Grant: Research Tools and Instruments

Equipment Requested: We are requesting support to establish a cryo-electron microscopy (cryo-EM) pipeline for protein structure elucidation. uOttawa is lacking a central component of this pipeline, an automated freeze-plunger for the preparation of vitrified protein grids. This equipment is not available in Roger Guindon Hall, the laboratory space of the participating co-applicants. The absence of such equipment is not only having a detrimental effect on the progress of uOttawa NSERC-funded research programs but is placing these programs at risk. A freeze-plunger is urgently needed to facilitate the structural characterization of biological macromolecules and for the training of our HQP in state-of-the-art structural biology methods. Research Activities to Support: Recent advancements have made it possible for scientists to visualize biological molecules at atomic resolution by single particle cryo-EM. Cryo-EM allows researchers to elucidate the structures of macromolecules, such as membrane proteins and large protein complexes, that are extremely difficult to crystallize and that are thus not amenable to traditional x-ray crystallographic approaches. The four co-applicants on this grant each have NSERC-funded research programs that will exploit this transformative technology to address fundamental questions regarding the structure and function of membrane proteins or protein complexes. Each of these labs is poised to make major breakthroughs in our understanding of protein structure-function relationships but in each case the inability to prepare cryo-EM grids at uOttawa is preventing or limiting the application of cryo-EM to their chosen targets. This application will support research focused on pentameric ligand-gated ion channels, lipid transporters, G-protein coupled hormone sensors, histone proteins, bacteriophages, and lipid metabolism enzymes, many of which are not stable enough for the long-distance transportation to remote cryo-EM facilities. There is thus an urgent need for the freeze-plunger for the preparation of vitrified protein sample grids at uOttawa. Expected Outcomes: The cryo-EM freeze plunger will allow researchers to elucidate structures of membrane proteins or protein complexes that previously were inaccessible by crystallographic methods. The in-house freeze plunger will allow uOttawa researchers to screen vitrification conditions and, once appropriate conditions are obtained, transport the prepared grids in liquid nitrogen to single-particle data collection facilities anywhere in the world. The freeze-plunger will be used in both graduate and undergraduate courses, which will expose our HQP to this state-of-the-art technology and thus equip them with the necessary research skills for career development and biotech jobs in Canada. The establishment of a cryo-EM pipeline will be transformative for several NSERC-funded research programs at uOttawa and will strengthen the training of over 100 HQP.