ジャーナル: EMBO Rep / 年: 2017 タイトル: Molecular architecture of the N-type ATPase rotor ring from . 著者: Sarah Schulz / Martin Wilkes / Deryck J Mills / Werner Kühlbrandt / Thomas Meier / 要旨: The genome of the highly infectious bacterium harbors an operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N- ...The genome of the highly infectious bacterium harbors an operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the NN-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c ring is H specific, demonstrating that the ATPase is proton-coupled. The c ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes.
全体 : Rotor ring of the N-type ATPase from B. pseudomallei
全体
名称: Rotor ring of the N-type ATPase from B. pseudomallei
要素
複合体: Rotor ring of the N-type ATPase from B. pseudomallei
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超分子 #1: Rotor ring of the N-type ATPase from B. pseudomallei
超分子
名称: Rotor ring of the N-type ATPase from B. pseudomallei タイプ: complex / ID: 1 / 親要素: 0 詳細: We determined the molecular architecture of the N-type ATPase rotor ring in the melioidosis-causing bacterium Burkholderia pseudomallei by electron cryo-microscopy. The structure shows an ...詳細: We determined the molecular architecture of the N-type ATPase rotor ring in the melioidosis-causing bacterium Burkholderia pseudomallei by electron cryo-microscopy. The structure shows an unusually large c-ring rotor with 17 subunits that, in the acidic environment of the host phagosome, is driven predominantly by protons.
由来(天然)
生物種: Burkholderia pseudomallei (類鼻疽菌)
組換発現
生物種: Escherichia coli (大腸菌) / 組換プラスミド: pt7-7
分子量
実験値: 8.3 kDa/nm
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実験情報
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構造解析
手法
クライオ電子顕微鏡法
解析
単粒子再構成法
試料の集合状態
particle
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試料調製
緩衝液
pH: 7.5
グリッド
モデル: Quantifoil R2/2 / 材質: COPPER
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 283 K / 装置: FEI VITROBOT MARK III
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電子顕微鏡法
顕微鏡
JEOL 3200FSC
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 平均電子線量: 1.8 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: 4.1 mm
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画像解析
最終 再構成
想定した対称性 - 点群: C17 (17回回転対称) / アルゴリズム: BACK PROJECTION / 解像度のタイプ: BY AUTHOR / 解像度: 6.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 47000