trajectory with WATsite. Water thermodynamics with (6) and with-
out (7) the presence of ligand can be calculated. We will then
explain how to import and analyze those results.3.1 Modify Settings The location paths to the required molecular modeling programs
need to be correctly specified. Select the menu item “Modify Paths
to Installed Programs” from the WATsite menu (Fig.2b). The
users need to specify or modify the paths to the programs according
to their installation. For example, the location of WATsite directory
should be defined forwatsite_home, and similarly the python path
associated with OpenMM, the path to Amber, PyMOL, and
Reduce installations. The settings only need to be modified for
the first time, and will be automatically read in subsequent sessions.
3.2 Prepare Protein/
Ligand System
The user can specify a protein structure from a file or a structure
already displayed in the current PyMOL session (Fig.3a). The user
can choose to perform protonation site analysis using Reduce [19],
or use a structure with previously predicted protonation states (see
Note 2). To define the protein binding site, the user needs to
provide a ligand molecule positioned within the binding site or a
“pseudo-ligand” using binding site residues. A margin (in A ̊) will
be used to define the binding pocket specifying a box surrounding
the ligand/pseudo-ligand. The minimum distance between any
ligand heavy atom and the edge of the box equals to the margin
value. Hydration sites can be predicted for both ligand-free (apo)Fig. 2(a) WATsite PyMOL plugin menu, and (b)step 1to specify the correct paths to the installed program
392 Ying Yang et al.