ジャーナル: EMBO J / 年: 2015 タイトル: X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding. 著者: Manatsu Morikawa / Hiroaki Yajima / Ryo Nitta / Shigeyuki Inoue / Toshihiko Ogura / Chikara Sato / Nobutaka Hirokawa / 要旨: The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state ...The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.
モード: BRIGHT FIELD / 倍率(公称値): 40000 X / 倍率(補正後): 40000 X / 最大 デフォーカス(公称値): 2600 nm / 最小 デフォーカス(公称値): 1200 nm / Cs: 3.3 mm
試料ホルダ
温度: 100 K / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 °
撮影
電子線照射量: 10 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
放射
プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray
放射波長
相対比: 1
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解析
EMソフトウェア
ID
名称
バージョン
カテゴリ
1
MODELLER
モデルフィッティング
2
UCSF Chimera
モデルフィッティング
3
IMAGIC
5
3次元再構成
4
MATLAB
3次元再構成
CTF補正
詳細: Each filament
対称性
点対称性: C1 (非対称)
3次元再構成
手法: Single Particle / 解像度: 8.1 Å / 粒子像の数: 302000 / ピクセルサイズ(公称値): 2.5 Å / ピクセルサイズ(実測値): 2.5 Å / クラス平均像の数: 18 / 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: Cross-correlation, Average map value, Atoms inside the contour 詳細: METHOD--Local refinement, Domain fitting REFINEMENT PROTOCOL--Rigid body refinement DETAILS--Initial local fitting was done using Chimera and for some loops Modeller was used.