A nano-positioning system for macromolecular structural analysis
A nano-positioning system for macromolecular structural analysis
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DOI：
10.1038/nmeth.1259
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发表时间：
2008-11-01
期刊：
影响因子：
48
通讯作者：
Michaelis, Jens
中科院分区：
文献类型：
--
作者：
Muschielok, Adam;Andrecka, Joanna;Michaelis, Jens
很多时候，大分子以及大分子复合物内柔性结构域的位置无法通过标准的结构生物学方法确定。为解决这一问题，我们开发了一种方法，该方法利用概率数据分析将单分子测量结果与X射线晶体学数据相结合。这种方法不仅能确定连接在结构域上的荧光染料分子最可能的位置，还能确定描述实验不确定性的完整三维概率分布。通过这种方法，单对荧光共振能量转移测量现在可作为一种定量工具，用于研究大分子复合物内柔性结构域的位置和动力学。我们应用这种方法找到了从酵母（酿酒酵母）RNA聚合酶II转录延伸复合物中出来的新生RNA的5'端位置，并研究了转录因子IIB对RNA位置的影响。
Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.