Direct visualization of secondary structures of F-actin by electron cryomicroscopy
Direct visualization of secondary structures of F-actin by electron cryomicroscopy
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DOI：
10.1038/nature09372
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发表时间：
2010-10-07
期刊：
影响因子：
64.8
通讯作者：
Namba, Keiichi
中科院分区：
文献类型：
--
作者：
Fujii, Takashi;Iwane, Atsuko H.;Namba, Keiichi
F - 肌动蛋白是肌动蛋白的螺旋组装体，它是肌肉纤维收缩所必需的成分，并且在众多细胞过程中起着关键作用，例如片状伪足和丝状伪足的形成（1,2），它作为细胞骨架最丰富的成分和调节因子，通过动态组装和去组装（从G - 肌动蛋白到F - 肌动蛋白，反之亦然）发挥作用。肌动蛋白是一种普遍存在的蛋白质，参与重要的生物学功能，但F - 肌动蛋白的确切高分辨率结构仍然未知。尽管最近的一个原子模型很好地重现了来自高度取向液晶溶胶标本的X射线纤维衍射强度数据（3），但在没有实验相位信息的情况下其精修存在一定局限性。然而，通过电子冷冻显微镜直接观察其结构一直很困难，因为它相对较薄且具有柔性。在此我们报道了通过电子冷冻显微镜的最新进展获得的6.6埃分辨率的F - 肌动蛋白结构。密度图清晰地分辨出G - 肌动蛋白的所有二级结构，例如α - 螺旋、β - 结构和环，使得明确的建模和精修成为可能。在F - 肌动蛋白模型中揭示了复杂的结构域运动，这些运动在F - 肌动蛋白形成时打开核苷酸结合口袋，特定的D - 环和末端构象，以及相对紧密的轴向但明显松散的原丝间相互作用（本质上是亲水的），并且所有这些似乎对肌动蛋白的动态功能都很重要。
F-actin is a helical assembly of actin, which is a component of muscle fibres essential for contraction and has a crucial role in numerous cellular processes, such as the formation of lamellipodia and filopodia(1,2), as the most abundant component and regulator of cytoskeletons by dynamic assembly and disassembly (from G-actin to F-actin and vice versa). Actin is a ubiquitous protein and is involved in important biological functions, but the definitive high-resolution structure of F-actin remains unknown. Although a recent atomic model well reproduced X-ray fibre diffraction intensity data from a highly oriented liquid-crystalline sol specimen(3), its refinement without experimental phase information has certain limitations. Direct visualization of the structure by electron cryomicroscopy, however, has been difficult because it is relatively thin and flexible. Here we report the F-actin structure at 6.6 angstrom resolution, made obtainable by recent advances in electron cryomicroscopy. The density map clearly resolves all the secondary structures of G-actin, such as alpha-helices, beta-structures and loops, and makes unambiguous modelling and refinement possible. Complex domain motions that open the nucleotide-binding pocket on F-actin formation, specific D-loop and terminal conformations, and relatively tight axial but markedly loose interprotofilament interactions hydrophilic in nature are revealed in the F-actin model, and all seem to be important for dynamic functions of actin.