Breaking barriers in CryoEM through computational protein design

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ABSTRACT Using single-particle cryogenic electron microscopy (cryoEM) we can study proteins and protein complexes to atomic resolution and elucidate dynamics and distinct functional states in native or near-native environments. But this is virtually impossible to do with small proteins. My lab is interested in understanding protein function by observing their structures at high-resolution in native states. We leverage expertise in cryoEM and computational protein design to study proteins, both soluble and membrane, and their complexes that play major roles in human diseases. Membrane proteins are cellular gatekeepers and one of the most important class of membrane proteins are the G protein-coupled receptors (GPCRs), which act as signal conduits through ligand-induced binding on outside cells to the recruitment of binding complexes inside cells to relay signals. As they are involved in most cellular processes they are of considerable interest in drug development. While cryoEM has gained momentum in structural biology, it has a fundamental limitation that proteins smaller than 40 kDa cannot be studied effectively because the signal in the images is too low. This means that most proteins in the human genome cannot be studied by cryoEM. Using a designed approach, I was recently able to solve the high-resolution structure of a 17kDa protein by cryoEM, almost 3 times smaller than current cryoEM size limits. I succeeded because we used computational design to attach the 17 kDa protein to a scaffold which helped imaging by increasing the mass of the particle and the higher symmetry afforded better reconstruction. This proof-of-principal experiment demonstrates the powerful combination of using computational design for cryoEM. While exciting, this scaffold approach is still at its infancy and further design and development are needed to realize its full potential. New scaffolds capable of displaying important membrane proteins like GPCRs will be developed, tested and optimized. These new nanomaterials will be specifically tailored to address cryoEM needs. With this approach we will investigate membrane protein structure, describe functional dynamics in near native environments and facilitate rapid structure-guided drug design to help against devastating diseases.