ジャーナル: Nature / 年: 2016 タイトル: Structure of mammalian respiratory complex I. 著者: Jiapeng Zhu / Kutti R Vinothkumar / Judy Hirst / 要旨: Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive ...Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner mitochondrial membrane. Mammalian complex I (ref. 1) contains 45 subunits, comprising 14 core subunits that house the catalytic machinery (and are conserved from bacteria to humans) and a mammalian-specific cohort of 31 supernumerary subunits. Knowledge of the structures and functions of the supernumerary subunits is fragmentary. Here we describe a 4.2-Å resolution single-particle electron cryomicroscopy structure of complex I from Bos taurus. We have located and modelled all 45 subunits, including the 31 supernumerary subunits, to provide the entire structure of the mammalian complex. Computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally dynamic regions and match biochemical descriptions of the 'active-to-de-active' enzyme transition that occurs during hypoxia. Our structures therefore provide a foundation for understanding complex I assembly and the effects of mutations that cause clinically relevant complex I dysfunctions, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.
名称: Bovine respiratory Complex I / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#44 詳細: Complex I is the first enzyme in the electron transport chain located in the inner membrane of mitochondria and in higher eukaryotes, it is a multi-subunit membrane protein complex, consisting of >40 subunits.
名称: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, NDUFA9 タイプ: protein_or_peptide / ID: 16 詳細: Residue range 187-199, 253-279 not modelled and none of the residues are assigned as the density doesn't allow it. The density for NAP is prominent and has been modelled. コピー数: 1 / 光学異性体: LEVO
pH: 8 / 構成要素 - 濃度: 150.0 mM / 構成要素 - 式: NaCl / 構成要素 - 名称: sodium chloride / 詳細: 20 mM TRIS-HCL, 150 mM NaCl, 0.03% CYMAL-7
グリッド
モデル: Quantifoil / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 75 sec. / 前処理 - 雰囲気: AIR 詳細: Holey carbon grids were placed on a filter paper in a glass petridish and pumped for around 5 minutes to remove any residual moisture. The glow of the plasma was observed and glow discharge when it was purple.
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277.15 K / 装置: HOMEMADE PLUNGER 詳細: The specimen was vitrified in an environmental plunge-freeze apparatus present in a cold room, blot for 12-15 seconds after the diameter of the blotted meniscus ceases to spread and plunged..
詳細
Enzyme was purified from bovine heart mitochondria. The detergent used for the final step is cymal-7.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
温度
最低: 87.5 K / 最高: 87.5 K
撮影
フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 検出モード: INTEGRATING / デジタル化 - サイズ - 横: 4096 pixel / デジタル化 - サイズ - 縦: 4096 pixel / デジタル化 - 画像ごとのフレーム数: 1-34 / 平均露光時間: 2.0 sec. / 平均電子線量: 35.0 e/Å2 詳細: Images were collected in movie mode at 17 frames per second. Thus each frame has ~1.0 e/A2
タイプ: OTHER / ソフトウェア - 名称: RELION (ver. 1.4) / ソフトウェア - 詳細: Maximum Likelihood
最終 3次元分類
クラス数: 8 / ソフトウェア - 名称: RELION (ver. 1.4) / ソフトウェア - 詳細: Maximum Likelihood 詳細: The data set with particles after 2D classification had 115,966 particles. After the angular assignment, movie refinement, B-factor weighting and refinement, the particles were subjected to ...詳細: The data set with particles after 2D classification had 115,966 particles. After the angular assignment, movie refinement, B-factor weighting and refinement, the particles were subjected to 3D classification with number of classes set to 8. Three classes were identified to contain high-resolution features and the map deposited with this is one of the classes.
FSC曲線 (解像度の算出)
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原子モデル構築 1
精密化
空間: RECIPROCAL / プロトコル: OTHER / 温度因子: 83.049 / 当てはまり具合の基準: Maximum likelihood
得られたモデル
PDB-5ldw: Structure of mammalian respiratory Complex I, class1