N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalysed by an eight-protein oligosaccharyltransferase (OST) complex that is embedded in the endoplasmic reticulum membrane. Our understanding of eukaryotic protein N-glycosylation has been limited owing to the lack of high-resolution structures. Here we report a 3.5 Å resolution cryo-electron microscopy structure of the Saccharomyces cerevisiae OST complex, revealing the structures of subunits Ost1–Ost5, Stt3, Wbp1 and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N-glycan mediates interactions with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST–Sec61 translocon interface, funnelling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides insights into co-translational protein N-glycosylation, and may facilitate the development of small-molecule inhibitors that target this process.
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Cryo-EM images were collected in the David Van Andel Advanced Cryo-Electron Microscopy Suite at Van Andel Research Institute. We thank Y. Harada for advice on yeast genetics and D. Nadziejka for proofreading. This work was partially supported by Van Andel Research Institute (to H.L.) and the US National Institutes of Health (GM111742 to H.L.).
The authors declare no competing financial interests.
Reviewer Information Nature thanks S. Withers and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, The Coomassie blue–stained SDS–PAGE gel of the purified OST complex. The small subunits Ost2, Ost4–Flag and Ost5 were not visible in this 12% acrylamide SDS–PAGE gel because of their weak density. b, Sequence coverage of tryptic digestion mass spectrometry of three bands at around 30 kDa that are labelled as Ost3, Ost6 and Swp1. The detected peptides are highlighted in blue. The lower bars under the sequences indicate matched peptides. Darker blue indicates more overlaps of peptides detected. c, Ost2, Ost4–Flag and Ost5 were seen in the 15% acrylamide SDS–PAGE gel that was run slower and stained longer. Experiments in a and c were repeated more than three times with similar results.
a, A representative electron micrograph of the OST complex imaged in the Titan Krios with a K2 detector. About 4,000 similar micrographs were recorded. b, Selected reference-free 2D class averages. c, 2D and 3D image classification procedure. d, Gold-standard Fourier correlation of two independent half maps, and the validation correlation curves of the atomic model by comparing the model with the final map or with the two half maps. e, Local resolution map of the OST complex structure.
Extended Data Figure 3 A gallery of selected regions in the OST structure, illustrating the fitting between the 3D density map and the atomic model.
Selected regions in the structure of the OST complex include 26 TMHs, several regions in the lumenal domains, four selected lipids and two N-glycans.
a, b, From 3D classification, one class (class I) contained stronger Ost3 TRX domain density than other classes. This map was further refined to 4.4 Å. Surface view of the map (left) and the corresponding cartoon view of the atomic model (right), coloured by subunit, are shown in two orthogonal side views. The N-terminal TRX domain of Ost3 is highlighted by a magenta disk and is visible in this low-threshold display. The detergent densities that surround the transmembrane region of OST are visible at this threshold, and are coloured in cyan. The structure of the homologous Ost6 TRX (PDB code 3G7Y) is tentatively placed for the purpose of domain location.
a, b, The TMHs of OST form a triangular shape and shown in cytoplasmic view (a) and lumenal view (b). The catalytic subunit Stt3 is in the centre, surrounded by the other subunits. There is a sizable cavity in the centre (red dotted circle). c, Superposition of the transmembrane region of Stt3 and PglB (PDB code 5OGL) viewed from the cytoplasmic side. The Stt3 TMH8–9 (light grey ellipse) moves towards the LLO biding surface relative to the TMH8–9 of PglB (light blue ellipse), creating space for the Ost2 TMHs. The Stt3 TMH1 and TMH13 also move apart, forming a space for the only TMH of Ost4.
PglB does not have the CTE sequence (underscored) found in the yeast Stt3 and human STT3B. Several conserved residues in the active site are highlighted in red. R331 in the PglB, which stabilizes the −2 position D of the acceptor peptide, is highlighted in blue. The asterisk indicates positions with identical residue, a colon indicates strong conservation and a full stop indicates weak conservation.
The CTE of human STT3A is shorter than those of STT3B and yeast Stt3. hs, Homo sapiens; sc, Saccharomyces cerevisiae.
An extra CTD in ribophorin I of complex eukaryotes such as flies, mice and humans is not present in the yeast proteins (shaded grey). NTD1 is shaded in light green, NTD2 in light magenta, TMH in light blue, and the CTD of ribophorin I of complex eukaryotes in light grey. dm, Drosophila melanogaster; pp, Pichia pastoris.
Ribophorin II of complex eukaryotes has evolved an extra N-terminal domain (NTD0, shaded in light orange) in the lumen that is not present in the two yeast proteins.
Surface-rendered cryo-EM 3D map of the OST complex segmented according to the eight individual subunits, which are coloured as in Fig. 1. (MOV 23844 kb)
Overall structure of the OST complex shown in cartoon. Individual subunits are coloured as in Fig. 1. (MOV 23807 kb)
Docking the structures of OST and Sec61 (PDB ID: 3JC2) into the cryo-electron tomogram of a mammalian Ribosome-Sec61-OST-TRAP complex (EMD-3069) reveals the interface between OST and Sec61. (MOV 14448 kb)
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Bai, L., Wang, T., Zhao, G. et al. The atomic structure of a eukaryotic oligosaccharyltransferase complex. Nature 555, 328–333 (2018). https://doi.org/10.1038/nature25755
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