Fig. 1: Preparation of KRAS/BRAF complexes for structural analysis.

a Schematic of domain structures of BRAF, KRAS, and MEK1. Key phosphorylation sites are indicated above the schematics. Binding sites for the 14-3-3 domain in BRAF are indicated in red. BRS BRAF-specific domain, RBD RAS-binding domain, CRD cysteine-rich domain, HVR RAS hypervariable region. b Size-exclusion chromatography of the KRAS/BRAF/MEK1/14-3-3 complex. Elution profile of the complex on Superdex S200 is shown on the left, and a Coomassie-stained SDS-PAGE gel of the indicated fractions is shown on the right. The experiment was performed more than three times with similar results. c Size-exclusion chromatography of a BS3-cross-linked KRAS/BRAF/MEK1/14-3-3 sample. The complex analyzed in (b) was subjected to cross-linking with BS3 and re-examined with size-exclusion chromatography. Elution profile of the complex on Superose 6 is shown on the left, and a Coomassie-stained SDS-PAGE gel of the indicated fractions is shown on the right. Aliquots of the input sample before and after approximately sevenfold concentration, but prior to cross-linking, are also shown on the gel. The experiment was performed three times with similar results.