Capturing eukaryotic ribosome dynamics in situ at high resolution

Abstract

Many protein complexes are highly dynamic in cells; thus, characterizing their conformational changes in cells is crucial for unraveling their functions. Here, using cryo-electron microscopy, 451,700 ribosome particles from Saccharomyces cerevisiae cell lamellae were obtained to solve the 60S region to 2.9-Å resolution by in situ single-particle analysis. Over 20 distinct conformations were identified by three-dimensional classification with resolutions typically higher than 4 Å. These conformations were used to reconstruct a complete elongation cycle of eukaryotic translation with elongation factors (eEFs). We found that compact eEF2 anchors to the partially rotated ribosome after subunit rolling and hypothesize that it stabilizes the local conformation for peptidyl transfer. Moreover, open-eEF3 binding to a fully rotated ribosome was observed, whose conformational change was coupled with head swiveling and body back-rotation of the 40S subunit. This work reveals a eukaryotic ribosome elongation cycle in situ, uncovers over 20 structural conformations, highlights the new role of compact eukaryotic elongation factor 2 (eEF2) during peptidyl transfer and discovers new conformations of open-eEF3.