Our research focuses on elucidating the structure and function of macromolecular assemblies that regulate cellular growth using electron cryo-microscopy (cryoEM) as a primary tool. Recent advances in sample preparation, direct electron detector devices, and image processing algorithms have made cryoEM the method of choice to study large and conformationally heterogenous complexes that can only be obtained at low abundance. The focus of my lab will be on complexes involved in mammalian target of rapamycin (mTOR) signaling, specifically mTOR complex 1 and 2 and tuberous sclerosis complex (TSC). We will biochemically purify these molecular machines from both immortal human cell lines such as HEK293F cells and model organisms yeast S. cerevisiae and S. pombe. The structural knowledge gained from the endogenous complexes will facilitate design of agonist and antagonist that may modulate the aberrant function of proteins harbouring disease mutations.
In addition to conventional protein expression and purification, our group will develop affinity-based electron microscopy grids that will enable direct capture of endogenous protein complexes from cell lysate. Affinity-based capture will allow for rapid purification of endogenously expressed complexes for cryoEM analysis. This platform will be used to study the conformational and compositional dynamics of regulatory scaffolding proteins such as kinase suppressor of Ras 1 (KSR1). The shorten time of purification is critical to preserve the native state of highly dynamic multi-component molecular complexes.