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

Visualization of biomacromolecules at atomic resolutions provides important information for 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 two available structural tools that can experimentally determine three-dimensional (3D) structures of

biomacromolecules at atomic levels. Recent advances in the instrumentation and methodology of the advanced microscopy technique called single-particle cryo-electron microscopy (cryo-EM) is a game-changer for molecular visualization. The cryo-EM technique has become the preferred technique for determining the 3D structure of large macromolecular complex or membrane

proteins that have been dauntingly challenging to either of the two conventional structural techniques. This fellowship will support the PI to develop the cryo-EM expertise at the Cryo-EM Center of the California Institute of Technology and apply the cryo-EM technique to the structural characterization of biomolecules at the atomic level. Adding cryo-EM to the PI’s structural toolbox

will not only stimulate new research directions in the PI’s research group at the University of Nebraska-Lincoln, but also benefit many research groups across the state of Nebraska, which is the 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 receive

comprehensive training on structural characterization of metalloproteins and assemblies by cryo- EM, from grid preparation, image collection to 3D map construction and model development. In particular, this project will focus on the structural investigation of two families of interconnected metalloproteins 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 to successfully achieve research goals in both the short and long term. The structural information resulted from the proposal will fill critical gaps in fundamental structural principles that govern the metalloproteins in the bacterial response to biotic and abiotic redox stresses and antibiotic

resistance. In the long run, adding the cryo-EM expertise to the PI’s interdisciplinary skillset will better position the PI to play a leading role in structure-mechanism investigations of metalloproteins in redox biology. From an educational aspect, this proposal will provide direct training 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 and training activities conducted by the PI at UNL, aligning with the educational goal in the PI’s NSF CAREER award. Furthermore, by working with the colleagues at the University of Nebraska system, the PI’s cryo-EM expertise will enable new research explorations and catalyze new

collaboration 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.