PSOMI WORKFLOW This workflow contains steps for analysis interaction between small ligand and protein by using ChemSketch, Open Babel, PRODRG and Gromacs on HPC cluster. Workflow uses complex of small ligand and protein as input file (in .gro format), and gives trajectories (.trr extension) that further can be analysed in VMD or similar software. In this example we examined interaction between small ligand (2R,3R,4R,5S)-5-hidroksi-5-((S)-5- metilen-2-okso-1,3-dioksolan-4-il)pentan-1,2,3,4-tetraacetat and protein lysozyme. ORTEP diagram of small molecule is shown in Fig 1. Figure 1. ORTEP diagram of 2R,3R,4R,5S)-5-hidroksi-5-((S)-5-metilen-2-okso-1,3-dioksolan-4- il)pentan-1,2,3,4-tetraacetat First, freeware software ChemSketch is used to draw the molecule and make 3D geometric optimisation. The resulting molecule (Fig. 2) is saved in .mol extension. H O O H H H O H O O O H H O H H H H H H H H O O O H HH H H O O H H Figure 2. Small ligand in ChemScetch After obtaining the 3D molecule in .mol fomat, the Open Babel is used for conversion to .pdb format. The resulting molecule, visualised in VMD, is shown in Fig. 3. Figure 3. Visualisation of ligand in VMD Finally, topology of ligand is generated by using online software PRODRG. The output "The GROMOS87/GROMACS coordinate file (polar/aromatic hydrogens)" is saved in file called "molecule.gro" and "The GROMACS topology" into a file called "drg.itp". The content of molecule.gro is listed below: PRODRG COORDS 29 1LIG CAY 1 1.559 -1.135 -0.290 1LIG CAX 2 1.604 -1.048 -0.172 1LIG OAZ 3 1.652 -1.101 -0.072 1LIG OAF 4 1.592 -0.913 -0.193 1LIG CAA 5 1.637 -0.831 -0.085 1LIG CAB 6 1.761 -0.746 -0.117 1LIG OAG 7 1.780 -0.663 -0.001 1LIG CBA 8 1.834 -0.540 -0.020 1LIG OBB 9 1.954 -0.517 -0.005 1LIG CBC 10 1.737 -0.428 -0.060 1LIG CAC 11 1.887 -0.834 -0.142 1LIG CAD 12 2.011 -0.751 -0.180 1LIG OAI 13 2.008 -0.705 -0.318 1LIG CAR 14 1.951 -0.583 -0.335 1LIG OAS 15 1.829 -0.565 -0.336 1LIG CAT 16 2.048 -0.465 -0.348 1LIG OAH 17 1.865 -0.933 -0.245 1LIG CAU 18 1.956 -1.034 -0.263 1LIG OAW 19 1.923 -1.106 -0.357 1LIG CAV 20 2.076 -1.063 -0.196 1LIG OAJ 21 2.123 -0.961 -0.105 1LIG CAE 22 2.143 -0.829 -0.163 1LIG CAK 23 2.236 -0.746 -0.073 1LIG OAO 24 2.262 -0.619 -0.134 1LIG CAN 25 2.385 -0.630 -0.193 1LIG OAP 26 2.425 -0.554 -0.280 1LIG OAM 27 2.451 -0.741 -0.150 1LIG CAL 28 2.364 -0.800 -0.064 1LIG CAQ 29 2.393 -0.899 0.020 1.04200 1.04200 1.04200 MD simulations are performed in Gromacs. For simulations we used cluster with 24 nodes. The simulation steps in Gromacs are described below. The resulting files from each phase (trajectories, .trj) can be further analysed with Gromacs commands (RMSD, energy minimisation curves, etc ) or in VMD or similar software. 1. Generate topology a) The protein structure 3HTB is processed by pdb2gmx, and GROMOS96 43A1 force field is chosen. gmx pdb2gmx -f 3HTB_clean.pdb -o 3HTB_processed.gro -water spc b) Next, a file called "3HTB_processed.gro", from pdb2gmx, is concatenated with "molecule.gro" in new file complex.gro. Also, a line that says #include "drg.itp" is inserted into topol.top. 2. Solvation After building the complex we used the following commands for adding the solvent and simulation of solvation: gmx editconf -f complex.gro -o newbox.gro -bt dodecahedron -d 1.0 gmx solvate -cp newbox.gro -cs spc216.gro -p topol.top -o solv.gro 3. Adding ions Solvated system contains a charged protein, so we added 6 CL ions by using the following commands: gmx grompp -f em.mdp -c solv.gro -p topol.top -o ions.tpr gmx genion -s ions.tpr -o solv_ions.gro -p topol.top -pname NA -nname CL - nn 6 4. Energy minimisation For energy minimisation we used em_real.mdp configuration file. This mdrun command is executed on the cluster. The content of pbs file is listed below. gmx grompp -f em_real.mdp -c solv_ions.gro -p topol.top -o em.tpr qsub 1.pbs 1.pbs #!/bin/bash #PBS -l nodes=24 #PBS -o test_opm.out #PBS -e test_omp.err #PBS -W group_list=margi #PBS -q margi #PBS -r n #PBS -V #PBS -t 1 cd $PBS_O_WORKDIR module avail /home/local/luka.filipovic/gromacs/gromacs-no-mpi/bin/gmx mdrun -deffnm em 5. Equilibration The next phase is an equilibration. In this phase we used nvt.mdp configuration files and the following set of commands: gmx genrestr -f molecule.gro -o posre_jz4.itp -fc 1000 1000 1000 gmx make_ndx -f em.gro -o index.ndx gmx grompp -f nvt.mdp -c em.gro -p topol.top -n index.ndx -o nvt.tpr qsub 2.pbs 2.pbs #!/bin/bash #PBS -l nodes=24 #PBS -o test_opm.out #PBS -e test_omp.err #PBS -W group_list=margi #PBS -q margi #PBS -r n #PBS -V #PBS -t 1 cd $PBS_O_WORKDIR module avail /home/local/luka.filipovic/gromacs/gromacs-no-mpi/bin/gmx mdrun -deffnm nvt 5. Equilibration Phase 2 Once the NVT simulation is complete, proceed to NPT with: npt.mdp gmx grompp -f npt.mdp -c nvt.gro -t nvt.cpt -p topol.top -n index.ndx -o npt.tpr qsub 3.pbs 3.pbs #!/bin/bash #PBS -l nodes=24 #PBS -o test_opm.out #PBS -e test_omp.err #PBS -W group_list=margi #PBS -q margi #PBS -r n #PBS -V #PBS -t 1 cd $PBS_O_WORKDIR module avail /home/local/luka.filipovic/gromacs/gromacs-no-mpi/bin/gmx mdrun -deffnm npt 6. MD Simulation Upon completion of the two equilibration phases, the system is well-equilibrated at the desired temperature and pressure. The MD simulation was run for 1ns (md.mdp). The mdrun command is also executed on the cluster, as in the previous cases. The commands are listed below. gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -n index.ndx -o md_0_1.tpr gmx mdrun -deffnm md_0_1 .