2.3 | Molecular Dynamics Simulation
In order to explore the structural and dynamic characteristics of the modeled complexes (OsL1/OsS1-ATP and OsL1:OsS1 heterotetramer), molecular dynamics (MD) simulations were conducted using GROMACS 5.139 with CHARRM36 force field40and periodic boundary conditions. The topology parameters for ATP were generated using the SwissParam server.41 The simulation systems were solvated using TIP3P water models and neutralized by adding Na+ and Cl-ions at a physiological concentration of 0.15 M to achieve electro-neutrality (Table. S1). Energy minimization of the systems were conducted using the steepest-descent method to avoid steric clashes and high energy interactions.42 Particle-mesh Ewald (PME) method was employed to handle long-range electrostatic interactions.43 Subsequently, the energy-minimized systems were position-restrained (equilibrated) in two different phases; NVT (constant volume and temperature) and NPT (constant pressure and temperature) for 0.1 and 1 ns, respectively. Then, the equilibrated monomeric systems (OsL1-/OsS1-ATP) were simulated up to 70 ns, and the heterotetrameric systems (C-I, C-II, and C-III) were subjected to 100 ns production run (in duplicate). The temperature and pressure of all simulation systems were maintained at 300 K and 1 bar, respectively. The built-in GROMACS tools were used for trajectory analysis. PDBe-PISA web interface (https://www.ebi.ac.uk/pdbe/pisa/picite.html) was used to analyze protein-protein interactions. PyMOL (https://pymol.org/) was used for structural visualization. 2D graphs were plotted in Grace 5.1.2 (http://plasma‐gate.weiz-mann.ac.il/Grace/).