Altering electron-induced radiolysis to optimize cryo-EM/ET imaging

PROJECT SUMMARY Living cells have a complex and often precise organization in space and time. Determining the three- dimensional structure of proteins and other biomolecules, as well as understanding how they form functional networks in vivo, is a major goal of modern biology. Answering these questions is paramount to understanding

both the normal functions of proteins, as well as their dysfunctions. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) are powerful imaging tools that enable visualization and structural determination of native macromolecular complexes in vitro and in situ. Researchers use cryo-EM to resolve

isolated (macro)molecules at near-atomic or atomic resolution, whereas cryo-electron tomography can visualize macromolecules and organelles inside unperturbed cells with molecular to near-atomic resolution. Together, cryo-EM and cryo-ET have the potential to reveal a more comprehensive and detailed (atomic-level)

picture of the spatiotemporal organization and inner workings of cells. However, to fully realize the potential of cryo-EM/ET imaging techniques, we need new tools and approaches that can address outstanding technical limitations, such as radiation damage of frozen-hydrated biological specimens and the localization of specific

molecules in cryo-tomograms. Therefore, we will develop a new sample preparation strategy that can reduce electron-induced radiolysis of frozen-hydrated specimens, thereby improving the resolution of cryo-EM/ET images and/or the speed of structure determination (Aim 1). Additionally, we plan to develop a cloneable,

hyper-bubbling protein tag that would allow the precise localization of target proteins in otherwise noisy, and difficult-to-parse, cryo-tomograms (Aim 2). We will then apply these new tools to biological model systems (i.e. rapidly frozen and cryo-FIB milled E. coli and yeast cells) as proof of principle. The successful fulfillment of our

research aims will further enhance the revelatory power of cryo-EM/ET techniques and illuminate the complex and dynamic relationship between molecular structure and function.