From 31c0b24317222e6e9fdcf41927ae0ec60eb3ea1a Mon Sep 17 00:00:00 2001
From: wiederm <marcus.wieder@univie.ac.at>
Date: Thu, 29 Oct 2020 13:30:29 +0100
Subject: [PATCH 01/11] updating scripts to run sampling for tautomers given
 two input smiles

---
 data/test_data/exp_results.pickle             | Bin 0 -> 261676 bytes
 neutromeratio/analysis.py                     | 101 +++++++++++++++++-
 neutromeratio/parameter_gradients.py          |   9 +-
 neutromeratio/plotting.py                     |   6 +-
 neutromeratio/tests/test_neutromeratio.py     |  56 +++++++++-
 neutromeratio/utils.py                        |  13 +++
 ...ing.py => Generate_equilibrium_samples.py} |   0
 ...ilibrium_samples_for_new_tautomer_pairs.py |  98 +++++++++++++++++
 .../generate_equilibrium_samples_in_vacuum.sh |  30 ++++++
 ...amples_in_vacuum_for_new_tautomer_pairs.sh |  30 ++++++
 ...gradient-opt.py => parameter_opt-kfold.py} |   0
 11 files changed, 333 insertions(+), 10 deletions(-)
 create mode 100644 data/test_data/exp_results.pickle
 rename scripts/{Generate_equilibrium_sampling.py => Generate_equilibrium_samples.py} (100%)
 create mode 100644 scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
 create mode 100644 scripts/generate_equilibrium_samples_in_vacuum.sh
 create mode 100644 scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
 rename scripts/{parameter_gradient-opt.py => parameter_opt-kfold.py} (100%)

diff --git a/data/test_data/exp_results.pickle b/data/test_data/exp_results.pickle
new file mode 100644
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HcmV?d00001

diff --git a/neutromeratio/analysis.py b/neutromeratio/analysis.py
index a9efc2ab..46b97e73 100644
--- a/neutromeratio/analysis.py
+++ b/neutromeratio/analysis.py
@@ -30,7 +30,7 @@
 )
 from .tautomers import Tautomer
 from .parameter_gradients import FreeEnergyCalculator
-from .utils import generate_tautomer_class_stereobond_aware
+from .utils import generate_tautomer_class_stereobond_aware, generate_new_tautomer_pair
 from .ani import (
     ANI1_force_and_energy,
     AlchemicalANI2x,
@@ -557,6 +557,105 @@ def setup_alchemical_system_and_energy_function(
     return energy_function, tautomer, flipped
 
 
+def setup_new_alchemical_system_and_energy_function(
+    env: str,
+    ANImodel: ANI,
+    checkpoint_file: str = "",
+    base_path: str = None,
+    diameter: int = -1,
+):
+
+    import os
+
+    if not (
+        issubclass(ANImodel, (AlchemicalANI2x, AlchemicalANI1ccx, AlchemicalANI1x))
+    ):
+        raise RuntimeError("Only Alchemical ANI objects allowed! Aborting.")
+
+    #######################
+
+    ####################
+    # Set up the system, set the restraints
+    tautomers = generate_new_tautomer_pair(t1_smiles, t2_smiles)
+    tautomer.perform_tautomer_transformation()
+
+    # if base_path is defined write out the topology
+    if base_path:
+        base_path = os.path.abspath(base_path)
+        logger.debug(base_path)
+        if not os.path.exists(base_path):
+            os.makedirs(base_path)
+
+    if env == "droplet":
+        if diameter == -1:
+            raise RuntimeError("Droplet is not specified. Aborting.")
+        # for droplet topology is written in every case
+        m = tautomer.add_droplet(
+            tautomer.hybrid_topology,
+            tautomer.get_hybrid_coordinates(),
+            diameter=diameter * unit.angstrom,
+            restrain_hydrogen_bonds=True,
+            restrain_hydrogen_angles=False,
+            top_file=f"{base_path}/{name}_in_droplet.pdb",
+        )
+    else:
+        if base_path:
+            # for vacuum only if base_path is defined
+            pdb_filepath = f"{base_path}/{name}.pdb"
+            try:
+                traj = md.load(pdb_filepath)
+            except OSError:
+                coordinates = tautomer.get_hybrid_coordinates()
+                traj = md.Trajectory(
+                    coordinates.value_in_unit(unit.nanometer), tautomer.hybrid_topology
+                )
+                traj.save_pdb(pdb_filepath)
+            tautomer.set_hybrid_coordinates(traj.xyz[0] * unit.nanometer)
+
+    # define the alchemical atoms
+    alchemical_atoms = [
+        tautomer.hybrid_hydrogen_idx_at_lambda_1,
+        tautomer.hybrid_hydrogen_idx_at_lambda_0,
+    ]
+
+    model = ANImodel(alchemical_atoms=alchemical_atoms).to(device)
+    # if specified, load nn parameters
+    if checkpoint_file:
+        logger.debug("Loading nn parameters ...")
+        model.load_nn_parameters(checkpoint_file)
+
+    # setup energy function
+    if env == "vacuum":
+        energy_function = ANI1_force_and_energy(
+            model=model,
+            atoms=tautomer.hybrid_atoms,
+            mol=None,
+        )
+    else:
+        energy_function = ANI1_force_and_energy(
+            model=model,
+            atoms=tautomer.ligand_in_water_atoms,
+            mol=None,
+        )
+
+    # add restraints
+    for r in tautomer.ligand_restraints:
+        energy_function.add_restraint_to_lambda_protocol(r)
+    for r in tautomer.hybrid_ligand_restraints:
+        energy_function.add_restraint_to_lambda_protocol(r)
+
+    if env == "droplet":
+        tautomer.add_COM_for_hybrid_ligand(
+            np.array([diameter / 2, diameter / 2, diameter / 2]) * unit.angstrom
+        )
+        for r in tautomer.solvent_restraints:
+            energy_function.add_restraint_to_lambda_protocol(r)
+        for r in tautomer.com_restraints:
+            energy_function.add_restraint_to_lambda_protocol(r)
+
+    return energy_function, tautomer, flipped
+
+
 def _error(x: np.ndarray, y: np.ndarray):
     """ Simple error """
     return x - y
diff --git a/neutromeratio/parameter_gradients.py b/neutromeratio/parameter_gradients.py
index ad9392af..a74c3fb1 100644
--- a/neutromeratio/parameter_gradients.py
+++ b/neutromeratio/parameter_gradients.py
@@ -37,7 +37,6 @@ def __init__(
         bulk_energy_calculation: bool,
         potential_energy_trajs: list,
         lambdas: list,
-        n_atoms: int,
         max_snapshots_per_window: int = 200,
         pickle_path: str = "",
     ):
@@ -1048,14 +1047,18 @@ def parse_lambda_from_dcd_filename(dcd_filename):
     assert len(lambdas) == len(energies)
     assert len(lambdas) == len(md_trajs)
 
