Ramey Vincent H, Wang Hong-Wei, Nogales Eva
Biophysics Graduate Program, UC Berkeley, Berkeley, CA, USA.
J Struct Biol. 2009 Aug;167(2):97-105. doi: 10.1016/j.jsb.2009.05.002. Epub 2009 May 15.
We describe modifications of the single particle helical reconstruction approach devised for the analysis of a sample that could not be processed with existing methods due to its variable and short range helical order. The added steps of reference-free two-dimensional image classification and alignment, and automated microtubule removal from images, have particular application to proteins or protein complexes that assemble around microtubules. The method was successfully applied to the Dam1 complex, an essential component of the yeast kinetochore that couples replicated chromosomes to spindle microtubules during mitosis. Because of its novel mode of binding, which does not involve a footprint on the microtubule lattice, new steps to deal with the disorder and heterogeneity of the Dam1 complex assembly were required to gain structural information about this complex both routinely and efficiently.
我们描述了对单粒子螺旋重建方法的改进，该方法是为分析一个因具有可变且短程螺旋顺序而无法用现有方法处理的样本而设计的。新增的无参考二维图像分类与对齐步骤，以及从图像中自动去除微管的步骤，特别适用于围绕微管组装的蛋白质或蛋白质复合物。该方法已成功应用于Dam1复合物，它是酵母动粒的一个重要组成部分，在有丝分裂期间将复制的染色体与纺锤体微管相连。由于其独特的结合模式，即不涉及在微管晶格上留下足迹，因此需要新的步骤来处理Dam1复合物组装的无序性和异质性，以便常规且高效地获取有关该复合物的结构信息。
