindon Hall是参与共同申请者的实验室空间，没有这个设备。这种设备的缺乏不仅对渥太华nserc资助的研究项目的进展产生了不利影响，而且使这些项目处于危险之中。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在最先进的结构生物学方法。研究活动支持：最近的进展使科学家能够通过单粒子冷冻电镜在原子分辨率上观察生物分子。Cryo-EM使研究人员能够阐明大分子的结构，例如膜蛋白和大蛋白复合物，这些大分子极难结晶，因此无法适应传统的x射线晶体学方法。该基金的四位共同申请者都有nserc资助的研究项目，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-功能关系的理解方面取得重大突破，但在每种情况下，渥太华大学无法制备冷冻电镜网格，这阻碍或限制了冷冻电镜在他们选择的目标上的应用。该应用程序将支持对五聚体配体门控离子通道、脂质转运体、g蛋白偶联激素传感器、组蛋白蛋白、噬菌体和脂质代谢酶的研究，其中许多蛋白不够稳定，无法远距离运输到远程低温电子显微镜设施。因此，渥太华大学迫切需要冷冻柱塞来制备玻璃化的蛋白质样品网格。预期结果：冷冻电镜冷冻柱塞将使研究人员能够阐明膜蛋白或蛋白质复合物的结构，这些结构以前是通过晶体学方法无法获得的。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，将准备好的液氮网格运输到世界任何地方的单颗粒数据收集设施。冷冻柱塞将在研究生和本科课程中使用，这将使我们的HQP接触到最先进的技术，从而为他们在加拿大的职业发展和生物技术工作提供必要的研究技能。低温电镜管道的建立将对渥太华大学nserc资助的几个研究项目产生革命性的影响，并将加强对100多名HQP的培训。

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.