+    if env == "vacuum":
+        pickle_path = f"{data_path}/{name}/{name}_{ANImodel.name}_{max_snapshots_per_window}_{len(tautomer.hybrid_atoms)}_atoms.pickle"
+    else:
+        pickle_path = f"{data_path}/{name}/{name}_{ANImodel.name}_{max_snapshots_per_window}_{diameter}A_{len(tautomer.ligand_in_water_atoms)}_atoms.pickle"
+
     # calculate free energy in kT
     fec = FreeEnergyCalculator(
         ani_model=energy_function,
         md_trajs=md_trajs,
         potential_energy_trajs=energies,
         lambdas=lambdas,
-        pickle_path=f"{data_path}/{name}/{name}_{ANImodel.name}_{max_snapshots_per_window}.pickle",
-        n_atoms=len(tautomer.hybrid_atoms),
+        pickle_path=pickle_path,
         bulk_energy_calculation=bulk_energy_calculation,
         max_snapshots_per_window=max_snapshots_per_window,
     )
diff --git a/neutromeratio/plotting.py b/neutromeratio/plotting.py
index 2b003df4..f10551c8 100644
--- a/neutromeratio/plotting.py
+++ b/neutromeratio/plotting.py
@@ -55,11 +55,13 @@ def plot_correlation_analysis(
                 Y,
                 yerr=error,
                 mfc="blue",
-                ms=3,
+                mec="blue",
+                ms=4,
                 fmt="o",
                 capthick=2,
+                capsize=2,
                 alpha=0.6,
-                ecolor="r",
+                ecolor="red",
             )
 
     else:
diff --git a/neutromeratio/tests/test_neutromeratio.py b/neutromeratio/tests/test_neutromeratio.py
index d2f0e792..e00f75b5 100644
--- a/neutromeratio/tests/test_neutromeratio.py
+++ b/neutromeratio/tests/test_neutromeratio.py
@@ -1273,6 +1273,57 @@ def test_setup_mbar():
     )
     assert np.isclose(-15.506057798507124, fec._end_state_free_energy_difference[0])
 
+    for testdir in [
+        f"data/test_data/droplet/{name}",
+        f"data/test_data/vacuum/{name}",
+    ]:
+        # remove the pickle files
+        for item in os.listdir(testdir):
+            if item.endswith(".pickle"):
+                os.remove(os.path.join(testdir, item))
+
+
+def test_setup_mbar_test_pickle_files():
+    # test the setup mbar function, write out the pickle file and test that everything works
+    from ..parameter_gradients import setup_mbar
+    from ..ani import AlchemicalANI2x, AlchemicalANI1x, AlchemicalANI1ccx
+
+    test = os.listdir("data/test_data/vacuum")
+
+    name = "molDWRow_298"
+    # remove the pickle files
+    for testdir in [
+        f"data/test_data/droplet/{name}",
+        f"data/test_data/vacuum/{name}",
+    ]:
+        # remove the pickle files
+        for item in os.listdir(testdir):
+            if item.endswith(".pickle"):
+                os.remove(os.path.join(testdir, item))
+
+    # vacuum
+    fec = setup_mbar(
+        name,
+        env="vacuum",
+        data_path="data/test_data/vacuum",
+        ANImodel=AlchemicalANI1ccx,
+        bulk_energy_calculation=False,
+        max_snapshots_per_window=20,
+    )
+    assert np.isclose(-3.2194223855155357, fec._end_state_free_energy_difference[0])
+
+    # vacuum
+    fec = setup_mbar(
+        name,
+        env="vacuum",
+        data_path="data/test_data/vacuum",
+        ANImodel=AlchemicalANI1ccx,
+        bulk_energy_calculation=False,
+        max_snapshots_per_window=20,
+    )
+    assert np.isclose(-3.2194223855155357, fec._end_state_free_energy_difference[0])
+
+
 
 def test_change_stereobond():
     from ..utils import change_only_stereobond, get_nr_of_stereobonds
@@ -1578,9 +1629,7 @@ def test_orca_input_generation():
     qm_results = pickle.load(open("data/results/QM/qm_results.pickle", "rb"))
     mol = qm_results[name][t1_smiles]["vac"][0]
 
-    orca_input = qmorca.generate_orca_script_for_solvation_free_energy(
-        mol, 0
-    )
+    orca_input = qmorca.generate_orca_script_for_solvation_free_energy(mol, 0)
     print(orca_input)
 
 
@@ -1785,7 +1834,6 @@ def test_parameter_gradient():
             potential_energy_trajs=potential_energy_trajs,
             lambdas=lambdas,
             bulk_energy_calculation=False,
-            n_atoms=len(tautomer.hybrid_atoms),
             max_snapshots_per_window=10,
         )
 
diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index 389312e7..cd8bab92 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -129,6 +129,19 @@ def get_stereotag_of_stereobonds(smiles: str) -> int:
     return stereo_tag
 
