Ndc80 Complex Coupling to T1S3 Scaffolds

Ndc80 complexes were coupled to streptavidin‐derived T₁S₃ scaffolds as described (Volkov et al, 2018 (link); Appendix Fig S1). In brief, T1S3 scaffolds were assembled from core traptavidin (T; addgene plasmid #26054) and Dead Streptavidin‐SpyCatcher (S; addgene plasmid #59547) (Chivers et al, 2010 (link); Fairhead et al, 2014 (link)). T₁S₃ scaffolds were incubated with an approximate 10‐fold molar excess of Ndc80 for 12–20 h at 10°C in the presence of PMSF (1 mM) and protease inhibitor mix (Serva). Sortase labeling was achieved in the same reaction, as described above. Reaction mixtures were applied to a Superose 6 increase 10/300 column (GE Healthcare) equilibrated in 20 mM TRIS pH 8.0, 200 mM NaCl, 2% v/v glycerol, 2 mM TCEP. Size‐exclusion chromatography was performed at 4°C under isocratic conditions at recommended flow rates and the relevant fractions were pooled and concentrated using 30 kDa molecular mass cut‐off Amicon concentrators (Millipore), flash‐frozen in liquid nitrogen, and stored at −80°C.