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:

Load the two proteins. In this case: system.gro and prot_lig.pdb.
Go to Extensions > Analysis > RMSD calculator. This will display a new window.
At the top left box, type name CA.
Unclick the option Backbone only.
Click Align to align the two systems.
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. Obtaining the membrane reference ligand: ref_lig_mem.pdb (optional).¶

Ligand partition on the membrane is a common problem in free energy calculations. If you are interested in the \(\Delta \Delta G_{\text{mem}}\) (involving the water and membrane systems) or \(\Delta \Delta G_{\text{int}}\) (involving the membrane and MEP systems), you might want to consider performing a simulation of the ligan partition into the membrane to obtain a reference ligand in the membrane.

As in the previous case, make sure that the membranes are correctly aligned and then extract the ligand pdb file.

If you are not interested in the membrane partition, you can skip this step and use the same reference ligand for all the systems.

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 after running align.sh:

ligands
  |  |
  |  |-- ligands
  |  |    |-- ligand1.pdb
  |  |    |-- ligand2.pdb
  |  |    |-- ...
  |  |
  |  |-- aligned
  |  |    |-- ligand1_a.pdb
  |  |    |-- ligand2_a.pdb
  |  |    |-- ...
  |  |    |-- mol2
  |  |    |    |-- ligand1.mol2
  |  |    |    |-- ligand2.mol2
  |  |    |    |-- ...
  |  |    |
  |  |    |-- sdf
  |  |    |    |-- ligand1.sdf
  |  |    |    |-- ligand2.sdf
  |  |    |    |-- ...
  |
  |-- ref_lig.pdb
  |-- ligands.sdf
  |-- align.sh
  |-- ligand1.mol2
  |-- ligand2.mol2
  |-- ...

Note

When starting a NEMAT run, some dummy files (starting with “INFO_”) 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.

If you have a file named “ref_lig_mem.pdb”, the align.sh script will also align the ligands to the reference ligand in the membrane and generate the corresponding mol2 files:

ligands
  |  |
  |  |-- ligands
  |  |    |-- ligand1.pdb
  |  |    |-- ligand2.pdb
  |  |    |-- ...
  |  |
  |  |-- aligned
  |  |    |-- ligand1_a.pdb
  |  |    |-- ligand2_a.pdb
  |  |    |-- ...
  |  |    |-- mol2
  |  |    |    |-- ligand1.mol2
  |  |    |    |-- ligand2.mol2
  |  |    |    |-- ...
  |  |    |
  |  |    |-- sdf
  |  |    |    |-- ligand1.sdf
  |  |    |    |-- ligand2.sdf
  |  |    |    |-- ...
  |  |
  |  |-- membrane
  |  |    |-- aligned
  |  |    |    |-- ligand1_a.pdb
  |  |    |    |-- ligand2_a.pdb
  |  |    |    |-- ...
  |  |    |    |-- mol2
  |  |    |    |    |-- ligand1.mol2
  |  |    |    |    |-- ligand2.mol2
  |  |    |    |    |-- ...
  |  |    |    |
  |  |    |    |-- sdf
  |  |    |    |    |-- ligand1.sdf
  |  |    |    |    |-- ligand2.sdf
  |  |    |    |    |-- ...
  |
  |-- ref_lig.pdb
  |-- ref_lig_mem.pdb
  |-- ligands.sdf
  |-- align.sh
  |-- ligand1.mol2
  |-- ligand2.mol2
  |-- ...

The mol2 files of the membrane must be inside the membrane/mol2 folder while at the ligands folder you must place the mol2 files of the MEP system.