Research in the Stewart lab is focused on applying cryoEM single particle reconstruction, as well as cryo electron tomography (cryoET) and hybrid structural methods, to a variety of macromolecular complexes. CryoEM single particle reconstruction has emerged as a powerful technique for determining atomic, or near-atomic, resolution structures of proteins and macromolecular complexes. Current cryoEM single particle research projects include human adenovirus and human papillomavirus interactions with host cell factors, bacterial heavy-metal efflux complexes, and engineered plant virus-based nanoparticles for therapeutic and imaging applications. The cryoEM single particle approach involves averaging multiple 2D projection images of different particles to generate a 3D structure and thus requires a specimen with a uniform or homogeneous structure. When a cryoEM specimen has a heterogeneous structure, for example an engineered viral particle decorated with an immune shielding protein attached via a flexible linker, cryoET provides an alternate way to generate a 3D structure. This approach involves the collection of a tilt-series of 2D projection images for a defined sample area and generation of a 3D tomogram. Recent cryoET projects include binary mixed polymer brush-grafted silica nanoparticles in aqueous and organic solvents; elongated plant virus-based nanoparticles for enhanced delivery of thrombolytic therapies; and serum albumin-camouflaged tobacco mosaic virus nanoparticles. Whenever possible the lab utilizes hybrid structural approaches, combining cryoEM single particle and cryoET data with information derived from X-ray crystallography, NMR, EPR, molecular dynamics flexible fitting guided by cryoEM structures, and de novo structure prediction methods.
Cleveland Center for Membrane and Structural Biology
Department of Pharmacology