Tutorial 3: A Protein and its Covalently Bonded Ligand 
=============================================================

.. contents::
  :local:

In this tutorial, we will show you how to use pdbtop to prepare a protein and its covalently bonded ligand for calculations.

Before we start, please make sure you have read :doc:`tutorial-1` and :doc:`tutorial-2`.

Combine Protein and Ligand
--------------------------

Now we combine the protein and ligand. For coordinates, we just paste the content of ``92v-1.pdb`` (see :doc:`tutorial-2`) at the end of ``5vbm-4.pdb`` (see :doc:`tutorial-1`) (before ``END`` statement), and save it to a new file, say ``5vbm-92v.pdb``.

For topology, we can mergy (``-m``) their PSF files:

.. code-block:: bash

  $ pdbtop psf -m "5vbm-4.psf 92v-2.psf" -o 5vbm-92v

Note that the order of PSF files must **MATCH*** the order of PDB files.

Now we look at the strucure:

.. image:: _static/figs/p5.png

So, we must treat the covalent bond between the protein and ligand.

To build a bond between ``SG`` of CYS72 (the 1081-th atom)  and ``S24`` (the 2686-th atom) of 92V. We have to ``modify`` the topology. Now we add a bond:

.. code-block:: bash

  $ pdbtop modify -i 5vbm-92v.pdb -t 5vbm-92v.psf --add-bonds "1081 2686" -o 5vbm-92v-1

Before proceeding, we note that ``HG1`` at CYS72 (the 1082-th atom) is to be deleted, so we adjust the charge to keep the total charge of the system constant. In ``5vbm-92v-1.psf``, we note that:

.. code-block:: bash
  :caption: 5vbm-92v-1.psf
  :emphasize-lines: 3-4
  :linenos:
  
  ...
  1080 A       72    CYS  HB2  HA        0.090000        1.0080           0
  1081 A       72    CYS  SG   S        -0.230000       32.0600           0
  1082 A       72    CYS  HG1  HS        0.160000        1.0080           0
  1083 A       73    ARG  N    NH1      -0.470000       14.0070           0
  1084 A       73    ARG  HN   H         0.310000        1.0080           0
  ...
  
So we add the charge of ``HG1`` to ``SG``, and set the charge of ``HG1`` to zero, and save it to a new file, say ``5vbm-92v-2.psf``:

.. code-block:: bash
  :caption: 5vbm-92v-2.psf
  :emphasize-lines: 3-4
  :linenos:
  
  ...
  1080 A       72    CYS  HB2  HA        0.090000        1.0080           0
  1081 A       72    CYS  SG   S        -0.070000       32.0600           0
  1082 A       72    CYS  HG1  HS        0.000000        1.0080           0
  1083 A       73    ARG  N    NH1      -0.470000       14.0070           0
  1084 A       73    ARG  HN   H         0.310000        1.0080           0
  ...

Next, we delete the hydrogen atom ``HG1`` at CYS72 (the 1082-th atom) and save it to a new file, say ``5vbm-92v-3.X``:

.. code-block:: bash

  $ pdbtop modify -i 5vbm-92v-1.pdb -t 5vbm-92v-2.psf --del-atom 1082 -o 5vbm-92v-3
  ...
  Write PDB: 5vbm-92v-3.pdb
  Write PSF: 5vbm-92v-3.psf
  Total charge: -5.00000

Now, we have a perfect covalently bonded protein and ligand:

.. image:: _static/figs/p6.png

``solvate`` System
--------------------------

Like introduced in :doc:`tutorial-1`, we need to add water to solvate the system, and add ions to neutralize it:

.. code-block:: bash

  $ pdbtop.exe solvate  -i 5vbm-92v-3.pdb -t 5vbm-92v-3.psf -o 5vbm-sol --box "70 70 70"

The output is:

.. code-block:: bash

  $ pdbtop.exe solvate -i 5vbm-92v-3.pdb -t 5vbm-92v-3.psf -o 5vbm-sol --box "70 70 70"
  ...
  Building water box: 70.000 x 70.000 x 70.000 Angstrom^3.
  12544 water molecules are added.
  Add ions:
   Target charge: 0
   Target ionic strength: 0.010 mol/L
  5 cations and 0 anions are added.
  Final ionic strength: 0.012 mol/L
  Write PDB: 5vbm-sol.pdb
  Write PSF: 5vbm-sol.psf
  Total charge: -0.00000

Now, we have a solvated, neutralized system, which is ready for calculations!

.. image:: _static/figs/p7.png