 
+def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str):
+
+    from neutromeratio import Tautomer
+
+    return Tautomer(
+        name=name,
+        initial_state_mol=generate_rdkit_mol(t1_smiles),
+        final_state_mol=generate_rdkit_mol(t2_smiles),
+        nr_of_conformations=1,
+        enforceChirality=True,
+    )
+
+
 def generate_tautomer_class_stereobond_aware(
     name: str,
     t1_smiles: str,
diff --git a/scripts/Generate_equilibrium_sampling.py b/scripts/Generate_equilibrium_samples.py
similarity index 100%
rename from scripts/Generate_equilibrium_sampling.py
rename to scripts/Generate_equilibrium_samples.py
diff --git a/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py b/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
new file mode 100644
index 00000000..19f4e51e
--- /dev/null
+++ b/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
@@ -0,0 +1,98 @@
+from simtk import unit
+import numpy as np
+import mdtraj as md
+import neutromeratio
+from neutromeratio.constants import _get_names, device, platform, kT
+import sys
+import os
+import torch
+from neutromeratio.analysis import setup_alchemical_system_and_energy_function
+from neutromeratio.ani import AlchemicalANI2x
+
+n_steps = int(sys.argv[2])
+base_path = str(sys.argv[3]) # where to write the results
+env = str(sys.argv[4])
+model_name = str(sys.argv[5])
+
+
+if model_name == 'ANI2x':
+    model = neutromeratio.ani.AlchemicalANI2x
+    print(f'Using {model_name}.')
+elif model_name == 'ANI1ccx':
+    model = neutromeratio.ani.AlchemicalANI1ccx
+    print(f'Using {model_name}.')
+elif model_name == 'ANI1x':
+    model = neutromeratio.ani.AlchemicalANI1x
+    print(f'Using {model_name}.')
+else:
+    raise RuntimeError(f'Unknown model name: {model_name}')
+
+
+
+if not (env in ('droplet','vacuum')):
+    raise RuntimeError('Env must be `droplet` or `vacuum`. Aborting.')
+# diameter
+if env == 'droplet':
+    diameter_in_angstrom = int(sys.argv[6])
+    if not diameter_in_angstrom or diameter_in_angstrom < 1:
+        raise RuntimeError('Diameter must be above 1 Angstrom')
+else:
+    diameter_in_angstrom = -1
+
+protocol = []
+names = neutromeratio.parameter_gradients._get_names()
+for name in names:
+    for lamb in np.linspace(0, 1, 11):
+        protocol.append((name, np.round(lamb, 2)))
+
+name, lambda_value = protocol[idx-1]
+print(name)
+print(lambda_value)
+
+base_path = f"{base_path}/{name}"
+os.makedirs(base_path, exist_ok=True)
+
+energy_function, tautomer, flipped = setup_alchemical_system_and_energy_function(
+    name=name,
+    ANImodel=model,
+    env=env,
+    diameter=diameter_in_angstrom,
+    base_path=base_path)
+
+energy_and_force = lambda x: energy_function.calculate_force(x, lambda_value)
+
+if env == 'droplet':
+    x0 = tautomer.get_ligand_in_water_coordinates()
+    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.ligand_in_water_atoms,
+                                        energy_and_force=energy_and_force)
+elif env == 'vacuum':
+    x0 = tautomer.get_hybrid_coordinates()
+    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.hybrid_atoms,
+                                        energy_and_force=energy_and_force)
+else:
+    raise RuntimeError()
+
+torch.set_num_threads(1)
+
+
+x0, e_history = energy_function.minimize(x0, maxiter=5000, lambda_value=lambda_value)
+equilibrium_samples, energies, restraint_contribution = langevin.run_dynamics(x0,
+                                                                    n_steps=n_steps,
+                                                                    stepsize=0.5*unit.femtosecond,
+                                                                    progress_bar=True)
+
+# save equilibrium energy values 
+for global_list, poperty_name in zip([energies, restraint_contribution], ['energy', 'restraint_energy_contribution']):
+    f = open(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_{poperty_name}_in_{env}.csv", 'w+')
+    for e in global_list[::20]:
+        e_unitless = e / kT
+        f.write('{}\n'.format(e_unitless))
+    f.close()   
+
+equilibrium_samples = [x[0].value_in_unit(unit.nanometer) for x in equilibrium_samples]
+if env == 'vacuum':
+    ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.hybrid_topology)
+else:
+    ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.ligand_in_water_topology)
+
+ani_traj.save(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_in_{env}.dcd", force_overwrite=True)
diff --git a/scripts/generate_equilibrium_samples_in_vacuum.sh b/scripts/generate_equilibrium_samples_in_vacuum.sh
new file mode 100644
index 00000000..07cb3ca7
--- /dev/null
+++ b/scripts/generate_equilibrium_samples_in_vacuum.sh
@@ -0,0 +1,30 @@
+########################################
+# idx has to be provided
+idx=${1}
+########################################
+# idx is an integer between 0 and 5000
+# idx is used to index in this code:
+
+#names = neutromeratio.parameter_gradients._get_names()
+#for name in names:
+#    for lamb in np.linspace(0, 1, 11):
+#        protocol.append((name, np.round(lamb, 2)))
+#name, lambda_value = protocol[idx-1]
+########################################
+
+n_steps=400000 # nr of steps 
+env='vacuum'
+potential_name='ANI1ccx' # which potential do you want to use? (ANI1ccx, ANI1x, ANI2x)
+echo 'Using potential ' ${potential_name}
+
+hostname
+echo ${idx}
+echo ${n_steps}
+echo ${env}
+
+#conda activate conda.env # fire up your conda environment
+
+base_path="./" # where do you want to save the ouput files
+mkdir -p ${base_path}
+
+python Generate_equilibrium_sampling.py ${idx} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
diff --git a/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh b/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
new file mode 100644
index 00000000..07cb3ca7
--- /dev/null
+++ b/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
@@ -0,0 +1,30 @@
+########################################
+# idx has to be provided
+idx=${1}
+########################################
+# idx is an integer between 0 and 5000
+# idx is used to index in this code:
+
+#names = neutromeratio.parameter_gradients._get_names()
+#for name in names:
+#    for lamb in np.linspace(0, 1, 11):
+#        protocol.append((name, np.round(lamb, 2)))
+#name, lambda_value = protocol[idx-1]
+########################################
+
+n_steps=400000 # nr of steps 
+env='vacuum'
+potential_name='ANI1ccx' # which potential do you want to use? (ANI1ccx, ANI1x, ANI2x)
+echo 'Using potential ' ${potential_name}
+
+hostname
+echo ${idx}
+echo ${n_steps}
+echo ${env}
+
+#conda activate conda.env # fire up your conda environment
+
+base_path="./" # where do you want to save the ouput files
+mkdir -p ${base_path}
+
+python Generate_equilibrium_sampling.py ${idx} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
diff --git a/scripts/parameter_gradient-opt.py b/scripts/parameter_opt-kfold.py
similarity index 100%
rename from scripts/parameter_gradient-opt.py
rename to scripts/parameter_opt-kfold.py

From 5dc66476fdbf9e0ce34c2b0b15da381985fdf4f1 Mon Sep 17 00:00:00 2001
From: wiederm <marcus.wieder@univie.ac.at>
Date: Thu, 29 Oct 2020 16:46:59 +0100
Subject: [PATCH 02/11] providing scripts to generate equilibrium samples with
 alchemical ANI potential for two tautomer pairs defined by SMILES strings

---
 neutromeratio/analysis.py                     |  59 +++++----
 neutromeratio/utils.py                        |   2 +-
 scripts/Analyse_equilibrium_samples.py        |  69 +++++-----
 scripts/Generate_equilibrium_samples.py       |  83 +++++++-----
 ...ilibrium_samples_for_new_tautomer_pairs.py | 123 ++++++++++--------
 .../generate_equilibrium_samples_in_vacuum.sh |   4 +-
 ...amples_in_vacuum_for_new_tautomer_pairs.sh |  35 ++---
 7 files changed, 205 insertions(+), 170 deletions(-)
 rename scripts/{ => backup}/generate_equilibrium_samples_in_vacuum.sh (82%)

diff --git a/neutromeratio/analysis.py b/neutromeratio/analysis.py
index 46b97e73..b3101e14 100644
--- a/neutromeratio/analysis.py
+++ b/neutromeratio/analysis.py
@@ -1,44 +1,46 @@
 import copy
 import logging
 import pickle
+from glob import glob
 
 import matplotlib.pyplot as plt
+import mdtraj as md
 import networkx as nx
 import numpy as np
 import pandas as pd
+import pkg_resources
+import scipy.stats as scs
 import seaborn as sns
+import torch
+import torchani
 from rdkit import Chem, Geometry
-from rdkit.Chem import AllChem
+from rdkit.Chem import AllChem, rdFMCS
 from scipy.special import logsumexp
 from simtk import unit
-import mdtraj as md
-import torchani
-import torch
-from rdkit.Chem import rdFMCS
-import pkg_resources
-import scipy.stats as scs
 
