ジャーナル: Nat Struct Mol Biol / 年: 2011 タイトル: Structure of green-type Rubisco activase from tobacco. 著者: Mathias Stotz / Oliver Mueller-Cajar / Susanne Ciniawsky / Petra Wendler / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl / 要旨: Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of ...Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of Nicotiana tabacum Rubisco activase (Rca), the enzyme that facilitates the removal of these inhibitors. Rca from tobacco has a classical AAA(+)-protein domain architecture. Although Rca populates a range of oligomeric states when in solution, it forms a helical arrangement with six subunits per turn when in the crystal. However, negative-stain electron microscopy of the active mutant R294V suggests that Rca functions as a hexamer. The residues determining species specificity for Rubisco are located in a helical insertion of the C-terminal domain and probably function in conjunction with the N-domain in Rubisco recognition. Loop segments exposed toward the central pore of the hexamer are required for the ATP-dependent remodeling of Rubisco, resulting in the release of inhibitory sugar.
濃度: 0.04 mg/ml / 包埋: NO / シャドウイング: NO / 染色: YES / 凍結: NO
染色
タイプ: NEGATIVE / 染色剤: uranyl acetate
試料支持
詳細: CARBON
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電子顕微鏡撮影
顕微鏡
モデル: FEI TECNAI 12 / 日付: 2011年6月22日
電子銃
電子線源: LAB6 / 加速電圧: 120 kV / 照射モード: FLOOD BEAM
電子レンズ
モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 950 nm / 最小 デフォーカス(公称値): 470 nm / Cs: 2 mm
撮影
電子線照射量: 20 e/Å2 / フィルム・検出器のモデル: FEI EAGLE (2k x 2k)
画像スキャン
デジタル画像の数: 11
放射波長
相対比: 1
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解析
EMソフトウェア
ID
名称
カテゴリ
1
UCSF Chimera
モデルフィッティング
2
IMAGIC
3次元再構成
3
MRC IMAGE PROCESSING PACKAGE
3次元再構成
4
SPIDER
3次元再構成
CTF補正
詳細: PHASE FLIPPING, EACH PARTICLE
対称性
点対称性: C6 (6回回転対称)
3次元再構成
手法: ANGULAR RECONSTITUTION / 解像度: 20 Å / 粒子像の数: 599 / ピクセルサイズ(公称値): 3.308 Å / ピクセルサイズ(実測値): 3.308 Å 詳細: A MODULE OF ALPHA HELICAL DOMAIN AND ALPHA-BETA DOMAIN OF THE NEIGHBOURING SUBUNIT WAS OVERLAID WITH THE P97 D2 (3CF3) STRUCTURE AND THE HEXAMER WAS FITTED INTO THE EM MAP SUBMISSION BASED ON ...詳細: A MODULE OF ALPHA HELICAL DOMAIN AND ALPHA-BETA DOMAIN OF THE NEIGHBOURING SUBUNIT WAS OVERLAID WITH THE P97 D2 (3CF3) STRUCTURE AND THE HEXAMER WAS FITTED INTO THE EM MAP SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1940. (DEPOSITION ID: 10169). 対称性のタイプ: POINT
原子モデル構築
プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: LOCAL MINIMISATION, FIT IN MAP / 詳細: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY