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Structural Basis of Human Kinetochore-Microtubule Coupling by the Ndc80 and Ska Complexes

Ju ZhouiD, Ipsita Saha, Valerie SiahaaniD, Yuanchang ZhaoiD, Carsten JankeiD, Ahmet YıldıziD, Eva NogalesiD

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Abstract

Faithful chromosome segregation depends on kinetochore-microtubule attachments that must be load-bearing, dynamic, and tightly regulated. In human cells, the Ska and Ndc80 complexes (SkaC and Ndc80C) cooperate to sustain these attachments, but how they are organized on microtubules and coupled to end dynamics remains unclear. Here, our cryo-EM structure shows that Ndc80C and SkaC form a coupled assembly across neighboring protofilaments. A phosphorylation-sensitive SKA3 tether binds a defined kink in the Ndc80 coiled-coil, positioning SKA1 beside Ndc80C. These two complexes jointly sandwich the -tubulin tail and sense its polyglutamylation state. SkaC recognizes the extended microtubule lattice and, through the SKA3 tether, confers this selectivity onto Ndc80C, enabling Ndc80C tip tracking while strengthening microtubule stabilization. Together, our findings define the structural basis of Ndc80C-SkaC cooperation and uncover a phosphorylation-regulated, polyglutamylation-sensitive, and lattice-state-dependent mechanism for robust and regulated KT-MT coupling during chromosome segregation in mitosis.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (10 Oct 2026) · CC BY 4.0

Cryo-EM shows human Ndc80 and Ska complexes bound to adjacent microtubule protofilaments, linked by a SKA3 tether that passes Ska's preference for GTP-like lattice and tip tracking to Ndc80.

Human kinetochores rely on the Ndc80 complex (Ndc80C) and Ska complex (SkaC) to stay attached to growing and shrinking microtubule ends under force, yet prior studies implicated different Ndc80C regions in SkaC binding and proposed differing ways SkaC contacts the lattice. This work resolves both complexes together on microtubules (2.8 to 6 Å) and connects that arrangement to tubulin-tail modification, lattice nucleotide state, microtubule dynamics and plus-end tracking in vitro.

Key results

  1. ShownNdc80C and SkaC never share a tubulin and are unlikely to occupy longitudinally neighbouring dimers; instead Ndc80C clusters along one protofilament at tubulin-monomer spacing while SkaC binds the interdimer junction on the adjacent protofilament.

    Cryo-EM of microtubules decorated with Ndc80C plus phosphomimetic SkaC-6D reached 2.8 Å (tubulin) to 6 Å (outer Ndc80C segment). Focused 3D classification: single-dimer masks showed no tubulin bound by both; two-longitudinal-dimer masks gave empty, Ndc80C-only (four monomers) or SkaC-only classes; two-lateral-dimer masks gave a class with both on neighbouring protofilaments. · Ndc80C and SkaC simultaneously bind adjacent…; Figure legends

  2. ShownThe α-tubulin C-terminal tail (residues 438-445) is ordered and clamped between the NDC80 calponin-homology domain and the SKA1 microtubule-binding domain; microtubule binding by both complexes depends on tubulin tails and is promoted by α-tubulin polyglutamylation.

    Tail modelled to E445; α-tubulin E445 and E443 salt-bridge NDC80 K115 and β-tubulin R390 respectively, with SKA1 K183/K184 opposite. Pelleting: subtilisin tail removal reduced binding of each complex alone and together; all bound HeLa microtubules more weakly than porcine brain ones. TIRF on mutant mouse brain microtubules: TTLL1-KO or TTLL1/TTLL7 double-KO strongly reduced binding; TTLL7-KO stayed near wild-type. · Ndc80C and SkaC sandwich the α-tubulin tail; Tubulin tails and polyglutamylation modulate…; Footnotes

  3. ShownThe SKA3 segment 355-380 tethers SkaC to a kink (NDC80 288-292/NUF2 170-174) in the Ndc80C coiled-coil that is absent in budding yeast; it is dispensable for SkaC's own microtubule binding but required for cooperative co-binding with Ndc80C.

    Unassigned density at the kink was attributed to SKA3 355-380 by combining cross-linking mass spectrometry (XL-MS) constraints with iterative AlphaFold3 (AF3) models (a tether helix carrying T358/T360). Pelleting: without Ndc80C, SkaC-6D lacking 355-380 (SkaC-6D_DEL) bound like full length; Ndc80C raised full-length but lowered SkaC-6D_DEL binding, and Ndc80C bound less alongside SkaC-6D_DEL. · SKA3 interacts with a vertebrate-conserved kink…; Figure legends

  4. ShownSkaC favours the extended, GTP-like lattice and, via the SKA3 tether, imposes that preference on Ndc80C; each complex also slows the other's dissociation from microtubules.

