# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 4 Methods  
Using the accession number 5 UHV [52] from the Protein Data Bank [62], the initial coordinates for the simulations were derived from crystal structures. Specifically, for the simulations involving the N-Ras-GTP complex, a refined crystal structure was selected as the reference. The N-Ras structure (residues 1-165) was altered using Chimera [63] to create a mutant structure for the N-Ras (G12V) G domain using the Dunbrack rotamer library [64], and the hypervariable region was created using the \"Interactive peptide chains modeling\" molefacture plugin from VMD [58]. The GTP molecule in the active site was left in the same location as the GTP analog in the original structure, and the \(\gamma\)-phosphate was manually substituted for the terminal group of the GTP analog found in the crystal structure to create a GTP ligand. Coordinates for crystal magnesium were overlaid on top of the magnesium ion center. The lipid bilayer structures were made using Membrane Builder [65]. Following the insertion of the protein into one leaflet, the system was neutralized and solvated with TIP3P water [66] to a physiological concentration of 0.15 mol/L of Na\({}^{+}\) Cl\({}^{-}\). N-Ras proteins form functional nanoclusters that are preferentially localized at the border of liquid-disordered (L\({}_{d}\)) and liquid-ordered (L\({}_{o}\)) domains [30]. The minimal membrane model required to obtain such phase separation is a ternary lipid bilayer with an unsaturated tailed phospholipid, a saturated tail phospholipid, and cholesterol [30]. For our mammalian membrane model, we used a ternary lipid bilayer formed of 160 DPPC (di-16:0-PC), 96 DOPC (di-18:1-PC), and 64 CHOL (cholesterol) (i.e., in 5:3:2 ratio), as in our previous studies [36; 37]. Two separate types of atomistic molecular dynamics simulations were conducted using the NAMD 2.9 simulation program [67] to study the GTP-bound full-length N-Ras wild-type (WT) and mutant (G12V) proteins (details are in Table S2). The CHARMM36(m) force field for both lipids [68] and proteins [69; 70] was employed in all our simulations. In our model systems, the guanosine triphosphate (GTP)-bound N-Ras protein model (see Figure 1A,B) was either solvated in water at a physiological ionic concentration (without the anchor and without the membrane) or inserted with its (natural) post-translationally lipid-modified anchors (palmitoylated Cys181-PAL and farnesylated Cys186-FAR) into our fully solvated membrane model (see Figure 1C) (membrane-bound) [55; 71]. The two preferred orientations of Ras proteins with respect to the membrane were used as starting configurations in our N-Ras simulations [22]. The simulations were carried out using a standardized protocol. Firstly, each system was minimized for 5000 steps using a conjugate gradient energy minimization-based algorithm [72]. Then, a 100 ps warm-up was conducted, increasing the temperature from 0 to 300 K at a rate of 3 K/ps, followed by a 50 ps 300 K temperature coupling. Next, a 350 ps constrained equilibration was performed, only along the normal to the membrane direction, at 300 K. Finally, a production run of hundreds of ns was executed for each simulation using a step cycle of 20 and a 2 fs integration timestep, in conjunction with constraints applied to all bonds involving hydrogen using the SHAKE algorithm [73; 74]. For unbound proteins (i.e., in a water solution only), the same protocol was used without inserting the protein into the membrane. For the calculation of long-range electrostatic interactions, the particle mesh Ewald method [75] was employed. A spacing of 1 A and a cutoff distance of 12 A were used to accurately account for the long-range interactions between charged particles. To maintain a constant temperature of 300 K, a Langevin thermostat [76] was utilized during all simulations, with a damping coefficient of 1 ps\({}^{-1}\). Additionally, the pressure was controlled using a Langevin piston [77] at 1 atm, allowing for the simulation of the system under constant pressure conditions.