Mapping was completed using the apo form of the proteins submitted to the FTMap server, http://ftmap.bu.edu.21 (link) (The FTMap site was accessed between June and October of 2011, and will be maintained to remain functional in the future.) For nNOS, both the heme and the cofactor were modeled with the apo structure during mapping; all other ligands were removed for all other systems. The FTMap algorithm21 (link) uses 16 small molecules as probes (ethanol, isopropanol, isobutanol, acetone, acetaldehyde, dimethyl ether, cyclohexane, ethane, acetonitrile, urea, methylamine, phenol, benzaldehyde, benzene, acetamide, and N,N dimethylformamide) and consists of four steps as follows.

The rotational/translational space of each probe is systematically sampled on a grid around the fixed protein, consisting of 0.8 Å translations and of 500 rotations at each location. The energy function includes a stepwise approximation of the Van der Waals energy with attractive and repulsive contributions, and an electrostatics/solvation term based on the Poisson-Boltzmann continuum model with dielectric constants of ε=4 and ε=80 for the protein and the solvent respectively61 . The energy expression is written as the sum of correlations functions, and hence it can be very efficiently evaluated using fast Fourier transforms.21 (link) The 2000 best poses for each probe are retained for further processing.

The 2000 complexes are refined by off-grid energy minimization during which the protein atoms are held fixed while the atoms of the probe molecules are free to move. The energy function includes the bonded and van der Waals terms of the CHARMM potential62 and an electrostatics/solvation term based on the Analytic Continuum Electrostatic (ACE) model63 as implemented in CHARMM.

The minimized probe conformations are grouped into clusters using a simple greedy algorithm. The lowest energy structure is selected and the structures within 4 Å RMSD are joined in the first cluster. The members of this cluster are removed, and the next lowest energy structure is selected to start the second cluster. This step is repeated until the entire set is exhausted. Clusters with less than 10 members are excluded from consideration. The retained clusters are ranked on the basis of their Boltzman averaged energies. Six clusters with the lowest average free energies are retained for each probe.

In order to identify consensus clusters where a number of probe clusters overlap19 (link) the probe clusters are themselves clustered using 4 Å distance between cluster centers as the clustering radius. The consensus clusters are ranked on the basis of the number of probe clusters contained.21 (link) To determine if any of the consensus clusters includes the core moiety from the fragment screening, the core-bound protein is superimposed on the unbound protein results using PyMol to obtain appropriate positioning and orientation of the core fragment. If a consensus cluster has 5 or more atoms within 1.25 Å of the core, it is considered to coincide with the core moiety, and is identified as “core consensus cluster” in Table 2. Similarly, if a (non-core) consensus cluster has 5 or more atoms within 1.25 Å of any atom of an extended ligand, which is not part of the core, it is defined as “extension consensus cluster”.