-from .constants import (
-    num_threads,
-    kT,
+from neutromeratio.ani import (
+    ANI,
+    AlchemicalANI1ccx,
+    AlchemicalANI1x,
+    AlchemicalANI2x,
+    ANI1_force_and_energy,
+)
+from neutromeratio.constants import (
+    device,
+    exclude_set_ANI,
     gas_constant,
-    temperature,
+    kT,
     mols_with_charge,
-    exclude_set_ANI,
     multiple_stereobonds,
-    device,
+    num_threads,
+    temperature,
 )
-from .tautomers import Tautomer
-from .parameter_gradients import FreeEnergyCalculator
-from .utils import generate_tautomer_class_stereobond_aware, generate_new_tautomer_pair
-from .ani import (
-    ANI1_force_and_energy,
-    AlchemicalANI2x,
-    AlchemicalANI1ccx,
-    ANI,
-    AlchemicalANI1x,
+from neutromeratio.parameter_gradients import FreeEnergyCalculator
+from neutromeratio.tautomers import Tautomer
+from neutromeratio.utils import (
+    generate_new_tautomer_pair,
+    generate_tautomer_class_stereobond_aware,
 )
-from glob import glob
 
 logger = logging.getLogger(__name__)
 
@@ -306,7 +308,7 @@ def compare_confomer_generator_and_trajectory_minimum_structures(
     mol.RemoveAllConformers()
 
     # generate energy function, use atom symbols of rdkti mol
-    from .ani import ANI1ccx, ANI1_force_and_energy
+    from .ani import ANI1_force_and_energy, ANI1ccx
 
     model = ANI1ccx()
     energy_function = ANI1_force_and_energy(
@@ -558,11 +560,14 @@ def setup_alchemical_system_and_energy_function(
 
 
 def setup_new_alchemical_system_and_energy_function(
+    name: str,
+    t1_smiles: str,
+    t2_smiles: str,
     env: str,
     ANImodel: ANI,
-    checkpoint_file: str = "",
     base_path: str = None,
     diameter: int = -1,
+    checkpoint_file: str = "",
 ):
 
     import os
@@ -576,7 +581,7 @@ def setup_new_alchemical_system_and_energy_function(
 
     ####################
     # Set up the system, set the restraints
-    tautomers = generate_new_tautomer_pair(t1_smiles, t2_smiles)
+    tautomer = generate_new_tautomer_pair(name, t1_smiles, t2_smiles)
     tautomer.perform_tautomer_transformation()
 
     # if base_path is defined write out the topology
@@ -653,7 +658,7 @@ def setup_new_alchemical_system_and_energy_function(
         for r in tautomer.com_restraints:
             energy_function.add_restraint_to_lambda_protocol(r)
 
-    return energy_function, tautomer, flipped
+    return energy_function, tautomer
 
 
 def _error(x: np.ndarray, y: np.ndarray):
diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index cd8bab92..07f1bf6e 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -9,7 +9,7 @@
 from rdkit.Chem import AllChem
 from simtk import unit
 
-from .constants import kT
+from neutromeratio.constants import kT
 
 logger = logging.getLogger(__name__)
 
diff --git a/scripts/Analyse_equilibrium_samples.py b/scripts/Analyse_equilibrium_samples.py
index 1e907793..ac986df8 100644
--- a/scripts/Analyse_equilibrium_samples.py
+++ b/scripts/Analyse_equilibrium_samples.py
@@ -4,10 +4,17 @@
 import pickle
 import sys
 import torch
-from neutromeratio.parameter_gradients import FreeEnergyCalculator, setup_mbar
-from neutromeratio.constants import kT, device, exclude_set_ANI, mols_with_charge, _get_names
+from neutromeratio.parameter_gradients import setup_mbar
+from neutromeratio.constants import (
+    kT,
+    device,
+    exclude_set_ANI,
+    mols_with_charge,
+    _get_names,
+)
 from glob import glob
 from neutromeratio.ani import AlchemicalANI1ccx, AlchemicalANI2x
+
 #######################
 #######################
 # job idx
@@ -19,52 +26,54 @@
 #######################
 #######################
 # read in exp results, smiles and names
-exp_results = pickle.load(open('../data/exp_results.pickle', 'rb'))
+exp_results = pickle.load(open("../data/exp_results.pickle", "rb"))
 
 # name of the system
 names = _get_names()
 torch.set_num_threads(4)
 
-name = names[idx-1]
-print(name)
-print(base_path)
-print(potential_name)
+name = names[idx - 1]
+print(f"Analysing samples for tautomer pair: {name}")
+print(f"Saving results in: {base_path}")
+print(f"Using potential: {potential_name}")
 
-if potential_name == 'ANI1ccx':
+if potential_name == "ANI1ccx":
     AlchemicalANI = AlchemicalANI1ccx
-elif potential_name == 'ANI2x':
+elif potential_name == "ANI2x":
     AlchemicalANI = AlchemicalANI2x
 else:
-    raise RuntimeError('Potential needs to be either ANI1ccx or ANI2x')
-
+    raise RuntimeError("Potential needs to be either ANI1ccx or ANI2x")
 
 
-if env == 'droplet':
-    print('Env: droplet.')
-    fec = setup_mbar(name=name, 
-                    data_path=base_path,
-                    ANImodel=AlchemicalANI,
-                    bulk_energy_calculation=False,
-                    env='droplet',
-                    max_snapshots_per_window=300,
-                    diameter=18)
-elif env == 'vacuum':
-    print('Env: vacuum.')
-    fec = setup_mbar(name=name, 
-                    data_path=base_path,
-                    ANImodel=AlchemicalANI,
-                    bulk_energy_calculation=True,
-                    env='vacuum',
-                    max_snapshots_per_window=300
+if env == "droplet":
+    print("Simulating in environment: droplet.")
+    fec = setup_mbar(
+        name=name,
+        data_path=base_path,
+        ANImodel=AlchemicalANI,
+        bulk_energy_calculation=False,
+        env="droplet",
+        max_snapshots_per_window=300,
+        diameter=18,
+    )
+elif env == "vacuum":
+    print("Simulating in environment: vacuum.")
+    fec = setup_mbar(
+        name=name,
+        data_path=base_path,
+        ANImodel=AlchemicalANI,
+        bulk_energy_calculation=True,
+        env="vacuum",
+        max_snapshots_per_window=300,
     )
 else:
-    raise RuntimeError('No env specified. Aborting.')
+    raise RuntimeError("No env specified. Aborting.")
 
