Reconstitution and structural analysis of the RZZ complex. (A) Schematic organization of the three RZZ subunits. (B) Biological functions of RZZ and Spindly. IP, inner plate of kinetochore; KT, kinetochore; OP, outer plate of kinetochore. (C) SDS-PAGE analysis of various RZZ complexes used in this paper. M, molecular weight marker. (D) Sedimentation velocity AUC profiles of the recombinant RZZ complex. “Mass (app)” denotes the apparent mass of RZZ derived from the velocity runs; “s” denotes the sedimentation coefficient; “Frict. ratio” is the frictional ratio of the RZZ particle. The calculated molecular mass for a complex containing two copies of each subunit is shown. The sedimentation velocity absorbance profiles and residuals of the fit showing the deviation of the c(S) model from the observed signals are shown, for this and other AUC profiles in this paper, in Fig. S1 E. AU, arbitrary units. (E) Typical class averages of negatively stained RZZ complexes. A clear twofold symmetry is evident. (F) Typical class averages of vitrified RZZ complexes. Bars, 10 nm. (G) Cryo-EM 3D reconstruction of the RZZ complex. The complex is ∼42 nm long and 10 nm wide. The position of a twofold symmetry axis is indicated in the top panel. The density maps of RZZ (here) and R1–1,250ZZ (Fig. 4 B) have been deposited into EMDataBank with the accession codes EMD-4103 and EMD-4104, respectively.
