PROJECT SUMMARY Cryogenic electron microscopy can generate atomic resolution views of cells and is therefore a promising technology to study the molecular mechanisms underlying key cellular processes in cells. However, cryo-EM images cannot be interpreted directly to atomic resolution because cryo-EM imaging introduces radiation damage to biological macromolecules. Current strategies to address radiation damage require combining multiple copies to generate a single 3D reconstruction representing an average of the individual molecules. This not suitable for cellular imaging because only a select few macromolecules are present in sufficiently high numbers to generate an interpretable average. As a postdoc I contributed to the development of an alternate, single molecule strategy to characterize the structure, interactions, and organization of macromolecular complexes in cryo-EM images called 2D template matching (2DTM). Since 2DTM does not require averaging, it has the potential to simultaneously visualize all cellular complexes individually, presenting a potentially revolutionary new understanding of cellular function in health and disease. However, the potential of 2DTM is currently limited by the difficulty to localize smaller complexes. With support from the NIH New Innovators Program, I will pursue new strategies to extend 2DTM to detect and characterize smaller structures. New technologies, particularly new imaging technologies, open new avenues of research because they allow us to ask questions and see things that were never previously possible. Opening the cell to visualization at the resolution of individual macromolecules will undoubtably reveal new, previously unanticipated biology.

PROEJCT NARRATIVE Cells are the fundamental unit of life. All cellular functions ultimately result from the structure, interactions, and organization of their component macromolecules. I will develop new methods using cryo-EM to visualize macromolecular structure within cells. This will provide previously inaccessible insight into the molecular mechanisms of cellular processes in health and disease.