Abstract
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.