Purified Cenp-C was mixed with either the Kmn1-Mis12-Nnf1a or Kmn1-Mis12-Nnf1b trimer, and interaction was assessed by gel filtration and analytical centrifugation.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Cenp-C can bind either the Kmn1-Mis12-Nnf1a or Kmn1-Mis12-Nnf1b trimer to form a complex that is 76.4 kDa or 75.6 kDa in size, respectively.

Purified Cenp-C was mixed with either the Kmn1-Mis12-Nnf1a or Kmn1-Mis12-Nnf1b trimer, then purified by gel filtration. The complex was then treated with BS2G bi-functional crosslinking reagent, and cross-links were mapped by mass spectrometry.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Interaction in vitro; proteins produced as a recombinant fusion protein in bacterial system.

A Nnf1a-Mis12-Cenp-C trimer ( 1:1:1 ) was analysed by mass spectrometry to identify regions protected from hydrogen-deuterium (H/D), indicative of interacting surfaces.

Interaction in vitro; proteins produced as a recombinant fusion protein in bacterial system.

Cenp-C, Mis12 and Nnf1a form a trimeric complex in this assay. The complex size was calculated by mass spectrometry to be 56.73 kDa, consistent with a 1:1:1 stoichiometry.

Cenp-C, Mis12 and Nnf1a were mixed in various combinations, and complex formation was observed by size exclusion chromatography.