Extended Data Fig. 7: Additional data on KMN cooperativity and MD simulations.

(a-c) BLI experiments on the indicated species and that generated binding data reported in Fig. 4e, (a) for Mis12C-Ndc80C, (b) for Mis12C-KNL1, (c) for Mis12C + KNL1-Ndc80C. (d) Root mean square deviation (RMSD) from the initial conformation recovered from molecular dynamics simulations of the four indicated systems. Each line represents one independent simulation replica (n = 5). (e) Per-residue root mean square fluctuations (RMSFs) of the Mis12C segments for the four systems indicated at the bottom. The four helical elements are separated by the dashed lines and their sequence coverage is shown on the X-axis. The colored rectangles indicate the binding site of the SPC24/SPC25 domains (red/green) and KNL1 (yellow). (f) Principal component analysis retrieved a main principal component (PC1) related to the bending motion of the C-terminus of the DSN1 domain (see cartoon representations). This component explained 33% of the positional fluctuations of the rigid part of the studied Mis12C fragment. Projection of the MD trajectories onto this PC component indicates whether the DSN1 unit adopted either a straight or a bent conformation. The normalized distribution of such projection is shown for the four studied systems (arbitrary units and same color as in Fig. 4f-g). (g) Normalized distribution of the exposed surface area of the Mis12C/Ndc80C (left) or Mis12C/KNL1 (right) binding interfaces for the indicated systems. (h) Normalized distribution of the number of atomic contacts (N_contacts), between the Mis12C/Ndc80C (left) or Mis12C/KNL1 (right) binding interfaces is presented for the indicated systems.