Purified ave-cnk heterodimer was mixed with an equimolar ratio of ksr-Dsor1 heterodimer, which eluted as a higher order complex captured as series of binary interactions that do not necessarily indicate direct protein-protein contact. Mutations in cnk or ksr interfere with the higher order cnk-ave-ksr-Dsor1 complex.

Interaction in vitro; ksr produced as a recombinant fusion protein in baculovirus and insect cell system; ave produced as a recombinant fusion protein in bacterial cell system.

Interaction in vitro; ksr produced as a recombinant fusion protein in a baculovirus and insect system; ave produced as a recombinant protein in bacterial system.

cryoEM structure of ave-cnk-ksr-Dsor1 complex captured as a series of binary interactions that are not all direct. cryoEM indicates direct interaction between ave-cnk, ksr-Dsor1, cnk-ksr and cnk-Dsor1.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected construct.

Interaction occurred with co-expression of Ras85D, Raf, cnk and ksr in Extended Data Fig2CFI, Fig2CDF, Fig3B. Interaction occurred with co-expression of cnk and Dsor1 in Extended data Fig7EG. Mutations in either ave or cnk that destabilize the ave-cnk scaffolding complex or mutations in Dsor1, decrease ave\'s ability to pull down ksr.