单分子簇分析剖析剪接途径的构象动力学。
Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics.
作者信息
Blanco Mario R, Martin Joshua S, Kahlscheuer Matthew L, Krishnan Ramya, Abelson John, Laederach Alain, Walter Nils G
机构信息
Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.
Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan, USA.
出版信息
Nat Methods. 2015 Nov;12(11):1077-84. doi: 10.1038/nmeth.3602. Epub 2015 Sep 28.
We report Single Molecule Cluster Analysis (SiMCAn), which utilizes hierarchical clustering of hidden Markov modeling-fitted single-molecule fluorescence resonance energy transfer (smFRET) trajectories to dissect the complex conformational dynamics of biomolecular machines. We used this method to study the conformational dynamics of a precursor mRNA during the splicing cycle as carried out by the spliceosome. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify the signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified an open conformation adopted late in splicing by a 3' splice-site mutant, invoking a mechanism for substrate proofreading. SiMCAn enables rapid interpretation of complex single-molecule behaviors and should prove useful for the comprehensive analysis of a plethora of dynamic cellular machines.
我们报告了单分子聚类分析（SiMCAn），该方法利用隐马尔可夫模型拟合的单分子荧光共振能量转移（smFRET）轨迹的层次聚类来剖析生物分子机器的复杂构象动力学。我们使用这种方法研究了剪接体在剪接循环过程中前体mRNA的构象动力学。通过对选择性阻断剪接反应产生的常见动态行为进行聚类，SiMCAn能够识别多种ATP依赖性中间体的特征构象和动态行为。此外，它还识别出了3'剪接位点突变体在剪接后期采用的开放构象，从而提出了一种底物校对机制。SiMCAn能够快速解读复杂的单分子行为，并且应该会被证明对大量动态细胞机器的综合分析有用。
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