Figure 1. Decoration of tubulin rings. (A) Representative images of tubulin rings alone (control [ctrl]) and decorated with the kinesin 13 M, NM, and FL constructs in the presence of AMPPNP. Undecorated rings have flexible shapes (dashed box), whereas decorated rings assume more fixed, rounded shapes (solid box). Arrows mark the stacks that are formed in the presence of NM and FL. The domain organization for each construct is depicted above each micrograph. (B) The mean num- ber of stacks per micrograph was determined for 20 randomly selected micrographs from each dataset. (C) For the NM and FL data- sets, the mean length of stacks counted in B was measured. Graphs display mean ± SEM. ***, P < 0.001. Bars, 1,000 Å. given concentration of tubulin rings was 1.28-fold larger than that of NM (P < 0.05) and 2.38-fold larger than that of FL (P < 0.001), yielding a statistically significant binding trend of M > NM > FL for dolastatin tubulin rings (Fig. 2 B). Significantly, the larger di- meric FL molecule is 1.86-fold less capable of occupying a given number of curved binding sites than NM (P < 0.01; Fig. 2 B, NM vs. FL). These data suggest that the additional domains present in FL, and to a lesser extent NM, interfere with the ability of these constructs to occupy binding sites on a curved protofilament. Single particle image analysis of M, NM, and FL interactions with rings To gain a better understanding of the structural interactions of kinesin 13 constructs and the curved protofilaments, we used single particle image processing to align and average images of rings and thereby gain increased signal to noise. Such averaging allowed the resolution of individual tubulin monomers, and an arrowhead-shaped protein density bound to tubulin hetero- dimers along the inner periphery of the ring (Fig. 3 A). Similar to previous observations (Moores and Milligan, 2008), the ring and motor complex revealed handedness, as indicated by the di- rectionality of the tubulin on the outer perimeter and the arrow- headed motor density along the inner perimeter. Averages of construct ring complexes revealed that NM and FL rarely occupied adjacent sites on the curved protofilament, whereas the M construct consistently occupied every heterodimer- binding site (Fig. 3 A and Fig. S3). Only 4.4% of rings processed in the M dataset passed our cross-correlation threshold for stable alignment to alternate site-binding templates (Fig. S2 and see Materials and methods) compared with 69.4% and 53.9% for the NM and FL datasets. Nonadjacent heterodimer binding was observed for multiple class averages within the NM and FL data- sets and support sedimentation assay (Fig. 2) observations that the additional domains in NM and FL interfere with these con- structs’ abilities to bind curved protofilaments. Furthermore, a dimeric construct lacking the N terminus also showed alternate KINESIN 13 BINDING TO CURVED PROTOFILAMENTS • Mulder et al. 53