 DeltaF_ji, dDeltaF_ji = fec._end_state_free_energy_difference
 if fec.flipped:
     DeltaF_ji *= -1
 print(DeltaF_ji)
 
-f = open(f"{base_path}/{potential_name}_{env}_rfe_results_in_kT_300snapshots.csv", 'a+')
+f = open(f"{base_path}/{potential_name}_{env}_rfe_results_in_kT_300snapshots.csv", "a+")
 f.write(f"{name}, {DeltaF_ji}, {dDeltaF_ji}\n")
 f.close()
diff --git a/scripts/Generate_equilibrium_samples.py b/scripts/Generate_equilibrium_samples.py
index cabb6e89..bf9e1f8d 100644
--- a/scripts/Generate_equilibrium_samples.py
+++ b/scripts/Generate_equilibrium_samples.py
@@ -11,32 +11,31 @@
 
 idx = int(sys.argv[1])
 n_steps = int(sys.argv[2])
-base_path = str(sys.argv[3]) # where to write the results
+base_path = str(sys.argv[3])  # where to write the results
 env = str(sys.argv[4])
 model_name = str(sys.argv[5])
 
 
-if model_name == 'ANI2x':
+if model_name == "ANI2x":
     model = neutromeratio.ani.AlchemicalANI2x
-    print(f'Using {model_name}.')
-elif model_name == 'ANI1ccx':
+    print(f"Using {model_name}.")
+elif model_name == "ANI1ccx":
     model = neutromeratio.ani.AlchemicalANI1ccx
-    print(f'Using {model_name}.')
-elif model_name == 'ANI1x':
+    print(f"Using {model_name}.")
+elif model_name == "ANI1x":
     model = neutromeratio.ani.AlchemicalANI1x
-    print(f'Using {model_name}.')
+    print(f"Using {model_name}.")
 else:
-    raise RuntimeError(f'Unknown model name: {model_name}')
+    raise RuntimeError(f"Unknown model name: {model_name}")
 
 
-
-if not (env in ('droplet','vacuum')):
-    raise RuntimeError('Env must be `droplet` or `vacuum`. Aborting.')
+if not (env in ("droplet", "vacuum")):
+    raise RuntimeError("Env must be `droplet` or `vacuum`. Aborting.")
 # diameter
-if env == 'droplet':
+if env == "droplet":
     diameter_in_angstrom = int(sys.argv[6])
     if not diameter_in_angstrom or diameter_in_angstrom < 1:
-        raise RuntimeError('Diameter must be above 1 Angstrom')
+        raise RuntimeError("Diameter must be above 1 Angstrom")
 else:
     diameter_in_angstrom = -1
 
@@ -46,9 +45,10 @@
     for lamb in np.linspace(0, 1, 11):
         protocol.append((name, np.round(lamb, 2)))
 
-name, lambda_value = protocol[idx-1]
-print(name)
-print(lambda_value)
+name, lambda_value = protocol[idx - 1]
+print(f"Generating samples for tautomer pair: {name}")
+print(f"Saving results in: {base_path}")
+print(f"Using potential: {model_name}")
 
 base_path = f"{base_path}/{name}"
 os.makedirs(base_path, exist_ok=True)
@@ -58,18 +58,21 @@
     ANImodel=model,
     env=env,
     diameter=diameter_in_angstrom,
-    base_path=base_path)
+    base_path=base_path,
+)
 
 energy_and_force = lambda x: energy_function.calculate_force(x, lambda_value)
 
-if env == 'droplet':
+if env == "droplet":
     x0 = tautomer.get_ligand_in_water_coordinates()
-    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.ligand_in_water_atoms,
-                                        energy_and_force=energy_and_force)
-elif env == 'vacuum':
+    langevin = neutromeratio.LangevinDynamics(
+        atoms=tautomer.ligand_in_water_atoms, energy_and_force=energy_and_force
+    )
+elif env == "vacuum":
     x0 = tautomer.get_hybrid_coordinates()
-    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.hybrid_atoms,
-                                        energy_and_force=energy_and_force)
+    langevin = neutromeratio.LangevinDynamics(
+        atoms=tautomer.hybrid_atoms, energy_and_force=energy_and_force
+    )
 else:
     raise RuntimeError()
 
@@ -77,23 +80,31 @@
 
 
 x0, e_history = energy_function.minimize(x0, maxiter=5000, lambda_value=lambda_value)
-equilibrium_samples, energies, restraint_contribution = langevin.run_dynamics(x0,
-                                                                    n_steps=n_steps,
-                                                                    stepsize=0.5*unit.femtosecond,
-                                                                    progress_bar=True)
-
-# save equilibrium energy values 
-for global_list, poperty_name in zip([energies, restraint_contribution], ['energy', 'restraint_energy_contribution']):
-    f = open(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_{poperty_name}_in_{env}.csv", 'w+')
+equilibrium_samples, energies, restraint_contribution = langevin.run_dynamics(
+    x0, n_steps=n_steps, stepsize=0.5 * unit.femtosecond, progress_bar=True
+)
+
+# save equilibrium energy values
+for global_list, poperty_name in zip(
+    [energies, restraint_contribution], ["energy", "restraint_energy_contribution"]
+):
+    f = open(
+        f"{base_path}/{name}_lambda_{lambda_value:0.4f}_{poperty_name}_in_{env}.csv",
+        "w+",
+    )
     for e in global_list[::20]:
         e_unitless = e / kT
-        f.write('{}\n'.format(e_unitless))
-    f.close()   
+        f.write("{}\n".format(e_unitless))
+    f.close()
 
 equilibrium_samples = [x[0].value_in_unit(unit.nanometer) for x in equilibrium_samples]
-if env == 'vacuum':
+if env == "vacuum":
     ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.hybrid_topology)
 else:
-    ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.ligand_in_water_topology)
+    ani_traj = md.Trajectory(
+        equilibrium_samples[::20], tautomer.ligand_in_water_topology
+    )
 
-ani_traj.save(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_in_{env}.dcd", force_overwrite=True)
+ani_traj.save(
+    f"{base_path}/{name}_lambda_{lambda_value:0.4f}_in_{env}.dcd", force_overwrite=True
+)
diff --git a/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py b/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
index 19f4e51e..f0b811bd 100644
--- a/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
+++ b/scripts/Generate_equilibrium_samples_for_new_tautomer_pairs.py
@@ -6,93 +6,112 @@
 import sys
 import os
 import torch
-from neutromeratio.analysis import setup_alchemical_system_and_energy_function
+from neutromeratio.analysis import setup_new_alchemical_system_and_energy_function
 from neutromeratio.ani import AlchemicalANI2x
 
