directory ($HOME). WATsite plugin can be installed using “Plugin
Manager” under Plugin menu, and choose the fileWATsite.py
under the “Install New Plugin” tab. The WATsite program needs
to be compiled on the platform by entering into the WATsite
directory and run “make –f makefile”.
The source code of WATsite compatible with OpenMM is
located under “openmm-watsite” and must be compiled with the
CUDA platform (seeNote 1).2.3 Input Data The inputs for WATsite are a protein structure and a binding site
definition both in pdb format. When predicting hydration site at the
protein–ligand interface, a ligand file in the mol2 format is required.
2.4 Hydration Site
Calculation
Here, we briefly describe the individual computational steps of the
WATsite procedure and the associated programs. A detailed
description of the WATsite method can be found in previous pub-
lications [18]. First, the side-chain conformations of ASN, GLN,
HIS, tautomers, and protonation states of HIS protein residues can
be adjusted by the Reduce program [19]. The user can also choose
to use a protein structure with previously predicted protonation
states (seeNote 2). Next, the protein will be solvated in an ortho-
rhombic box of user-selected explicit water model (seeNote 3) with
a user-specified minimum distance between any protein atom and
the faces of the orthorhombic box. Chloride and sodium ions will
be added to neutralize the systems. MD simulation will be per-
formed using OpenMM [15] with the user-selected amber force
field [20](seeNote 3). The system will be energy minimized and
equilibrated before production run. MD simulations can be per-
formed for a user-specified length (seeNote 4).
After the MD simulation, a 3D grid is placed over the user-
defined binding site. The occupancy of the water molecule is
distributed onto the 3D grid using all snapshots generated
throughout the production run of the MD simulation. The occu-
pancy distribution was then averaged over the production run and a
user-specified clustering algorithm (seeNote 5) was used to identify
the pronounced peaks that define the hydration site locations. For
each identified hydration site, WATsite tools will then be used to
compute the enthalpic (ΔHhs) and entropic (ΔShs) change of trans-
ferring a water molecule from the bulk solvent to the hydration site
of the protein binding site.3 Methods and Results
The WATsite PyMOL plugin contains six menu items as shown in
Fig.2a. This section will first describe the steps of using the plugin
to (1) modify settings, (2) prepare the system, (3) set parameters
for simulation, (4) run simulation with OpenMM, and (5) analyzeCalculation of Water Thermodynamics 391