Kmn1, Mis12 and either Nnf1a or Nnf1b were coexpressed in E. coli (otherwise proteins were insoluble), then purified by gel filtration. The complex was then treated with BS2G bi-functional crosslinking reagent, and cross-links were mapped by mass spectrometry.

Within either the Kmn1-Mis12-Nnf1a or Kmn1-Mis12-Nnf1b trimeric complexes, all three subunits make extensive contacts with each other, extending along their entire sequences.

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

Kmn1, Mis12, Nnf1a and Nnf1b were co-expressed (otherwise proteins were insoluble), then pulled down via either tagged Nnf1a or Nnf1b.

Kmn1 interacts with either Nnf1a or Nnf1b in distinct complexes.

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

Kmn1, Mis12 and either Nnf1a or Nnf1b were coexpressed in E. coli (otherwise proteins were insoluble), then analyzed by gel filtration to assess complex formation.

Kmn1 and Mis12 interact as part of a trimeric complex with either Nnf1a (64.5 kDa) or Nnf1b (67.1 kDa).

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