Kinetochores

To investigate if all the subunits are homologous to one another, a profile-versus-profile search was performed (fig. 4A), using the database of Pfam 31, pdb70, a database of profiles of kinetochore proteins and of the merged alignments of the subunit pairs of the Dam1-C. The merged alignments were aligned using MAFFT merge E-INS-i (MAFFT v7.271) and filtered using trimAl with parameter gt = 0.05. The alignments were manually curated for gene prediction problems. HHsuite (version 3.3.0, tool hhsearch) was used for profile-versus-profile search using the merged protein alignments of the coupled paralogous subunits and the separate protein alignments for each subunit. The FASTA files used to create the merged profiles can be found in the supplementary files.

We modelled a 13-protofilament MT based on the cryoEM structure of a yeast tubulin dimer polymerized with GTP in vitro (PDB-ID 5W3F) [65 (link)]. As published, we used lattice parameters for the MT tube with a helical rise of 83.3 Å and a rotation of 0.43° between dimers within a protofilament, and a helical rise of 9.65 Å and a rotation of −27.6° between protofilaments. The majority of kinetochore MTs in tomograms recorded from budding yeast were observed in in ‘ram's horn’ geometry [66 (link)], and we modelled the flaring of protofilaments at the MT plus end by matching the average curvature of the tomograms with the curvature observed in various structures of bent tubulin protofilaments (PDB-IDs 3J6H, 3RYH, 4HNA, 4FFB, 6MZG).
To model a MT-bound yeast Ndc80c, we first docked the AF2 prediction of Ndc80 : Nuf2 up to a few residues beyond the hinge (Ndc80^115–444, Nuf2^1–277) onto the MT. For this, we used the structure of the human Ndc80 : Nuf2 head domain bound to the MT lattice (PDB-ID 3iZ0) [7 (link)] for superposition of tubulin and the Ndc80 head domain (H. sapiens residues 110–202). Next, we placed a composite model of two AF2 predictions, comprising sequence from Ndc80 : Nuf2 just before the hinge all the way to the Spc24 : Spc25 head domains (Ndc80^413–691, Nuf2^252–451, Spc25^1–89, Spc25^1–83; and Ndc80^619–691, Nuf2^404–451, Spc24^1–213, Spc25^1–221) with a hinge angle such that the coiled-coils of Ndc80 : Nuf2 (after the hinge) and Spc24 : Spc25 were approximately parallel to the microtubule axis. Finally, we re-modelled the hinge residues in plausible conformation using RosettaRemodel [67 (link)].
For the DASH/Dam1c, we manually placed one heterodecamer of the S. cerevisiae full-length AF2 prediction, including only the well-structured regions of the core complex with high confidence scores (Ask1^2–69, Dad1^14–73, Dad2^{2–85, 116–133}, Dad3^6–94, Dad4^2–72, Dam1^54–162, Duo1^61–180, Hsk3^2–69, Spc19^2–106, Spc34^{2–118,157–264}), into the cryoEM map of a DASH/Dam1c ring assembled around a MT [68 (link)] (note that the deposited map, EMD-5254, has the wrong hand and needs to be inverted), followed by rigid-body fitting with phenix.real_space_refine [69 (link)]. The full-length complex was then placed on the fitted core complex and 17-fold rotationally expanded around the MT axis. The whole DASH/Dam1c ring was then rotated around and translated along the MT axis such that interaction C [1 (link),19 (link)] between the protrusion domain of one DASH/Dam1c heterodecamer and the MT-bound Ndc80c could be established. This juxtaposed Thr199 of Spc34, the residue that is phosphorylated by Ipl1 and regulates interaction C [19 (link)], and residue 583 of Ndc80, the position of a five amino acid mutation (insertion) that abrogates interaction C [1 (link)]. It also allows for establishment of the interaction between Spc19 residues 128–165 and Nuf2 residues 399–429 [70 (link)]. The C termini of Spc19 and Sp34 form a coiled-coil at the tip of the DASH/Dam1c protrusion domain, which was not observed in the cryoEM reconstruction of the C. thermophilum DASH/Dam1c complex (because of its flexible attachment) but was inferred from sequence analysis [16 (link)]; AF2 also predicts it now. We used HADDOCK 2.4 [71 (link)] to dock the C-terminal Spc19 : Spc34 coiled-coil onto Ndc80 : Nuf2 and re-modelled the residues that connect it to the protrusion domain with RosettaRemodel [67 (link)]. The model of the DASH/Dam1c ring shown in figure 5 contains the following residues for each subunit: Ask1^1–70, Dad1^15–77, Dad2^{1–73, 119–133}, Dad3^{6–35, 49–94}, Dad4^3–70, Dam1^53–156, Duo1^58–179, Hsk3^3–66, Spc19^1–165, Spc34^1–295. We added the C-terminal Dam1 segment (residues 254–270, 290–305) from the crystal structure determined here by superposition of the Ndc80 head domains.