Organization of the KMN network. (A) Schematic illustration of the organization of the human MIS12C complex. (B) Summary of interactions identified in this study. The black dots connected by a line represent cross-links. Yellow dots represent defined binding sites. Yellow stars represent undefined binding sites. The orange cylinder represents a predicted C-terminal helix in the MIS12 subunit. (C) Summary of interactions in KMN complexes in different species. Black arrows represent established interactions. Blue arrows represent established binding requirements that have not been mapped at the molecular level. Purple arrows represent putative interactions. In C. elegans, KBP-4 and KBP-3 (SPC24 and SPC25 homologues, respectively) are 97- and 134-residue proteins lacking the globular domains of SPC24 and SPC25 in other species. A globular domain at the C-terminal end of KNL1 is also missing in this organism (see alignment in Fig. S4). In S. cerevisiae, Mtw1 (MIS12 homologue) does not have a PVIHL motif or a positively charged C-terminal domain, and the binding site for Spc24p–Spc24p and Spc105p (KNL1 homologue) is therefore unknown. The four-color scheme for MIS12C in C. elegans and S. cerevisiae conveys that the binding sites have not been mapped and could therefore be anywhere on these structures. (D) An extension of the two-hand model (Cheeseman et al., 2008) for kinetochore recruitment of the KMN network based on our experiments. MT, microtubule.