PROJECT SUMMARY Understanding how proteins interact within the cell to perform specific functions is a major goal of modern biology, and vital for understanding the diverse roles these molecules play in biomedicine. Cryo-electron tomography (cryo-ET) combined with sub-volume averaging (SVA) is currently the only imaging technology that allows imaging macromolecules within their unperturbed native environment at nanometer resolutions. Most successful studies, however, have been of large complexes or supramolecular assemblies, and at resolutions that are too low to reveal molecular level interactions. The overall objective of this Technology Development project is to design computational tools to improve the resolution of cryo-ET/SVA and extend its applicability to a wider class of biomedically relevant targets. The specific aims are: (1) we will develop strategies to improve the accuracy of the tilted contrast transfer function determination from low-dose tomographic projections, (2) we will design algorithms to improve the accuracy of sub-volume alignment, reconstruction and classification aimed at reducing the computational B-factors associated with data processing, and (3) we will optimize imaging and data processing parameters to enable high-resolution studies of a wider class of targets including small complexes. As proof of principle, we implemented a first-generation prototype of our platform and tested it on monodisperse samples imaged by cryo-ET. The preliminary results demonstrate that our platform: (1) improves the state-of-the-art in terms of achievable resolution, and (2) can be used to determine the structure of a 300kDa enzyme at 3.9 Å resolution, representing a ground-breaking achievement for the field. Our research is innovative because it seeks to overcome fundamental technical challenges in cryo-ET needed to realize the full potential of this emerging imaging technology. The proposal is significant because it will be the first demonstration that low- molecular weight targets can be imaged at near-atomic resolution using cryo-ET, indicating that this technique is the most promising route for imaging important biomolecules in-situ. Ultimately, by closing the “resolution gap” between strategies for studying monodisperse samples at high-resolution (X-ray, NMR and single-particle cryo- EM) and techniques to study proteins in their native environments, our methods will allow the visualization of protein complexes in their functional state at unprecedented levels of detail.