Molecular docking of Ap4A on STING was performed using ZDOCK version 3.0 using the STING crystal structure (PDB: 4KSY). RDOCK was used to refine and quickly evaluate the results obtained by ZDOCK. Energy minimization was performed to remove the geometrical strain from the top-ranking poses of Ap4A during the docking experiments. Top hit complexes of Ap4A and STING were subjected to extensive energy minimization run using the Amber99 force field as it is implemented into Gromacs, version 4.5.5, via the Gromita graphical interface, version 1.07. An implicit generalized born solvation was chosen at this stage, in an attempt to speed up the energy minimization process.

The interaction space and binding potential of each docking conformation were further explored by subjecting the molecular complexes to unrestrained molecular dynamics simulations using the Gromacs suite, version 4.5.5. Molecular dynamics took place in a periodic environment, which was subsequently solvated with sphingosylphosphoryl choline water using the truncated octahedron box extending to 7 Å from each molecule. Partial charges were applied, and the molecular systems were neutralized with counter ions as required. The temperature was set to 300 K, and the step size was set to 2 fs. The total run of each molecular complex was 100 ns, using the NVT ensemble in a canonical environment. NVT stands for number of atoms, volume, and temperature that remain constant throughout the calculation. The results of the molecular dynamics simulations were collected into a molecular trajectory database for further analysis.