3. Prepare the membrane-embedded protein (MEP) system.

3.1. Creating the MEP system using CHARMM-GUI.

Prepare the membrane-embedded protein system using CHARMM-GUI, making sure that the protein is correctly placed in the membrane. Use only POPC for the membrane for a simple yet effective membrane. While the lipid is characterized in AMBER, it should be no problem to use a more complex membrane (i.e., add cholesterol).

Remember to align the protein to the Z axis and use the AMBER FF. The membrane must be parametrized with AMBER for a better reliability with GAFF2, with which the small molecules are parametrized.

Note

The protein must be embedded in the membrane at the correct depth to avoid spending simulation time on achieving it. You can use nemat cm to automatically color the B-factor of your protein based on whether the residue is inside (I) or outside (O) of the membrane or at the membrane itself (M). This will also provide membrane.tcl which is useful to visualize the MEP system.

  1. Provide the FASTA of your protein to the DeepTMHMM-1.0 <https://services.healthtech.dtu.dk/services/DeepTMHMM-1.0/> server.

  2. Copy the IMO sequence (i.e. IIIOMMMMMMOOOIIIIIII…)``.

  3. Download step3_packing.pdb from the 3rd step of CHARMM-GUI. Place it on the NEMAT working directory along with the protein pdb.

  4. Use nemat cm, which will ask the user for the protein pdb file and the corresponding IMO sequence.

  5. Use vmd -e membrane.tcl to visualize the newly colored MEP system.

  6. Use Extensions > Visualization > Ruler to have an idea of which is the shift in Amstrongs of the proposed membrane. If the white region does not match the proposed membrane, you will have to move the protein in step 2 of CHARMM-GUI.

You can manually align the systems if something goes wrong: Use the Extensions > Analysis > RMSD calculator, uncheck the backbone only option, and write name CA and segname PROA A (A is the name of the chain of the original protein). Then, click “Align”.

Use only the gromacs folder of the output generated by CHARMM-GUI. Rename the following files provided by CHARMM-GUI:

  1. step5_input.gro –> system.gro

  2. topol.top –> system.top

The proteins folder should contain at least the following files per protein (prot1, prot2, etc):

proteins
  |  |
  |  |-- prot1
  |  |    |
  |  |    |-- toppar
  |  |    |-- system.gro
  |  |    |-- system.top
  |  |
  |  |-- prot2
  |  |    |
  |  |    |-- toppar
  |  |    |-- system.gro
  |  |    |-- system.top
  |  |
  |  |-- ...

3.2. Using a different method.

NEMAT searches for the files system.top (which references files inside the toppar folder), system.gro, and the folder toppar. Therefore, any set of files following this structure is a valid input for NEMAT.

Note

Actually, you could just change a force field folder’s name to toppar and update the #include inside the topology file. This would still provide a valid input for NEMAT.

If you are using a custom force field, it is recommended to use the same for the membrane system.