Extended Data Fig. 10: S. cerevisiae CCAN–Cenp-ANuc comprises two CCAN complexes in solution.
From: Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome
a–c, The predicted mass of (CCAN)2–Cenp-ANuc is 1.31 MDa, (CCAN)1–Cenp-ANuc is 0.77 MDa and that of a CCAN dimer 1.09 MDa (Extended Data Table 2). Representative SEC–MALS data for crosslinked S. cerevisiae CCAN–Cenp-ANuc complex (a), run independently in triplicate with similar results, average molecular mass is 1.23 MDa ((CCAN)2–Cenp-ANuc); uncrosslinked S. cerevisiae CCAN–Cenp-ANuc complex (b), run independently in triplicate with similar results, with average masses of 1.38 MDa ((CCAN)2–Cenp-ANuc) and 526 kDa (CCAN); and S. cerevisiae CCAN alone (c), run independently in duplicate with similar results, with average masses of 839 kDa for the leading edge (green) and 650 kDa for the trailing edge (magenta), suggesting a non-resolved monomer–dimer equilibrium. d, e, Velocity analytical ultracentrifugation of crosslinked (d) and uncrosslinked (e) S. cerevisiae CCAN–Cenp-ANuc complexes with residuals to the fits shown in f and g. f, g, Fit of a c(s) distribution model for the crosslinked complex (f), the major species sediments at 15.8S (Sw,20 = 26.1S) with a minor species at 12.1S (Sw,20 = 20.0S) that corresponds to calculated masses of 1.34 MDa ((CCAN)2–Cenp-ANuc) and 896 kDa (possibly (CCAN)1–Cenp-ANuc), respectively, with a fitted value of 1.761 for the frictional ratio. g, Fit for uncrosslinked samples, the major species is resolved into two species that sediment at 14.3S (Sw,20 = 22.6S) and 15.7S (Sw,20 = 24.9S) with a minor species at 12.3S (Sw,20 = 19.4 S), which gave masses of 1.32 MDa ((CCAN)2–Cenp-ANuc) and 1.15 MDa ((CCAN)2) for the major species and 716 kDa ((CCAN)1–Cenp-ANuc) for the minor species. The experiments shown in d–g were performed independently in triplicate with similar results. h, Examples of two 2D class averages showing the (CCAN)2–Cenp-ANuc particles viewed in the plane of the C2 symmetry axis (red outline) (data from Extended Data Fig. 2c) and the 2D reprojections of a modelled (CCAN)2–Cenp-ANuc based on the CCAN–Cenp-ANuc cryo-EM reconstruction (yellow outline) (shown in i). There is a close correspondence in shape and dimensions between the calculated reprojections and the observed 2D classes. The two-fold symmetry axes of the (CCAN)2–Cenp-ANuc complex are shown as dashed arrows. i, j, Two alternative models for how CCAN assembled onto a Cenp-A nucleosome would interact with the outer kinetochore–microtubule interface (Supplementary Video 2). i, In scenario (1), CCAN interacts with the outer kinetochore from the same side as the DNA-binding surface. Microtubules attached to the outer kinetochore would hoist CCAN from below the over-lying nucleosome and out-stretched DNA. j, In scenario (2), the microtubule-outer kinetochore interface contacts CCAN from the opposite side to the CCAN DNA-binding surface. Outer-kinetochore (outer-KT): KMN network and microtubule-attachment complexes, Dam1–DASH (budding yeast) and Ska proteins of vertebrates. The combined dimension of (CCAN)2–Cenp-ANuc (32 nm) matches that of the hub at the centre of the yeast kinetochore63.
