ジャーナル: Nature / 年: 2014 タイトル: Structure of the AcrAB-TolC multidrug efflux pump. 著者: Dijun Du / Zhao Wang / Nathan R James / Jarrod E Voss / Ewa Klimont / Thelma Ohene-Agyei / Henrietta Venter / Wah Chiu / Ben F Luisi / 要旨: The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of ...The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.
名称: AcrB-AcrA-TolC-AcrZ multidrug efflux pump / タイプ: sample / ID: 1000 集合状態: One homotrimer of TolC and one homotrimer of AcrB bound to a homohexamer of AcrA; three AcrZ molecules are bound to the AcrB Number unique components: 1
pH: 7.5 / 詳細: 50 mM HEPES pH 7.5, 400 mM NaCl, 0.03% DDM
グリッド
詳細: 200 mesh copper grid with holey carbon film
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV / 手法: Blot 1 second at force 0
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電子顕微鏡法
顕微鏡
JEOL 3200FSC
温度
最低: 78 K / 最高: 80 K / 平均: 79 K
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at 100k magnification
特殊光学系
エネルギーフィルター - 名称: JEOL
日付
2013年5月20日
撮影
カテゴリ: CCD フィルム・検出器のモデル: DIRECT ELECTRON DE-12 (4k x 3k) デジタル化 - サンプリング間隔: 6 µm / 実像数: 1281 / 平均電子線量: 25 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: DARK FIELD / 最大 デフォーカス(公称値): 3.5 µm / 最小 デフォーカス(公称値): 0.5 µm / 倍率(公称値): 20000
試料ステージ
試料ホルダーモデル: JEOL 3200FSC CRYOHOLDER
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画像解析
詳細
Particle were selected by eman2 e2boxer. Initial model was generated by eman2 base on reference free 2d class averages. Further refinements were done by eman2.07, followed by Relion at the end.
CTF補正
詳細: per image
最終 再構成
解像度のタイプ: BY AUTHOR / 解像度: 15.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: EMAN2, RELION 詳細: Resolution was estimated by gold standard eotest from two independent reconstructions. 使用した粒子像数: 7000