    On dynamic microtubules grown from GMPCPP seeds, SkaC alone preferred seeds (more so with Ndc80C); Ndc80C alone slightly favoured GDP lattice but was enriched on GMPCPP lattice with SkaC-6D, a bias lost with SkaC-6D_DEL and reduced with SkaC-6D_2R (R358/R360). On GMPCPP microtubules Ndc80C slowed SkaC dissociation over an order of magnitude; SkaC cut Ndc80C's dissociation rate 3-fold. · SkaC biases Ndc80C toward the extended MT…; The SKA3 tether couples SkaC lattice selectivity…

  5. ShownCoupling through the SKA3 tether boosts joint stabilization of dynamic microtubule ends and lets SkaC confer depolymerizing-tip tracking on Ndc80C, which showed no tip tracking alone.

    Alone, each complex raised growth rate and rescue frequency and lowered shrinkage rate and catastrophe frequency (SkaC more so at assay concentrations); combined stabilization rose with SkaC-WT, more with SkaC-6D_FL, was largely lost with SkaC-6D_DEL and intermediate with SkaC-6D_2R. SkaC tracked depolymerizing tips; Ndc80C alone did not, but did with SkaC-6D_FL and largely not with SkaC-6D_DEL. · SKA3 tethering couples Ndc80C-SkaC to MT…; The SKA3 tether couples SkaC lattice selectivity…

Methods, data and reproducibility

Cryo-EM of microtubules co-decorated with recombinant human Ndc80C and phosphomimetic SkaC-6D (focused 3D classification, local refinement), with AF3 plus XL-MS constraints placing SKA3. Structure-guided mutants were tested by microtubule pelleting and total internal reflection fluorescence (TIRF) assays of binding, dynamics and tip tracking, using HeLa, porcine brain and TTLL-knockout mouse brain tubulin.

  • Recombinant full-length human Ndc80C and SkaC (wild-type, phosphomimetic SkaC-6D, SKA3 Δ355-380 and R358/R360 variants) · human
  • Cryo-EM maps (composite, consensus and focused) of Ndc80C and SkaC-6D on microtubules, plus atomic model · human complexes; tubulin source not stated in text · EMDB 75942 (composite), 75938 (consensus), 75939, 75940, 75941 (focused); PDB 11QA
  • Porcine brain microtubules (highly post-translationally modified tubulin) · pig (porcine brain)
  • HeLa microtubules (low tubulin-tail modification) · human (HeLa cell line)
  • Brain microtubules from wild-type, TTLL1-KO, TTLL7-KO and TTLL1/TTLL7 double-KO mice · mouse
  • TIRF titrations of Ndc80C and SkaC on GMPCPP microtubules from wild-type mouse brain or HeLa tubulin (Extended Data Fig. 7) · mouse, human · n = n=32, 39, 43, 47 (Ndc80C) and n=31, 39, 36, 36 (SkaC) MTs, 1 experiment each
  • Tubulin sequences used for HeLa versus porcine alignment · human, pig · UniProt P68363, Q9BQE3, P07437, Q2XVP4, P02554
  • Yeast NDC80/NUF2 cryo-EM map used for comparison of the coiled-coil kink · yeast · EMDB 18304
Methods & tools
cryo-EM, AlphaFold3, cross-linking mass spectrometry (XL-MS), TIRF microscopy, microtubule pelleting assay, mass photometry, DeepEMhancer, ClustalW, ESPRIPT, EMDB, PDB, UniProt
Reproducibility gaps
The text includes no code, analysis scripts or Methods section, and no processing software is named apart from DeepEMhancer (visualization). Maps (EMDB 75942, 75938, 75939, 75940, 75941) and model (PDB 11QA) are deposited, but no URLs are given.
Limitations (6)
  • A ~40-residue disordered linker separates the SKA1 microtubule-binding domain (MTBD) from the rest of SkaC, so the map cannot show how the two MTBDs of a SkaC dimer sit on the microtubule. · stated by the authors
  • The extra density at the coiled-coil kink was too low in resolution to assign directly; the SKA3 placement rests on XL-MS-guided AF3 models, which match the interface but differ from the map in kink angle. · stated by the authors
  • Ordered density extends only to about 550 Å diameter, with flexible outer regions unresolved, and SKA1 contacts with α1- and α2-tubulin are less well resolved than those with β2-tubulin. · stated by the authors
  • The straight-lattice structure does not capture SKA1 contacts with curved tubulin implied by earlier XL-MS data, so the proposed curved-end engagement by a second SKA1 MTBD is not resolved here. · stated by the authors
  • All functional data come from in vitro reconstitutions, mostly using the aspartate phosphomimetic SkaC-6D; the stepwise mitotic model and the anaphase release are proposed in the Discussion and not tested in cells here. · noted in this summary
  • The text describes pelleting and dynamics results only qualitatively. The Extended Data Fig. 7 TIRF titrations come from one independent experiment per condition, and that legend names mouse brain tubulin, whereas the main text compares HeLa with porcine brain. · noted in this summary

Read this if you work on kinetochore-microtubule attachment, the tubulin code or mitotic phospho-regulation and want a structural account of how human Ndc80 and Ska complexes co-bind and track microtubule ends.

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