In eukaryotic cells, cotranslational protein translocation across the endoplasmic reticulum (ER) membrane requires an elaborate macromolecular machinery. While structural details of ribosomes bound to purified and solubilized constituents of the translocon have been elucidated in recent years, little structural knowledge of ribosomes bound to the complete ER protein translocation machinery in a native membrane environment exists. Here, we used cryoelectron tomography to provide a three-dimensional reconstruction of 80S ribosomes attached to functional canine pancreatic ER microsomes in situ. In the resulting subtomogram average at 31 Å resolution, we observe direct contact of ribosomal expansion segment ES27L and the membrane and distinguish several membrane-embedded and lumenal complexes, including Sec61, the TRAP complex and another large complex protruding 90 Å into the lumen. Membrane-associated ribosomes adopt a preferred three-dimensional arrangement that is likely specific for ER-associated polyribosomes and may explain the high translation efficiency of ER-associated ribosomes compared to their cytosolic counterparts.
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