ジャーナル: Nat Commun / 年: 2014 タイトル: Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon. 著者: Stefan Pfeffer / Johanna Dudek / Marko Gogala / Stefan Schorr / Johannes Linxweiler / Sven Lang / Thomas Becker / Roland Beckmann / Richard Zimmermann / Friedrich Förster / 要旨: In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting ...In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
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モデル: Titan Krios / 画像提供: FEI Company
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詳細
Candidate particles were localized using template matching and cross correlation peaks in areas containing rough ER were visually inspected to identify true positive matches. At the selected coordinates, unbinned subtomograms were reconstructed individually from the weighted back-projections and aligned to a template.
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 40.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: av3, tom, PyTom / 使用したサブトモグラム数: 295