5. Prepare the ligands.

5.1. Obtaining the reference ligand: ref_lig.pdb.

First, we need to align the reference protein-ligand system (i.e., a crystallographic structure from the PDB containing the protein and the docked ligand) with the newly generated GROMACS system. Use VMD or any aligning tool to align the two proteins. For VMD, you could do:

  1. Load the two proteins. In this case: system.gro and prot_lig.pdb.

  2. Go to Extensions > Analysis > RMSD calculator. This will display a new window.

  3. At the top left box, type name CA.

  4. Unclick the option Backbone only.

  5. Click Align to align the two systems.

  6. Save the new ligand coordinates.

Warning

It is important that the GROMACS system (system.gro) is marked with the option “T” on the VMD main page. If not, the whole system will be aligned to the protein and not the other way around.

Then extract the ligand, which will serve as a reference for aligning the other ligands. Save it as ref_lig.pdb.

5.2. Aligning the ligands.

Add hydrogens to the reference ligand and to the rest (for example, using CHARMM-GUI). You can manually align the ligands and directly save the mol2 files at the ligands folder.

If you have pdb files of the ligands, the align.sh file might be useful. Save all the pdb files of your ligands inside the “/ligands/ligands” folder and the ref_ligand.pdb on the “/ligands” folder. Then use:

cd ligands bash align.sh

This will generate ligands.sdf and all the necessary files: it will align the ligands with the reference ligand and then generate mol2 files for every ligand.

You can use the ligands.sdf file to both visualize all the ligands at once and, for example, to use some program such as openFE to generate a mapping of the ligands and determine the possible edges.

The ligands folder should contain (at least) the mol2 of all the ligands. The whole folder, including the reference ligand and the alignment script, should look like this:

ligands
  |  |
  |  |-- ligands
  |       |-- ligand1.pdb
  |       |-- ligand2.pdb
  |       |-- ...
  |
  |-- ref_lig.pdb
  |-- ligands.sdf
  |-- align.sh
  |-- ligand1.mol2
  |-- ligand2.mol2
  |-- ...

Note

When starting a NEMAT run, some dummy files are inside the ligands and lignads/ligands folders to help the user know where every kind of file should be placed. Remove them before aligning with align.sh.

Check that the ligand alignment is correct. If the molecule is small, it is possible that something went wrong. You can manually align the ligands and directly save the mol2 files at the ligands folder.