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3D结构对于理解其功能至关重要。从历史上看，一种称为X射线晶体学的技术被用来获得蛋白质的原子级结构。然而，晶体学对于研究大的和动态的蛋白质组装体或存在于脂质膜中的蛋白质是不期望的。但是，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（cryoEM）的先进显微镜形式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和研究团队获得西北太平洋冷冻EM中心（PNCC）专家的冷冻EM实践培训，PNCC是由俄勒冈州健康科学大学和西北太平洋国家实验室运营的最先进的用户设施。完成研究后，研究人员将成为cryoEM专家，并应用这些新知识来可视化他们在原子级细节中研究的大型膜嵌入蛋白质组装体的结构。此外，该奖学金将通过引进cryoEM专业知识来支持内布拉斯加大学（NU）分子生命科学的研究和教育。这种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，这些经验将向他们传授这种最先进的结构技术的理论和实践。由于该州没有机构拥有cryoEM能力，基础设施或专业知识，该奖学金有可能对内布拉斯加人产生变革性影响。PI的团队使用cryoEM的专业培训，以推动他们在新的和变革性方向上的结构生物学能力。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械原理。这些膜蛋白组装体值得深入理解，因为它们控制着动物体内两个关键的生物过程--上皮细胞间隙（称为紧密连接）的强化和脂肪酸的合成，脂肪酸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cryoEM工作流程的深入知识，其中包括样品制备;负染色EM;网格筛选和评估;图像收集;图像处理和颗粒分类; 3D模型构建和改进;以及cryoEM的结构确定。这种cryoEM专业知识将通过在内布拉斯加大学林肯分校（UNL）创建一个专家核心来促进内布拉斯加州新的独立和合作研究，该核心将培训更多的NU研究人员，使许多团体能够使用cryoEM和PNCC等国家中心来确定其他重要大分子系统的结构。教育目标，教导后代的理论和实践的国家的最先进的结构技术，如cryoEM也将通过整合到PI在UNL进行的咨询活动和本科生生物化学课程的培训实现。由于UNL、NU和其他地区机构目前缺乏冷冻EM能力，这个奖学金将提供一个基本的知识基础，将有广泛的-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进行评估，被认为值得支持的搜索.

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.