Project Summary/Abstract Overall, this project aims to develop methods to efficiently determine, at atomic-resolution, three-dimensional (3D) structures of large, native, symmetry-mismatched, locally dis-ordered or polymorphic biological complexes through cryo electron microscopy (cryoEM). The past funding cycle of this project (2014-2019) has witnessed a drastic transformation of the field of structural biology, popularly referred as “the cryoEM revolution”. Today, single-particle cryoEM has arguably become the default choice for structural biology. The funding from this project has enabled the PI’s group to contribute to this transformation in multiple fronts: we have developed novel imaging and processing methods and validated these methods by determining in situ structures of important—and sometimes fundamental—biological processes in large icosahedral and helical complexes, as well as atomic models of purified protein-nucleic acid complexes and membrane proteins that have been resistant to previous x-ray crystallography and NMR efforts. We hypothesize that combining electron counting (i.e., quantum) capabilities with cryoEM can advance the field by determining atomic models of native cellular complexes, viral genome packaging and transcription in situ and transiently stable catalytic intermediates. In the last funding period we aimed to, and succeeded in, derive atomic models using our cryoEM method alone for larger complexes, particularly those in icosahedral and helical viruses. Now, this renewal application aims to develop an integrative proteome cryoEM method for atomic structures of cellular complexes in their native, unpurified, and functional states. We will also use viruses as tools to address fundamental questions concerning genome packaging, compaction, supercoiling, replication and transcription. We will continue developing novel computational methods for sub-particle refinement and nucleic acid modeling, specifically cryoID, an integrative software package that allows near-atomic resolution cryoEM structures from many complexes in enriched cellular milieu to be determined, identified, and atomically modeled (Aim #1); sub-particle reconstructions and refinement for in situ atomic structures in large deformable or intrinsically structurally heterogeneous complexes such as those in large enveloped viruses and native cellular complexes (e.g., helical assemblies) (Aim #2); modeling genome structures in both RNA and DNA viruses, as well as cellular transcriptional/replicative complexes (Aim #3); and validate these new methods for atomic structure determination by application to red blood cell proteome, translocon bacteriocin nano-machines and membrane protein complexes, helical filamentous cellular (e.g., actin and axoneme) and viral assemblies and genome structures inside a number of ssRNA, dsRNA and dsDNA viruses (Aim #4). A successful outcome of this renewal project will further advance cryoEM in structural studies of large comple...