-n_steps = int(sys.argv[2])
-base_path = str(sys.argv[3]) # where to write the results
-env = str(sys.argv[4])
-model_name = str(sys.argv[5])
-
-
-if model_name == 'ANI2x':
+smiles1 = str(sys.argv[1])
+smiles2 = str(sys.argv[2])
+name = str(sys.argv[3])
+lambda_value = float(sys.argv[4])
+n_steps = int(sys.argv[5])
+base_path = str(sys.argv[6])  # where to write the results
+env = str(sys.argv[7])
+model_name = str(sys.argv[8])
+
+print(f"Generating samples for tautomer pair: {name}")
+print(f"With SMILES1: {smiles1}")
+print(f"With SMILES2: {smiles2}")
+print(f"Lambda coupling parameter: {lambda_value} (between 0. and 1.)")
+print(f"Saving results in: {base_path}/{name}")
+print(f"Using potential: {model_name}")
+print(f"Simulating in environment: {env}")
+
+if model_name == "ANI2x":
     model = neutromeratio.ani.AlchemicalANI2x
-    print(f'Using {model_name}.')
-elif model_name == 'ANI1ccx':
+    print(f"Using {model_name}.")
+elif model_name == "ANI1ccx":
     model = neutromeratio.ani.AlchemicalANI1ccx
-    print(f'Using {model_name}.')
-elif model_name == 'ANI1x':
+    print(f"Using {model_name}.")
+elif model_name == "ANI1x":
     model = neutromeratio.ani.AlchemicalANI1x
-    print(f'Using {model_name}.')
+    print(f"Using {model_name}.")
 else:
-    raise RuntimeError(f'Unknown model name: {model_name}')
+    raise RuntimeError(f"Unknown model name: {model_name}")
 
 
-
-if not (env in ('droplet','vacuum')):
-    raise RuntimeError('Env must be `droplet` or `vacuum`. Aborting.')
-# diameter
-if env == 'droplet':
+# INPUT CHECKS
+if not (env in ("droplet", "vacuum")):
+    raise RuntimeError("Env must be `droplet` or `vacuum`. Aborting.")
+if env == "droplet":
     diameter_in_angstrom = int(sys.argv[6])
     if not diameter_in_angstrom or diameter_in_angstrom < 1:
-        raise RuntimeError('Diameter must be above 1 Angstrom')
+        raise RuntimeError("Diameter must be above 1 Angstrom")
 else:
     diameter_in_angstrom = -1
 
-protocol = []
-names = neutromeratio.parameter_gradients._get_names()
-for name in names:
-    for lamb in np.linspace(0, 1, 11):
-        protocol.append((name, np.round(lamb, 2)))
-
-name, lambda_value = protocol[idx-1]
-print(name)
-print(lambda_value)
-
+# Generating base path
 base_path = f"{base_path}/{name}"
 os.makedirs(base_path, exist_ok=True)
 
-energy_function, tautomer, flipped = setup_alchemical_system_and_energy_function(
+# setting up energy function
+energy_function, tautomer = setup_new_alchemical_system_and_energy_function(
     name=name,
-    ANImodel=model,
+    t1_smiles=smiles1,
+    t2_smiles=smiles2,
     env=env,
+    ANImodel=model,
     diameter=diameter_in_angstrom,
-    base_path=base_path)
+    base_path=base_path,
+)
 
 energy_and_force = lambda x: energy_function.calculate_force(x, lambda_value)
 
-if env == 'droplet':
+if env == "droplet":
     x0 = tautomer.get_ligand_in_water_coordinates()
-    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.ligand_in_water_atoms,
-                                        energy_and_force=energy_and_force)
-elif env == 'vacuum':
+    langevin = neutromeratio.LangevinDynamics(
+        atoms=tautomer.ligand_in_water_atoms, energy_and_force=energy_and_force
+    )
+elif env == "vacuum":
     x0 = tautomer.get_hybrid_coordinates()
-    langevin = neutromeratio.LangevinDynamics(atoms=tautomer.hybrid_atoms,
-                                        energy_and_force=energy_and_force)
+    langevin = neutromeratio.LangevinDynamics(
+        atoms=tautomer.hybrid_atoms, energy_and_force=energy_and_force
+    )
 else:
     raise RuntimeError()
 
+# pytorch number of threads to be used
 torch.set_num_threads(1)
 
-
+# start minimization
 x0, e_history = energy_function.minimize(x0, maxiter=5000, lambda_value=lambda_value)
-equilibrium_samples, energies, restraint_contribution = langevin.run_dynamics(x0,
-                                                                    n_steps=n_steps,
-                                                                    stepsize=0.5*unit.femtosecond,
-                                                                    progress_bar=True)
 
-# save equilibrium energy values 
-for global_list, poperty_name in zip([energies, restraint_contribution], ['energy', 'restraint_energy_contribution']):
-    f = open(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_{poperty_name}_in_{env}.csv", 'w+')
+# start sampling
+equilibrium_samples, energies, restraint_contribution = langevin.run_dynamics(
+    x0, n_steps=n_steps, stepsize=0.5 * unit.femtosecond, progress_bar=True
+)
+
+# save equilibrium energy values
+for global_list, poperty_name in zip(
+    [energies, restraint_contribution], ["energy", "restraint_energy_contribution"]
+):
+    f = open(
+        f"{base_path}/{name}_lambda_{lambda_value:0.4f}_{poperty_name}_in_{env}.csv",
+        "w+",
+    )
     for e in global_list[::20]:
         e_unitless = e / kT
-        f.write('{}\n'.format(e_unitless))
-    f.close()   
+        f.write("{}\n".format(e_unitless))
+    f.close()
 
+# save trajectory
 equilibrium_samples = [x[0].value_in_unit(unit.nanometer) for x in equilibrium_samples]
-if env == 'vacuum':
+if env == "vacuum":
     ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.hybrid_topology)
 else:
-    ani_traj = md.Trajectory(equilibrium_samples[::20], tautomer.ligand_in_water_topology)
+    ani_traj = md.Trajectory(
+        equilibrium_samples[::20], tautomer.ligand_in_water_topology
+    )
 
-ani_traj.save(f"{base_path}/{name}_lambda_{lambda_value:0.4f}_in_{env}.dcd", force_overwrite=True)
+ani_traj.save(
+    f"{base_path}/{name}_lambda_{lambda_value:0.4f}_in_{env}.dcd", force_overwrite=True
+)
diff --git a/scripts/generate_equilibrium_samples_in_vacuum.sh b/scripts/backup/generate_equilibrium_samples_in_vacuum.sh
similarity index 82%
rename from scripts/generate_equilibrium_samples_in_vacuum.sh
rename to scripts/backup/generate_equilibrium_samples_in_vacuum.sh
index 07cb3ca7..3ba3a6e1 100644
--- a/scripts/generate_equilibrium_samples_in_vacuum.sh
+++ b/scripts/backup/generate_equilibrium_samples_in_vacuum.sh
@@ -22,9 +22,7 @@ echo ${idx}
 echo ${n_steps}
 echo ${env}
 
