a, Cryo-EM density map in the region of the BRAF active site showing bound ATP-γ-S. b, Cryo-EM density map in the region of the MEK1 active site indicating bound ADP, which is probably hydrolysed from ATP-γ-S. Maps in a and b are contoured at the same level. c, Superposition of the BRAF–MEK1 component of the present autoinhibited cryo-EM structure (green and dark blue) with the previously reported crystal structure of a BRAF and MEK1 kinase domain complex (yellow and light blue; PDB ID: 4MNE). The superposition is based on the MEK component of the structures, and it reveals a relative rotation of BRAF of approximately 5° about the C-lobe contact. d, Superposition of the BRAF kinase domain from the present structure with that of previously isolated BRAF–MEK kinase complex (PDB ID: 4MNE). Note that the present structure (dark blue, with C-helix coloured purple and the activation segment orange) exhibits key features of an autoinhibited state (C-helix out, with an inhibitory turn in the activation segment), whereas the previous structure (light blue) adopts an overall active conformation. e, Detailed view of a portion of the C-lobe contact between BRAF (blue) and MEK1 (green). f, Portions of the BRAF (blue) and MEK1 (green) activation segments interact in an anti-parallel orientation. Activating phosphorylation sites in the MEK1 activation loop are substituted with alanine in this structure (S218A/S222A), but neither residue is positioned appropriately for phosphorylation by BRAF. Note that our discussion of these interactions relies in part on the crystal structures referenced to build the atomic model, as the cryo-EM map in this region does not unambiguously define all sidechain conformations. g–j, Comparison of BRAF kinase domain conformations and relative N- and C-lobe orientations. g, Sulfonamide-containing BRAF inhibitors perturb the inactive conformation of BRAF. The BRAF kinase domain in the present structure (blue ribbon, with C-helix coloured red and the activation segment orange) is superimposed on the structure of the BRAF kinase domain crystallized as a monomer with PLX4720 (grey, PDB ID: 4WO5). The superposition is based on the C-lobes of both kinases, revealing an altered orientation of the N-lobe in the inhibitor-bound structure (a rotation of around 15°). Note also that the inhibitory turn in the activation segment helix is replaced by a short helix in the PLX4720 complex. h, Alternative view of the superposition shown in g, highlighting the axis of rotation (pink arrow) between the N-lobes. i, As in h, but with a representative inhibitor-bound dimeric BRAF structure superimposed (PDB ID: 5CSW, a dabrafenib complex). The rotation axes for N-lobe rotations of dimer structures are shown as green arrows. Note that the orientation of the rotation axis is similar for all of the dimer structures, but almost orthogonal to that of the monomer structure in h. In both h and i, the Cα atoms of K522 are shown as spheres as a point of reference. j, Relative N-lobe rotation of wild-type and BRAF(V600E) crystal structures available in the Protein Data Bank (PDB) are compared with the present nucleotide-bound, autoinhibited structure. As illustrated in h and i, C-lobes of the BRAF kinase domains were superimposed, and the rotation required to bring the kinase N-lobes into register were calculated using PyMOL. With the exception of 4MNE, all structures compared were determined in complex with inhibitors.