Quantitative Analysis of Late-Stage Ribosome Assembly with Cryo-EM

Abstract

Despite the “resolution revolution” in cryo-electron microscopy (cryo-EM), compositionally and conformationally dynamic systems still remain a challenge for traditional cryo-EM workflows. Recently, we developed a novel cryo-EM workflow to harvest all of the information content possible with current computational tools from compositionally and conformationally dynamic ribosome assembly intermediates. This workflow includes modifications of conventional classification strategies--adding steps for iterative sub-classification, a statistically significant difference analysis derived from the signal-to-noise ration from cryo-EM maps, a quantitative strategy for deciding on the number of classes, and a catalog of features to facilitate interpretation of the massive amounts of data gathered from cryo-EM data collection. We developed this strategy to interrogate the process of ribosome assembly. The ribosome is a large, cellular machine responsible for protein biosynthesis. While much is known about how ribosomes translate mRNA into proteins, comparatively little is known about how this machine assembles in the first place. Furthermore, what is known about ribosome assembly focuses on protein binding networks and excludes information about rRNA folding. Recently, the Williamson lab has developed a novel genetic approach to put the r-protein bL17 under the control of a titratable promoter that controls the level of r-protein available to the host (bL17-lim). As a consequence, ribosome assembly intermediates build up and can be analyzed by cryo-EM. Here, we present our latest work applying our workflow to new r-protein limitation strains to delineate the effects that were specific to bL17 depletion from a more general ribosome assembly pathway. We use this data to build a map of interdependence between r-protein binding and rRNA folding downstream of bL17 and build on our knowledge of these interactions to determine a potential mechanism for ribosome assembly in vivo.