RII Track-4: Molecular Visualization by Single Particle Cryo-Electron Microscopy

原子分辨率的生物大分子可视化为深入了解基本的生物过程和生命规律提供了重要的信息。传统上，x射线晶体学和核磁共振是仅有的两种可用的结构工具，可以在原子水平上实验确定生物大分子的三维（3D）结构。先进的显微镜技术——单粒子冷冻电子显微镜（cryo-EM）的仪器和方法的最新进展改变了分子可视化的游戏规则。低温电镜技术已成为确定大型大分子复合物或膜蛋白三维结构的首选技术，这对两种传统结构技术中的任何一种都是巨大的挑战。该奖学金将支持PI在加州理工学院冷冻电子显微镜中心发展冷冻电子显微镜专业知识，并将冷冻电子显微镜技术应用于原子水平上生物分子的结构表征。将冷冻电镜添加到PI的结构工具箱中，不仅会激发内布拉斯加大学林肯分校PI研究小组的新研究方向，而且还会使内布拉斯加州的许多研究小组受益，内布拉斯加州是唯一一个拥有10强大学但没有冷冻电镜专业知识或仪器的州。在该奖学金的支持下，PI及其小组成员将接受cryo-EM金属蛋白和组装体结构表征的全面培训，从网格制备，图像收集到3D地图构建和模型开发。特别是，该项目将重点研究两个相互连接的金属蛋白家族，分别参与细菌氧化还原应激反应和过渡金属稳态。从这次培训中获得的低温电镜专业知识将推动PI的团队在短期和长期内成功实现研究目标。该提案所产生的结构信息将填补在细菌对生物和非生物氧化还原应激和抗生素耐药性反应中控制金属蛋白的基本结构原理的关键空白。从长远来看，将低温电镜专业知识添加到PI的跨学科技能中，将更好地使PI在氧化还原生物学中金属蛋白的结构-机制研究中发挥主导作用。在教育方面，本提案将为研究生和博士后提供使用低温电镜技术的直接培训机会。此外，获得的冷冻电镜应用知识将被整合到PI在UNL进行的教学和培训活动中，与PI的NSFCAREER奖的教育目标保持一致。此外，通过与内布拉斯加大学系统的同事合作，PI的低温电子显微镜专业知识将使新的研究探索和催化跨校园的新合作，促进创新研究。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。

Visualization of biomacromolecules at atomic resolutions provides important informationfor the in-depth understanding of the fundamental biological processes and the rules of life.Traditionally, X-ray crystallography and nuclear magnetic resonance have been the only twoavailable structural tools that can experimentally determine three-dimensional (3D) structures ofbiomacromolecules at atomic levels. Recent advances in the instrumentation and methodology ofthe advanced microscopy technique called single-particle cryo-electron microscopy (cryo-EM) isa game-changer for molecular visualization. The cryo-EM technique has become the preferredtechnique for determining the 3D structure of large macromolecular complex or membraneproteins that have been dauntingly challenging to either of the two conventional structuraltechniques. This fellowship will support the PI to develop the cryo-EM expertise at the Cryo-EMCenter of the California Institute of Technology and apply the cryo-EM technique to the structuralcharacterization of biomolecules at the atomic level. Adding cryo-EM to the PI’s structural toolboxwill not only stimulate new research directions in the PI’s research group at the University ofNebraska-Lincoln, but also benefit many research groups across the state of Nebraska, which isthe only state that hosts a Big10 university but without expertise or instrumentation for cryo-EM.With the support of this fellowship, the PI and her group member will receivecomprehensive training on structural characterization of metalloproteins and assemblies by cryo-EM, from grid preparation, image collection to 3D map construction and model development. Inparticular, this project will focus on the structural investigation of two families of interconnectedmetalloproteins involved in bacterial redox stress response and transition metal homeostasis,respectively. The expertise in cryo-EM acquired from this training will propel the PI’s group tosuccessfully achieve research goals in both the short and long term. The structural informationresulted from the proposal will fill critical gaps in fundamental structural principles that governthe metalloproteins in the bacterial response to biotic and abiotic redox stresses and antibioticresistance. In the long run, adding the cryo-EM expertise to the PI’s interdisciplinary skillset willbetter position the PI to play a leading role in structure-mechanism investigations ofmetalloproteins in redox biology. From an educational aspect, this proposal will provide directtraining opportunities to graduate students and postdocs using the cryo-EM technique. In addition,the acquired knowledge of the application of cryo-EM will be integrated into the teaching andtraining activities conducted by the PI at UNL, aligning with the educational goal in the PI’s NSFCAREER award. Furthermore, by working with the colleagues at the University of Nebraskasystem, the PI’s cryo-EM expertise will enable new research explorations and catalyze newcollaboration across the campus and promote innovation research.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.