of the restraint is constant, and the restraint is applied to the
distance between C and L atoms. Beside this additional
restraint, the conditions in the targeted MD step are the same
as in the last part of the above-described equilibration step. The
restraint force constantkused is 200 kcal/(mol A ̊^2 ) and the
length of the targeted MD stepttMDis 4 ns (seeNote 13).- The complex obtained in the previous step is simulated further
without any restraints under the same conditions for 10 ns (see
Note 14). - Such procedure is repeated 100 times (seeNote 15).
- One hundred frames from the last 200 ps of each free MD
trajectory are taken for MM-PBSA/MM-GBSA analysis yield-
ing docking poses final scores and single residue energy decom-
position. Averaged from repeated DMD runs single residue
energy decomposition defines the anchoring residues in the
whole ensemble of docking solutions. The poses with
MM-PBSAΔG>1 kcal/mol are considered not to represent
binding and, therefore, are excluded from the further analysis. - DBSCAN algorithm [19] is used for clustering the obtained
docking solutions. Instead of RMSD as a metric for clustering,
we use a distance metric as the root-mean-square of pairwise
atomic distances while pairing up the spatially closest atoms of
the same type. This distance metric accounts for periodicity of
GAGs and so allows avoiding periodicity-related artifacts
obtained when classical RMSD is used.
3 Notes
- After the complete water placement procedure is carried out,
these molecules can be used for comparison with the obtained
predicted water molecules, which could give an idea about the
reproducibility of the experimental data (if the receptor is not
energy minimized prior to the water placement) or about the
changes in the hydration pattern associated with the energy
minimization of the receptor structure (in case the receptor is
minimized). Note that although there are water molecules
explicitly obtained in X-ray structures, in most of the cases
the information about the hydration is far from being
complete. - This step should be done carefully: some divalent ions, for
example, could be crucial for the local protein structure and
its hydration properties and, therefore, substantially affect fur-
ther docking results obtained using this receptor structure. In
case there is direct literature evidence for this, such ions should
be taken into account either being included into water
450 Sergey A. Samsonov