Structural details of interactions between Ndc80c and Mis12c ^Mtw1c . (A) Cryo-EM density map (Fig. S2 Diii) highlighting the interactions between Mis12c^Mtw1c and Spc24 and Spc25 RWD domains. [C] and [D] highlight regions of the map that are shown close-up in C and D for interface 3 and interface 4, respectively. (B) Model of Spc24 and Spc25 RWD domains interacting with motifs from Mis12c^Mtw1c, highlighting the Mis12^{Mtw1_Spc24-helix} and Dsn1^Cterm-helix helical motifs from Mis12c^Mtw1c that interact with Spc24 and Spc25 RWD domains at interface (3). The interaction between the CC3 region of the Mis12c^Mtw1c stalk domain and the Spc24 RWD domain is also featured. (C) Structural details of the interactions between the CC3 region of the Mis12c^Mtw1c stalk domain and the solvent-exposed hydrophobic patch on the Spc24 RWD domain at interface (4). (D) Structural details of the interactions between Dsn1^Cterm-helix and Mis12^{Mtw1_Spc24-helix} motifs and the Spc24 and Spc25 RWD domains. (E and F) Comparison of S. cerevisiae (E) and human (F) KMN junction complexes at the Mis12c interface with Spc24:Spc25^RWD domains. In both S. cerevisiae and human KMN junction complexes, the Dsn1^Cterm-helix contacts Spc24:Spc25^RWD interface. The comparison highlights the interchangeable roles played by the C termini of Mis12^Mtw1 in S. cerevisiae (Mis12^{Mtw1_Spc24 helix}) and Nsl1 in human in contacting the Spc24^RWD. In the S. cerevisiae KMN junction complex, the longer Mis12^{Mtw1_Spc24 helix} contacts Dsn1^Cterm-helix. Structures superimposed on the Spc24^RWD domain. Human KMN junction (PDB: 8PPR) from Yatskevich et al. (2024). Views in E and F are similar to (B).