Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy
Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy
复制标题
DOI：
10.1073/pnas.2001546117
复制
发表时间：
2020-07-21
影响因子：
11.1
通讯作者：
Sindelar, Charles V.
中科院分区：
文献类型：
--
作者：
Debs, Garrett E.;Cha, Michael;Sindelar, Charles V.
微管是管状聚合物，在许多细胞活动中发挥重要作用。冷冻电镜已经以越来越高的分辨率捕获了微管的结构。然而，微管的动态特性是其功能的关键，并且由于现有结构测定方法的限制，这种行为很难在结构水平上表征。我们开发了一种高分辨率冷冻电镜细化方法，可将成像微管分成其组成原丝，从而能够在单亚基水平上量化和校正螺旋性和其他异质性来源的偏差。我们证明，与利用螺旋对称平均的方法相比，该方法提高了微管 3D 重建的分辨率，并大大减少了各向异性模糊伪影。此外，我们还发现了 m 环的一种意想不到的离散行为，它介导相邻原丝之间的横向相互作用，并充当它们之间的灵活铰链。铰链角度采用对应于与螺旋对称性不相容的m环的不同构象的优选值。这些铰链角度以随机方式波动，因此完美的圆柱形微管构象受到能量和熵的惩罚。铰链角度可以进一步偏离微管接缝处的螺旋对称性，从而产生高度扭曲的微管亚群。然而，在微管稳定剂紫杉醇存在下，接缝扭曲的亚群消失。这些观察结果为微管灵活性和动力学的结构起源提供了线索，并强调了结构多态性在定义微管行为中的作用。
Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties of the microtubule are key to its function, and this behavior has proved difficult to characterize at a structural level due to limitations in existing structure determination methods. We developed a high-resolution cryo-EM refinement method that divides an imaged microtubule into its constituent protofilaments, enabling deviations from helicity and other sources of heterogeneity to be quantified and corrected for at the single-subunit level. We demonstrate that this method improves the resolution of microtubule 3D reconstructions and substantially reduces anisotropic blurring artifacts, compared with methods that utilize helical symmetry averaging. Moreover, we identified an unexpected, discrete behavior of the m-loop, which mediates lateral interactions between neighboring protofilaments and acts as a flexible hinge between them. The hinge angle adopts preferred values corresponding to distinct conformations of the m-loop that are incompatible with helical symmetry. These hinge angles fluctuate in a stochastic manner, and perfectly cylindrical microtubule conformations are thus energetically and entropically penalized. The hinge angle can diverge further from helical symmetry at the microtubule seam, generating a subpopulation of highly distorted microtubules. However, the seam-distorted subpopulation disappears in the presence of Taxol, a microtubule stabilizing agent. These observations provide clues into the structural origins of microtubule flexibility and dynamics and highlight the role of structural polymorphism in defining microtubule behavior.