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

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.