In this Project we will develop transformative tools for determining 3D structures of biomolecules, imaging cellular assemblies, and delivering cargos into target cells. Nanotechnology and structural biology, particularly cryo-EM, are advancing very rapidly, and we aim to contribute to these revolutions. Specifically, we will: 1) Develop macromolecular platforms that will enable the structures of even small proteins to be determined by cryo-EM single particle methods, The platforms will be large, rigid objects like microtubules, ribosomes, or antibody complexes that will bind the small target protein rigidly and add the mass needed to precisely align particles and in some case (like microtubules), introduce high symmetry. 2) Implement and develop micro- electron diffraction (microED)1-4 and racemic crystallographic methods for determining structures of challenging targets,5 MicroED can be applied to micron-sized crystals that are too small for standard X-ray crystallography. Racemic crystallography has promise for small proteins (<300 residues) that resist initial crystallization screens. 3) Develop CLEM methods for visualizing viruses and virus-associated complexes in their native state within cells, 4) Use enveloped protein nanocages6 to explore the fundamental biology of virus budding and develop systems for delivering specific cargoes into cells.