-#conda activate conda.env # fire up your conda environment
-
 base_path="./" # where do you want to save the ouput files
 mkdir -p ${base_path}
 
-python Generate_equilibrium_sampling.py ${idx} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
+python Generate_equilibrium_samples.py ${idx} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
diff --git a/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh b/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
index 07cb3ca7..96d09aa3 100644
--- a/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
+++ b/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
@@ -1,30 +1,23 @@
-########################################
-# idx has to be provided
-idx=${1}
-########################################
-# idx is an integer between 0 and 5000
-# idx is used to index in this code:
 
-#names = neutromeratio.parameter_gradients._get_names()
-#for name in names:
-#    for lamb in np.linspace(0, 1, 11):
-#        protocol.append((name, np.round(lamb, 2)))
-#name, lambda_value = protocol[idx-1]
+# User specified runtime variables
 ########################################
-
-n_steps=400000 # nr of steps 
-env='vacuum'
+SMILES1='OC1=CC=C2C=CC=CC2=N1'
+SMILES2='O=C1NC2=C(C=CC=C2)C=C1'
+lambda_value=0.0   # alchemical coupling parameter (between 0. and 1.) --> we recommend using 11 lambda_values
+name='SAMP2Lmol4'
 potential_name='ANI1ccx' # which potential do you want to use? (ANI1ccx, ANI1x, ANI2x)
-echo 'Using potential ' ${potential_name}
+n_steps=200 # nr of steps (dt = 0.5fs)
+########################################
+########################################
 
-hostname
-echo ${idx}
-echo ${n_steps}
-echo ${env}
 
-#conda activate conda.env # fire up your conda environment
+env='vacuum' 
+echo 'Using potential ' ${potential_name}
+echo 'Nr of steps : ' ${n_steps}
+echo 'Simulating in : ' ${env}
+echo 'Lambda value: ' ${lambda_value}
 
 base_path="./" # where do you want to save the ouput files
 mkdir -p ${base_path}
 
-python Generate_equilibrium_sampling.py ${idx} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
+python Generate_equilibrium_samples_for_new_tautomer_pairs.py ${SMILES1} ${SMILES2} ${name} ${lambda_value} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file

From 26d706ce52b30103117f7bfe1235a0c8dbf62ead Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:07:32 -0400
Subject: [PATCH 03/11] parameters --> parameter_path in logger

---
 neutromeratio/ani.py | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/neutromeratio/ani.py b/neutromeratio/ani.py
index 8c1f36fe..d6850f30 100644
--- a/neutromeratio/ani.py
+++ b/neutromeratio/ani.py
@@ -87,7 +87,7 @@ def load_nn_parameters(
             else:
                 self.tweaked_neural_network.load_state_dict(parameters)
         else:
-            logger.info(f"Parameter file {parameters} does not exist.")
+            logger.info(f"Parameter file {parameter_path} does not exist.")
 
     def _from_neurochem_resources(self, info_file_path, periodic_table_index):
         (

From 688c6aa81a4541cceb16c62ff1974670e054b516 Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:10:05 -0400
Subject: [PATCH 04/11] float32 --> float64 in ani.py

TODO: set default precision throughout?
---
 neutromeratio/ani.py | 4 ++--
 1 file changed, 2 insertions(+), 2 deletions(-)

diff --git a/neutromeratio/ani.py b/neutromeratio/ani.py
index d6850f30..f45cb0a4 100644
--- a/neutromeratio/ani.py
+++ b/neutromeratio/ani.py
@@ -775,7 +775,7 @@ def calculate_force(
             raise RuntimeError(f"Shape of coordinates: {x.shape} is wrong. Aborting.")
 
         coordinates = torch.tensor(
-            x, requires_grad=True, device=self.device, dtype=torch.float32
+            x, requires_grad=True, device=self.device, dtype=torch.float64
         )
 
         energy_in_kT, restraint_energy_contribution = self._calculate_energy(
@@ -921,7 +921,7 @@ def calculate_energy(
             coordinate_list.value_in_unit(unit.nanometer),
             requires_grad=requires_grad_wrt_coordinates,
             device=self.device,
-            dtype=torch.float32,
+            dtype=torch.float64,
         )
 
         logger.debug(f"coordinates tensor: {coordinates.size()}")

From 2d06a7570f0783c80ee1e9ee3e17eae23edaa234 Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:14:38 -0400
Subject: [PATCH 05/11] add docstring to generate_new_tautomer_pair

---
 neutromeratio/utils.py | 8 ++++----
 1 file changed, 4 insertions(+), 4 deletions(-)

diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index 07f1bf6e..0e340df1 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -8,6 +8,7 @@
 from rdkit import Chem
 from rdkit.Chem import AllChem
 from simtk import unit
+from neutromeratio import Tautomer
 
 from neutromeratio.constants import kT
 
@@ -129,10 +130,9 @@ def get_stereotag_of_stereobonds(smiles: str) -> int:
     return stereo_tag
 
 
-def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str):
-
-    from neutromeratio import Tautomer
-
+def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str) -> Tautomer:
+    """Constructs and returns a Tautomer pair object, generating RDKit mols
+    from t1 and t2 smiles"""
     return Tautomer(
         name=name,
         initial_state_mol=generate_rdkit_mol(t1_smiles),

From 3735132e77b8befbb2c5f9abcda607f1a8555fea Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:21:41 -0400
Subject: [PATCH 06/11] update documentation and type hints in
 geenerate_tautomer_class_stereobond_aware

---
 neutromeratio/utils.py | 9 +++++++--
 1 file changed, 7 insertions(+), 2 deletions(-)

diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index 0e340df1..fb6b84a7 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -1,6 +1,7 @@
 import logging
 import os
 import random
+from typing import List, Tuple, Any, Union
 
 import mdtraj as md
 import nglview
@@ -10,6 +11,8 @@
 from simtk import unit
 from neutromeratio import Tautomer
 
