Computational separation of conformational heterogeneity using cryo-electron tomography and 3D sub-volume averaging.
Computational separation of conformational heterogeneity using cryo-electron tomography and 3D sub-volume averaging.
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DOI：
10.1016/j.jsb.2012.01.004
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发表时间：
2012-05
影响因子：
3
通讯作者：
Subramaniam S
中科院分区：
文献类型：
--
作者：
Frank GA;Bartesaghi A;Kuybeda O;Borgnia MJ;White TA;Sapiro G;Subramaniam S
我们以前使用冷冻电子断层扫描结合子体积平均和分类，以获得大分子组件的三维结构的情况下，一个单一的优势物种是本，并应用这些方法来分析各种三聚体HIV-1和SIV包膜糖蛋白（Env）。在这里，我们通过展示自动化，迭代，缺失楔形校正的3D图像对齐和分类方法来区分同时存在的多种构象来扩展这些研究。我们提出了一种方法，用于测量代表不同构象状态的Env的矢量元素的空间分布。我们确定的数据处理策略，允许明确分离的先前表征的封闭和开放的构象，以及unliganded和抗体配体状态的Env时，它们存在于混合物中。我们发现，识别和去除尖峰最低的信噪比提高了整体精度的个别Env子卷之间的对齐，对齐精度，反过来，决定了成功的图像分类在评估异构混合物的构象异质性。我们通过成功分离和重建不同的3D结构的unliganded和抗体配体，以及开放和封闭的构象的Env同时存在于混合物中，验证这些程序的计算分离。
We have previously used cryo-electron tomography combined with sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by demonstrating automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. We present a method for measuring the spatial distribution of the vector elements representing distinct conformational states of Env. We identify data processing strategies that allow clear separation of the previously characterized closed and open conformations, as well as unliganded and antibody liganded states of Env when they are present in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. We validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures.
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影响因子：
5.4
作者：
White, Tommi A.;Bartesaghi, Alberto;Subramaniam, Sriram
通讯作者：
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影响因子：
64.8
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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作者：
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