Assembly and Purification of Phosphorylated Ndc80 Complexes

T₁S₃ and T₃S₁ assemblies were assembled from Traptavidin (T; addgene plasmid #26054) and Dead Streptavidin-SpyCatcher (S; addgene plasmid # 59547). Both plasmids were kind gifts from Mark Howarth (Chivers et al., 2010 (link); Fairhead et al., 2014 (link)). T₁S₃-[Ndc80]₃ modules were prepared as described previously (Volkov et al., 2018 (link)). In brief, a mixture of Ndc80 (or Ndc80^Δ80) and T₁S₃ (1.8 μM) with an approximate 10-fold molar excess of Ndc80 was incubated for 12–16 hr at 10°C in the presence of protease inhibitor mix (Serva). Sortase (4 μM) and GGGGK^TMR (137 μM) were included in the reaction to fluorescently label SPC25. In order to phosphorylate Ndc80 (19 μM), CDK1:Cyclin-B (75 nM), Aurora B (2.1 μM), ATP (1.25 mM) and MgCl₂ (10 mM) were also included in the overnight reaction. Reaction mixtures were applied to a Superose 6 increase 10/300 column (GE Healthcare) equilibrated in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% v/v glycerol, 2 mM TCEP. Relevant fractions were pooled and concentrated using 50 kDa molecular mass cut-off. Amicon concentrators (Millipore), flash-frozen in liquid nitrogen, and stored at −80°C. Efficient phosphorylation of NDC80 was confirmed by mass spectrometry and phostag-SDS-PAGE.