2D class averages of CTNMAMP-PNP complex

Images of ring-like particles of KLP10A NM construct in complex with curved tubulin protofilaments in the presence of AMP-PNP (Supplementary Fig. ^1d) were aligned and classified using Relion⁵⁷ (v 2.0). CTF was estimated using CTFFIND4⁶⁰. First 10,016 ring-like structures were picked manually from 464 cryo-EM images by marking their centers and masking them with a circular mask of 540 Å. This set of particles was subjected to three rounds of alignment classification and averaging with 100 class averages in each round. Only particles assigned to classes showing distinct tubulin rings with resolved tubulin dimers were kept for the next cycle. In each cycle the coordinates of the particles in the original images were recalculated based on the position of the average-image particle. This was done by applying the inverse transformation used to align the particles and any additional displacement necessary to re-center the particle in the class-average-images. The updated coordinates were used to generate a new set of particles for the next round of alignment classification and averaging. This procedure resulted in three class averages with well centered ring like structures with 15, 14, and 13 tubulin dimers (Fig. 5c). The number of particles in each of these classes were respectively 469, 2171, and 920. We then calculated the coordinates in the original images corresponding to each of two contiguous tubulin dimers in these three class averages. With these coordinate three new sets of particle images (one per each of the three ring-like class average, Fig. 5c) were generated. Each particle-image was masked with a circular mask to included only two tubulin heterodimers (176 Å mask diameter). These three data sets were subjected to three rounds of alignment classification and averaging done as before (centering the particles on the inter-dimer interface of the two tubulin heterodimers approximately at the location where the tip of KLP10A loop-2 would contact tubulin). This procedure resulted in three independently aligned groups of class average images. Two distinct types of class average images could be recognized in each of the three groups. One in which there is a kinesin density associated with each tubulin heterodimer (two kinesins and two tubulin heterodimers) and another with one kinesin per two tubulin heterodimers (Fig. 5d, e). The number of particles associated to each of these two class-average images for each of the three groups (15, 14, and 13 tubulin heterodimers ring like structures) were respectively (954, 1441), (610, 567), and (1109, 530) with the first number in parenthesis corresponding to the class with two motor-domains bound and the second number corresponding to the class with one motor domain bound.