ジャーナル: Proc Natl Acad Sci U S A / 年: 2009 タイトル: Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system. 著者: Michael J Tarry / Eva Schäfer / Shuyun Chen / Grant Buchanan / Nicholas P Greene / Susan M Lea / Tracy Palmer / Helen R Saibil / Ben C Berks / 要旨: The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the ...The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.
カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: ZEISS SCAI / デジタル化 - サンプリング間隔: 7 µm 詳細: Micrographs were digitized on a Zeiss SCAI scanner at a pixel size of 7 microns, corresponding to 1.667 angstroms on the specimen. Subsequently, adjacent pixels were 3 x 3 averaged to yield a ...詳細: Micrographs were digitized on a Zeiss SCAI scanner at a pixel size of 7 microns, corresponding to 1.667 angstroms on the specimen. Subsequently, adjacent pixels were 3 x 3 averaged to yield a pixel size of 5 angstroms.
Tilt angle min
0
電子線
加速電圧: 120 kV / 電子線源: TUNGSTEN HAIRPIN
電子光学系
照射モード: OTHER / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 1.5 µm / 倍率(公称値): 42000
試料ステージ
試料ホルダー: eucentric / 試料ホルダーモデル: OTHER / Tilt angle max: 50
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画像解析
CTF補正
詳細: phase flipping
最終 再構成
想定した対称性 - 点群: C1 (非対称) / ソフトウェア - 名称: SPIDER 詳細: The tilted images were corrected for the effects of the contrast transfer function (CTF) by phase flipping, taking into account the defocus gradient across the micrographs and the position of ...詳細: The tilted images were corrected for the effects of the contrast transfer function (CTF) by phase flipping, taking into account the defocus gradient across the micrographs and the position of each particle. Images were processed using SPIDER version 11.12 and 15.06. Three-dimensional reconstruction was performed by the random conical tilt method in SPIDER. The particles were windowed into 64 x 64 pixel boxes. 使用した粒子像数: 1358