High-resolution electron microscopy of helical specimens:: A fresh look at Tobacco Mosaic Virus
High-resolution electron microscopy of helical specimens:: A fresh look at Tobacco Mosaic Virus
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DOI：
10.1016/j.jmb.2007.05.088
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发表时间：
2007-08-17
影响因子：
5.6
通讯作者：
Grigorieff, Nikolaus
中科院分区：
文献类型：
--
作者：
Sachse, Carsten;Chen, James Z.;Grigorieff, Nikolaus
将螺旋状物体作为一串单个粒子处理已经成为使用电子低温显微镜解析其三维（3D）结构的既定技术。它可以应用于广泛的螺旋颗粒，如病毒，微管和螺旋丝。我们对这种方法进行了改进，使用烟草花叶病毒（TMV）作为测试样品，并获得了分辨率优于5 A的210，000个不对称单位的图谱。通过对显微镜的对比度传递函数进行完全校正，这成为可能。螺旋片段的排列受到来自病毒螺旋对称性的约束的帮助。此外，在三维重建中通过多次包含与图像相关的视图来实现对称化。我们使用密度图来建立TMV的原子模型。该模型进行了改进，使用一个真正的空间细化策略，可容纳多个构象。原子模型显示出显着的偏差，从沉积模型的螺旋形式的TMV在较低的半径区域（残基88至109）。与早期的螺旋结构相比，该区域似乎更有序，具有明确的二级结构。RNA磷酸骨架夹在两个精氨酸侧链之间，稳定RNA和外壳蛋白之间的相互作用。两个或三个羧酸根的簇被埋在疏水环境中，将其与相邻的亚基隔离。这些羧酸盐可以代表所谓的卡斯帕羧酸盐，其形成用于病毒分解的亚稳态开关。总的来说，观察到的差异表明，与早期发表的模型相比，新模型代表了一种不同的、更稳定的病毒状态。(c)2007爱思唯尔有限公司保留所有权利。
The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 A. This was made possible by performing a full correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side-chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model. (c) 2007 Elsevier Ltd. All rights reserved.