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.
Our lab studies the structure of engineered adenovirus vectors and their complexes with immune molecules. More specifically we focus on understanding the molecular mechanisms underlying systemic inflammation and how targeted viral capsid mutations might alter host-pathogen interactions. We utilize cryo-electron microscopy (cryo-EM) single particle reconstruction and tomography, molecular dynamics and molecular modeling.