2. Setup of the solvated protein system

• Setup all directories:

  mkdir coord top solvation emin posres MD analysis

• Get 4AKE and only keep chain A:

  Note

  The starting structure coord/4ake_a.pdb has been provided as part of the tutorial package so the instructions here are simply telling you what you would need to do if the file hadn’t been provided.

  • download from the protein databank through the webinterface (pdb: 4AKE) or use pdb_downloader.sh:

    pdb_downloader.sh -d coord 4ake

    (You wrote the pdb_downloader.sh script as part of an assignment for Practical 2.)

  • modify the structure; in simple cases such as the one here, you can just open the PDB file in a text editor and remove all the lines that are not needed. In more difficult cases you might have to use molecular modelling software.

    • remove all comment lines (but keep TITLE, HEADER)
    • remove all crystal waters (HOH) [1]
    • remove all chain B ATOM records.
    • save as coord/4ake_a.pdb.

• Generate a topology for the CHARMM27 force field together with the TIP3P water model with pdb2gmx tool:

  cd top
  pdb2gmx -f ../coord/4ake_a.pdb -o protein.pdb -p 4ake.top -i protein_posre.itp -water tip3p -ff charmm27 -nochargegrp

  Note: total charge -4.000e (in the next step we will add ions to neutralize the system; we need a net-neutral system)

• solvation: create a simulation box with editconf and add solvent with genbox:

  cd solvation
  editconf -f ../top/protein.pdb -o boxed.pdb -d 0.5 -bt dodecahedron -c
  genbox -cp boxed.pdb -cs spc216 -p ../top/4ake.top -o solvated.pdb

  Note

  In order to reduce the system size and make the simulations run faster we are choosing a very tight box (minimum protein-edge distance 0.5 nm, -d 0.5); for simulations you want to publish this number should be 1.2...1.5 nm so that the electrostatic interactions between copies of the protein across periodic boundaries are sufficiently screened.

  genbox updates the number of solvent molecules (“SOL”) in the topology file (check the [ system ] section in top/system.top) [2].

• add ions with the genion program.

  First we need a basic TPR file (an empty file is sufficient, just ignore the warnings that grompp spits out by setting -maxwarn 10), then run genion (which has convenient options to neutralize the system and set the concentration (check the help!); genion also updates the topology’s [ system ] section if the top file is provided [2]; it reduces the “SOL” molecules by the number of removed molecules and adds the ions, e.g. “NA” and “CL”).

  touch ions.mdp
  grompp -f ions.mdp -p ../top/4ake.top -c solvated.pdb -o ions.tpr
  echo "SOL" | genion -s ions.tpr -random -pname NA -pq 1 -nname CL  -nq -1 -conc 0.1 -neutral -p ../top/4ake.top -o ionized.pdb

  The final output is solvation/ionized.pdb. Check visually in VMD (but note that the dodecahedral box is not represented properly.)

Footnotes

[1]

Often you would actually want to retain crystallographic water molecules as they might have biological relevance. In our example this is likely not the case and by removing all of them we simplify the preparation step somewhat. If you keep them, pdb2gmx in the next step will actually create entries in the topology for them.

[2]

(1, 2) The automatic modification of the top file by genbox and genion can become a problem if you try to run these commands multiple times and you get error messages later (typically from grompp) that the number of molecules in structure file and the topology file do not agree. In this case you might have to manually delete or adjust the corresponding lines in :file”system.top file.