Netherlands Organisation for Scientific Research (NWO)
724.016.001
Netherlands
Netherlands Organisation for Scientific Research (NWO)
212.152
Netherlands
Citation
Journal: Nature / Year: 2023 Title: Visualization of translation and protein biogenesis at the ER membrane. Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C ...Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C Howes / Abhay Kotecha / Juliette Fedry / Friedrich Förster / Abstract: The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane ...The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 7-7 / Number grids imaged: 8 / Average exposure time: 0.7 sec. / Average electron dose: 2.3 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
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Image processing
Extraction
Number tomograms: 869 / Number images used: 134350 Reference model: Subtomogram average of ER membrane-associated ribosome Method: Template matching / Software - Name: PyTom (ver. 0.994)
Final 3D classification
Software - Name: RELION (ver. 3.1.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.1)
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 3884
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