Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules
Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules
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DOI：
10.1016/j.jsb.2004.04.004
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发表时间：
2004-09-01
影响因子：
3
通讯作者：
Knight, PJ
中科院分区：
文献类型：
--
作者：
Burgess, SA;Walker, ML;Knight, PJ
柔性大分子给用晶体学或核磁共振测定结构带来了特殊的困难。电子显微镜可以取得进展，但电子冷冻显微镜对未染色的水合标本的观察由于其固有的低信噪比而局限于较大的大分子。为了确定三维结构，单个粒子必须在结构上是不变的。在这里，我们描述了我们如何使用阴性染色和单粒子图像处理技术来探索两种运动蛋白：肌球蛋白和动力蛋白的单分子结构和灵活性。负染色成功的关键是一层亲水的薄碳膜，因为它在每个分子周围产生低噪音背景，并稳定分子免受染色的破坏。单粒子图像处理所采用的策略充分利用了SPIDER软件套件的灵活性。我们说明了连续几轮图像对齐和分类的好处，以及使用全分子平均和电影来分析和显示dynein马达的结构和灵活性。(C) 2004爱思唯尔公司版权所有。
Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor. (C) 2004 Elsevier Inc. All rights reserved.