ジャーナル: Nature / 年: 2005 タイトル: Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. 著者: Hong-Wei Wang / Eva Nogales / 要旨: The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are ...The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.
pH: 6.8 / 詳細: 80 mM PIPES, 1 mM MgCl2, 1mM GDP, 30 mM MnCl2
グリッド
詳細: 400 Quantifoil
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 90 K / 装置: OTHER / 詳細: Vitrification instrument: Vitrobot 手法: Blot for 1.8 seconds before plunging with Vitrobot offset as -2 mm
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電子顕微鏡法
顕微鏡
FEI/PHILIPS CM200FEG
温度
平均: 90 K
アライメント法
Legacy - 非点収差: objective lens astigmatism was corrected at 200K mag
撮影
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: OTHER / デジタル化 - サンプリング間隔: 12.7 µm / 実像数: 200 / 平均電子線量: 15 e/Å2 / ビット/ピクセル: 14
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 12.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: MRC and home-made programs / 詳細: final maps were calculated from 19 tube images