## 3 Results
### Raf-CRD Links to the Active Site through Loop 8 across the Dimer Interface  
To study Ras-Raf in the context of the dimer and determine possible roles for the CRD, we modelled the HRas-CRaf-RBD_CRD dimer utilizing a two-fold symmetry axis conserved across many Ras crystal structures, including our structure of the HRas-CRaf-RBD complex (PDB ID 4G0N) containing the \(\alpha\)4-\(\alpha\)5 dimer interface [7; 50; 51]. In this model, the Raf-CRD is positioned for interaction with both Ras protomers in the Ras dimer, bridging the interswitch region of one to loop 8 of the other at the base of the Ras dimerization interface (Figure 2a), as is found in the recently published KRas-CRaf-RBD_CRD crystal structure containing the dimer (PDB ID 6 XI7) [20]. We performed three independent replicates of molecular dynamics (MD) simulations, each for a duration of 350 ns, totaling just over 1 us. The model equilibrated quickly and was stable throughout the trajectories, with average RMSD near 2.5 A from the starting model in each case (Figure S3a). The simulations show greater fluctuations for Raf-CRD than observed for the rest of the complex (Figure S3b), with sampling of interactions seen in the KRas-CRaf-RBD_CRD crystals where the dimer is present. Figure 2: HRas–CRaf-RBD_CRD dimer complex. (**a**) Modelled HRas–CRaf-RBD_CRD dimer complex. Approximately perpendicular orientation of helices 3, 4 and 5 with respect to the membrane does not allow the Raf-CRD to interact with the membrane. (**b**) Frame taken from the replicate 2 HRas–CRaf-RBD_CRD simulation after 60 ns shows sample Ras–Raf-CRD’ (**left**) and Ras’/Raf-CRD (right) interactions. (**c**) Frame taken from replicate 3 HRas–CRaf-RBD_CRD simulation after 140 ns shows sample Ras–Raf-CRD’ (**left**) and Ras’/Raf-CRD (**right**) interactions. Right and left panels in b and c illustrate the asymmetry introduced through the Raf-CRDs’ dynamic interaction with the opposing Ras protomers. One protomer of HRas in the dimer is shown in pink and the other, HRas’ in yellow. Both CRDs in the dimer, CRD and CRD’ are shown in dark green. In the starting model for simulations of the HRas-CRaf-RBD_CRD dimer, Raf-CRD residues R143, N161, and E174 are within interacting distance of loop 8 residue R123, the nucleotide-binding residue D119, and Q150 at loop 10 immediately preceding \(\alpha\)5 in the opposing Ras protomer. Ras D119 is part of the NKxD motif (residues 116-119), and directly binds the guanine base in the Ras active site (Figure 2b,c). This motif is followed by loop 8, containing residue R123 which makes a salt bridge interaction with E143 of the ExSAK motif (residues 143-147), linking two highly conserved regions important for active site stability [52]. The Ras R123-E143 salt bridge remains present throughout the simulations, even as R143 of the Raf-CRD moves to interact with Ras E143 (Figure 2b,c, Figure S4a (replicate 1), Figure S5a (replicate 2) and Figure S6a (replicate 3)), contributing to the interface with the opposing Ras promoter. Raf-CRD residue N161 samples various states in which it can make contacts with the side chains of Ras T148 and Q150 in loop 10 (Figure 2b,c, Figures S4b, S5b and S6b) or with the backbone of loop 8 residues C118, D119, and L120 (Figure 2b,c, Figures S4c, S5c and S6c). Ras Q150 also interacts with Raf-CRD E174 and H175 (Figure 2b,c, Figures S4d, S5d and S6d). Raf-CRD T145 is also observed to interact with the loop 8 residues D119 and L120 (Figure 2b,c). Various interactions revealed in the simulations, although not initially present in the model built from our structure of the HRas-CRaf-RBD_CRD in which the dimer is not present (PDB ID 7 JHP), are observed in the recently published structure of the KRas-CRaf-RBD_CRD dimer (PDB ID 6 XI7). Most notably, the interactions between the Raf-CRD and both the glutamate and lysine of the ExSAK nucleotide-binding motif are observed both in our simulations and KRas-CRaf-RBD_CRD dimer structure (PDB ID 6 XI7).