ジャーナル: Elife / 年: 2014 タイトル: A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family. 著者: Qing Yao / Qiuhe Lu / Xiaobo Wan / Feng Song / Yue Xu / Mo Hu / Alla Zamyatina / Xiaoyun Liu / Niu Huang / Ping Zhu / Feng Shao / 要旨: A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. ...A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex.
凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV 手法: 10 ug/ml bacitracin (Sigma) was added to the purified protein to obtain monodispersed particles and make the orientation distribution more anisotropic. Blot for 4 sec using blotting force 2 before plunging.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at 155,000 times magnification
詳細
Energy filter turned-off
日付
2013年5月1日
撮影
カテゴリ: CCD / フィルム・検出器のモデル: OTHER / 平均電子線量: 18 e/Å2
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 倍率(公称値): 81000
試料ステージ
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
CTF補正
詳細: CTF parameters calculated from whole micrograph using CTFFIND3
最終 再構成
想定した対称性 - 点群: C6 (6回回転対称) / 解像度のタイプ: BY AUTHOR / 解像度: 9.7 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Relion / 使用した粒子像数: 53303