RII Track-4:NSF: Structures of Membrane Protein Assemblies Resolved by Cryogenic Electron Microscopy

以原子分辨率可视化蛋白质的三维结构对于理解其功能至关重要。从历史上看，一种叫做x射线晶体学的技术更倾向于获得蛋白质的原子水平结构。然而，晶体学是不希望研究大的和动态的蛋白质组装或蛋白质驻留在脂质膜。但由于具有这些特征的蛋白质是许多生物过程的关键，因此需要新的技术来可视化它们的原子结构，以了解它们的细胞功能。一种称为低温电子显微镜（cryogenic electron microscopy, cryoEM）的先进显微镜是研究这些具有挑战性的蛋白质的理想选择，最近的进展使其能够实现原子分辨率。该奖学金将支持PI和研究团队获得太平洋西北低温技术中心（PNCC）专家的低温技术实践培训，该中心是由俄勒冈健康与科学大学和太平洋西北国家实验室运营的最先进的用户设施。完成奖学金后，研究人员将成为低温专家，并将这些新知识应用于他们在原子水平上研究的大型膜嵌入蛋白质组装的可视化结构。此外，该奖学金将通过引进冷冻冷冻技术，加强内布拉斯加大学（NU）分子生命科学的研究和教育。这些专业知识将通过实验室和课堂经验传递给内布拉斯加州的其他研究人员和学生，向他们传授这种最先进的结构技术的理论和实践。由于内布拉斯加州没有任何机构具备低温低温技术、基础设施或专业知识，因此这项奖学金有可能改变内布拉斯加州的面貌。PI的团队使用低温技术的专业培训来推动他们的结构生物学能力在新的和变革的方向。CryoEM将使PI的团队能够解析他们研究的由小膜蛋白组成的超分子机器的3D结构，这将有助于定义驱动这些蛋白质自组装的机制原理。这些膜蛋白组合需要深入了解，因为它们控制着动物的两个关键生物过程——上皮细胞间空间的强化（称为紧密连接）和脂肪酸的合成（产生对膜组织至关重要的脂质）。该奖学金将通过在PI小组和PNCC之间建立协作培训生态系统来发挥作用，其主要目标是获得对低温低温工作流程的深入了解，其中包括样品制备；阴性染色EM；网格筛选与评价；图像采集;图像处理与粒子分类；三维模型的构建和细化；并用低温电镜进行结构测定。通过在内布拉斯加大学林肯分校（UNL）建立一个专家核心，这些专家将培养更多的NU研究人员，使许多团体能够使用cryoEM和国家中心（如PNCC）来确定其他重要大分子系统的结构，从而促进内布拉斯加州新的独立和合作研究。通过将这种培训整合到PI在UNL开展的咨询活动和本科生物化学课程中，也将实现向后代传授最先进的结构技术（如cryoEM）的理论和实践的教育目标。由于UNL， NU和其他地区机构目前缺乏冷冻em的能力，该奖学金将提供一个基本的知识库，这将对内布拉斯加州和大平原地区许多个人和团体的研究和教育轨迹产生广泛的影响。该奖项反映了美国国家科学基金会的法定使命，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。

Visualizing a protein’s 3D structure at atomic resolution is essential to understand its function. Historically, a technique called X-ray crystallography was preferred to obtain atomic-level structures of proteins. However, crystallography is undesirable for studying large and dynamic protein assemblies or proteins that reside in lipid membranes. But because proteins with these traits are key to many biological processes, new techniques to visualize their atomic structures are required to understand their cellular functions. An advanced form of microscopy called cryogenic electron microscopy (cryoEM) is ideal for studying these challenging proteins, and recent advances have made it capable of achieving atomic resolution. This fellowship will support the PI and research team in acquiring hands-on training in cryoEM by experts at the Pacific Northwest Center for CryoEM (PNCC), a state-of-the-art user facility operated by the Oregon Health & Science University and the Pacific Northwest National Laboratory. Upon completion of the fellowship, the researchers will be cryoEM specialists, and apply this new knowledge to visualize structures of the large membrane-embedded protein assemblies they study in atomic-level detail. Further, the fellowship will bolster research and education in the molecular life sciences at the University of Nebraska (NU) by importing cryoEM expertise there. This expertise will be passed to other researchers and students across Nebraska via lab-based and in-class experiences that will impart the theory and practice of this state-of-the-art structural technique to them. As no institution in the state has cryoEM capability, infrastructure, or expertise, this fellowship has potential to be transformative to Nebraskans.The PI’s group use the specialized training in cryoEM to propel their structural biology capacities in new and transformative directions. CryoEM will enable the PI’s group to resolve 3D structures of the small membrane protein-comprised supramolecular machines they study that will help define the mechanistic principles that drive self-assembly of these proteins. These membrane protein assemblies warrant in-depth comprehension because they control two key biological processes in animals—fortification of epithelial intercellular spaces called tight junctions, and synthesis of fatty acids that produce lipids vital to membrane organization. The fellowship will function by establishing a collaborative training ecosystem between the PI’s group and PNCC, with the primary goal to acquire deep knowledge of the cryoEM workflow, which includes sample preparation; negative stain EM; grid screening and evaluation; image collection; image processing and particle classification; 3D model building and refinement; and structure determination by cryoEM. This cryoEM expertise will foster new independent and collaborative research in Nebraska by creating a core of specialists at the University of Nebraska-Lincoln (UNL) that will train additional NU researchers, allowing many groups to use cryoEM and national centers like PNCC to determine structures of other important macromolecular systems. Educational objectives to teach future generations the theory and practice of state-of-the-art structural techniques like cryoEM will also be realized by integrating this training into the advising activities and undergraduate biochemistry courses the PI conducts at UNL. Because UNL, NU, and other regional institutions currently lack abilities in cryoEM, this fellowship will provide an essential knowledgebase that will have wide-reaching impacts on the research and educational trajectories of many individuals and groups throughout Nebraska and the Great Plains.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.