# Comment lines starting with hashtags will be ignored
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# Your brief name for this protocol
Name: alchemical free energy protocol with average network analysis same charge
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# All software packages used and their versions:
Software: FESetup release 1.3dev, SUI version: 0.8.1, somd-freenrg 2016.1.1, pymbar version 3.0.0.dev0.dev-Unknown, Networkx v.1.11

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# List any number of key computational parameters in the following lines:
Parameter: forcefield amber, ff14sb, tip3p, hfe
Parameter: gaff2
Parameter: mdengine, amber pmemd
Parameter: AFE.type sire
Parameter: AFE.separate_vdw_elec false
Parameter: box.type = rectangular
Parameter: box.length = 12.0
Parameter: neutralize = False
Parameter: temperature = 300 K
Parameter: pressure = 1 atm
Parameter: nmoves = 20000
Parameter: ncycles = 100
Parameter: buffered coordinates frequency = 5000
Parameter: save coordinates = True
Parameter: timestep = 2 * femtosecond
Parameter: constraint = hbonds-notperturbed
Parameter: hydrogen mass repartitioning factor = 1.0
Parameter: cutoff type = cutoffperiodic
Parameter: cutoff distance = 10*angstrom
Parameter: barostat = True
Parameter: andersen = True
Parameter: energy frequency = 250
Parameter: precision = mixed
Parameter: minimise = True
Parameter: equilibrate = False
Parameter: equilibration iterations = 5000
Parameter: center solute = True
Parameter: reaction field dielectric = 82.0
Parameter: minimal coordinate saving = True
Parameter: lambda array varying between 9-27 evenly spaced lambda windows
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# Provide free text description of your method below. Use as many lines as you need.
# The level of detail should be about what you would write for a publication.
Method: The ligands were prepared for relative free energy calculations from the output of the docking protocols. Each ligand was parameterized using GAFF inside FESetup. Ligands were then solvated in TIP3P water and minimized and equilibrated for 100 ps. For the relative free energy perturbations, a series of alchemical transformation was proposed and the necessary perturbation protocol was computed with FESetup, resulting in  Sire compatible input for the solvated ligands as well as vacuum ligands. FESetup, calling AMBERtoolswas used to solvated the protein and ligand complexes without adding any counter ions resulting in a charged simulation box. The solvated complexes were minimized and equilibrated for 100 ps with all but solvent restraint. Alchemical morphs are obtained with FESetup with explicit mapping of atoms for different binding poses of the same ligand. Production simulations were carried out using Sire somd-frenrg. Prior to the production run, Sire was also used for a further minimization and 2 ps equilibration to each of the intermediate lambda values using an annealing protocol. All simulations were run with a 2 fs timestep. Reduced gradients and energies were saved every 400 fs with a total simulation time of 4 ns. The simulation temperature of 298.15 K was kept constant using an anderson thermostat and the pressure of 1 atm was maintained using a MC barostat as implemented in OpenMM. The barostat frequency is set to 25 and the collision frequency of the anderson thermostat is 1 /ps. Furthermore, multi state Bennet's acceptance ratio (MBAR) was also as a second free energy analysis method. All submitted results come from the MBAR estimator including the error estimates. 5% of the initial data was discarded to equilibration in the MBAR analysis. The individually estimated free energy differences were then read into a networkx digraph, but removing any edges that involve the not charged nodes FXR_38, FXR_41, FXR_73, FXR_75. Estimates for these compounds were arbitrarily set to 100. Backward and forward simulations were averaged, as well as repeated runs. All possible paths within the graph were estimated and weighted averages of all possible paths between two compounds for the relative free energy computation were computed. Standard errors were computed based on forward and backward averages as well as repeated runs and then propagated along the paths using standard error propagations. Each path was then weighted based on the standard error along the path, the same was done for averaged path errors. Multiple binding modes were averaged over for all compounds other than FXR_76, FXR_89 and FXR_78 with only one binding mode simulated. The poses submitted do not necessarily correspond to the actual binding mode but were selected by hand based on lower computed free energies.
