BPS2026 – Cryo-EM investigations of the structure and mechanism of the R-body, a pH-sensitive protein machine

R-Bodies are microns-long protein ribbons assembled from thousands of copies of small Reb subunits. These ribbons adopt a squat, cylindrical coiled conformation in neutral pH environments, but rapidly extend into helical spears when exposed to acidic conditions. R-bodies occur in evolutionarily diverse bacteria and were originally discovered in endosymbionts of Paramecia where they are used by the host to kill competitors. The pH-dependent conformational switch is fully reversible over hundreds of cycles of extension and contraction, generates enough force to rupture membranes, and can be tuned by point mutations, making R-bodies attractive potential targets for bioengineering applications. Prior work in our lab has revealed that the R-body ribbon consists of a two-dimensional lattice of Reb subunits, the core of which is stable across pH. Thus, R-body extension appears not to be driven by subunit rearrangement. Instead, extension occurs via a pH-dependent disorder-to-order transition in the C termini of the major Reb subunits. The molecular mechanism of how this subunit-scale transition drives the extension of the R-body as a whole remains unclear. Our current work utilizes cryo-electron microscopy (cryo-EM) methods to investigate the structure of the R-body in its contracted and extended states to map subunit-level changes to the overall mechanism of extension. Using both cryo-EM single particle analysis and cryo-electron tomography (cryo-ET) we are beginning to resolve the features of the Reb subunits within the R-body ribbon. Additionally, we are investigating how these massive structures are assembled from individual subunits via cryo-EM analyses of subcomplexes.