Generating initial configurations of Ras-SOS complex

To construct initial configurations, we obtained the crystal structures of SOS1 (PDB: 4NYJ) (15), GDP-bound KRas4B^C118S (PDB: 4EPT) (24), and GTP-bound KRas4B^Q61H (PDB: 3GFT) from the Protein Data Bank (PDB). In 4NYJ, both Ras binding sites (allosteric and catalytic) in SOS1 are occupied by HRas. At the allosteric site, the Ras protein is a GNP-bound HRas, and it is a nucleotide-free HRas at the SOS1 catalytic site. After replacing the mutants with the wild-type residues, KRas4B was superimposed onto HRas in complex with SOS1, generating the coordinates for the KRas4B-SOS1 complex. At the catalytic site of SOS1, the switch I region of both GDP- and GTP-bound KRas4B clashed with the αF helix because of the switch I open conformation of the nucleotide-free Ras. To avoid this steric clash, we re-modeled the switch I region using the Modeler server (29), ensuring that the modeling of switch I does not affect the positions of GDP, GTP, and Mg²⁺. A total of 12 SOS1 systems were constructed: an apo-SOS1 monomer, SOS1⁰⁰; four dimeric systems, SOS1^D0, SOS1^T0, SOS1^0D, and SOS1^0T; four ternary systems, SOS1^DD, SOS1^DT, SOS1^TD, and SOS1^TT; and four ternary systems in the exchange event, SOS1^TD∗, SOS1^TR, and SOS1^TT∗ (details of the notations are given in the Results).