+StereoBondType = Union[Any, str]
+
 from neutromeratio.constants import kT
 
 logger = logging.getLogger(__name__)
@@ -133,6 +136,7 @@ def get_stereotag_of_stereobonds(smiles: str) -> int:
 def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str) -> Tautomer:
     """Constructs and returns a Tautomer pair object, generating RDKit mols
     from t1 and t2 smiles"""
+    # TOOD: should this also accept nr_of_conformations, enforceChirality?
     return Tautomer(
         name=name,
         initial_state_mol=generate_rdkit_mol(t1_smiles),
@@ -148,7 +152,7 @@ def generate_tautomer_class_stereobond_aware(
     t2_smiles: str,
     nr_of_conformations: int = 1,
     enforceChirality=True,
-) -> list:
+) -> Tuple[StereoBondType, List[Tautomer], bool]:
     """
     If a stereobond is present in the tautomer pair we need to transform from the molecule
     with the stereobond (e.g. enol) to the tautomer without the stereobond (e.g. keto). This
@@ -167,6 +171,8 @@ def generate_tautomer_class_stereobond_aware(
         nr of conformations
     Returns
     ----------
+    stereobond_type: StereoBondType
+        one of [None, "generic", "Imine"]
     tautomers: list
         a list of tautomer(s)
     flipped: bool
@@ -174,7 +180,6 @@ def generate_tautomer_class_stereobond_aware(
     """
 
     tautomers = []
-    from neutromeratio import Tautomer
 
     flipped = False
     stereobond_type = None

From feaf360dbf9bb33b1e77f0102ad3fb3e9cd98109 Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:42:00 -0400
Subject: [PATCH 07/11] start to refactor sterebond handler

---
 neutromeratio/utils.py | 77 ++++++++++++++++++++++++++++++------------
 1 file changed, 55 insertions(+), 22 deletions(-)

diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index fb6b84a7..5f0b6ffe 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -159,6 +159,7 @@ def generate_tautomer_class_stereobond_aware(
     function makes sure that this happens and returns a list of tautomers.
     If there is no stereobond present in either tautomer, the list contains only one tautomer.
     If there is a stereobond present the list contains two tautomers (one with cis, one with trans configuration).
+
     Parameters
     ----------
     name: str
@@ -169,6 +170,7 @@ def generate_tautomer_class_stereobond_aware(
         a SMILES string
     nr_of_conformations: int
         nr of conformations
+
     Returns
     ----------
     stereobond_type: StereoBondType
@@ -195,11 +197,18 @@ def generate_tautomer_class_stereobond_aware(
             t1_kappa_1 = change_stereobond_in_imine_to_trans(
                 generate_rdkit_mol(t1_smiles)
             )
+
+            t1_a = t1_kappa_0
+            t2_a = generate_rdkit_mol(t2_smiles)
+
+            t1_b = t1_kappa_1
+            t2_b = generate_rdkit_mol(t2_smiles)
+
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=t1_kappa_0,
-                    final_state_mol=generate_rdkit_mol(t2_smiles),
+                    initial_state_mol=t1_a,
+                    final_state_mol= t2_a,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -207,8 +216,8 @@ def generate_tautomer_class_stereobond_aware(
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=t1_kappa_1,
-                    final_state_mol=generate_rdkit_mol(t2_smiles),
+                    initial_state_mol=t1_b,
+                    final_state_mol=t2_b,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -222,11 +231,18 @@ def generate_tautomer_class_stereobond_aware(
             t2_kappa_1 = change_stereobond_in_imine_to_trans(
                 generate_rdkit_mol(t2_smiles)
             )
+
+            t1_a = t2_kappa_0
+            t2_a = generate_rdkit_mol(t1_smiles)
+
+            t1_b = t2_kappa_1
+            t2_b = generate_rdkit_mol(t1_smiles)
+
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=t2_kappa_0,
-                    final_state_mol=generate_rdkit_mol(t1_smiles),
+                    initial_state_mol=t1_a,
+                    final_state_mol=t2_a,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -234,8 +250,8 @@ def generate_tautomer_class_stereobond_aware(
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=t2_kappa_1,
-                    final_state_mol=generate_rdkit_mol(t1_smiles),
+                    initial_state_mol=t1_b,
+                    final_state_mol=t2_b,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -245,6 +261,9 @@ def generate_tautomer_class_stereobond_aware(
             raise RuntimeError("Stereobonds present in both tautomers ... aborting!")
 
     elif get_nr_of_stereobonds(t1_smiles) == get_nr_of_stereobonds(t2_smiles):
+        t1 = generate_rdkit_mol(t1_smiles)
+        t2 = generate_rdkit_mol(t2_smiles)
+
         if (
             get_nr_of_stereobonds(t1_smiles) == 0
             and get_nr_of_stereobonds(t2_smiles) == 0
@@ -255,8 +274,8 @@ def generate_tautomer_class_stereobond_aware(
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=generate_rdkit_mol(t1_smiles),
-                    final_state_mol=generate_rdkit_mol(t2_smiles),
+                    initial_state_mol=t1,
+                    final_state_mol=t2,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -266,8 +285,8 @@ def generate_tautomer_class_stereobond_aware(
             tautomers.append(
                 Tautomer(
                     name=name,
-                    initial_state_mol=generate_rdkit_mol(t1_smiles),
-                    final_state_mol=generate_rdkit_mol(t2_smiles),
+                    initial_state_mol=t1,
+                    final_state_mol=t2,
                     nr_of_conformations=nr_of_conformations,
                     enforceChirality=enforceChirality,
                 )
@@ -275,18 +294,24 @@ def generate_tautomer_class_stereobond_aware(
 
         else:
             # stereobonds on both endstates
-            # we need to add a torsion bias to make sure that the lambda protocol stopp at the correct torsion
-
+            # we need to add a torsion bias to make sure that the lambda protocol stops at the correct torsion
             raise RuntimeError("Two stereobonds ... aborting")
     elif get_nr_of_stereobonds(t1_smiles) > get_nr_of_stereobonds(t2_smiles):
         stereobond_type = "generic"
         t1_smiles_kappa_0 = t1_smiles
         t1_smiles_kappa_1 = change_only_stereobond(t1_smiles)
+
+        t1_a = generate_rdkit_mol(t1_smiles_kappa_0)
+        t2_a = generate_rdkit_mol(t2_smiles)
+
+        t1_b = generate_rdkit_mol(t1_smiles_kappa_1)
+        t2_b = generate_rdkit_mol(t2_smiles)
+
         tautomers.append(
             Tautomer(
                 name=name,
-                initial_state_mol=generate_rdkit_mol(t1_smiles_kappa_0),
-                final_state_mol=generate_rdkit_mol(t2_smiles),
+                initial_state_mol=t1_a,
+                final_state_mol=t2_a,
                 nr_of_conformations=nr_of_conformations,
                 enforceChirality=enforceChirality,
             )
@@ -294,8 +319,8 @@ def generate_tautomer_class_stereobond_aware(
         tautomers.append(
             Tautomer(
                 name=name,
-                initial_state_mol=generate_rdkit_mol(t1_smiles_kappa_1),
-                final_state_mol=generate_rdkit_mol(t2_smiles),
+                initial_state_mol=t1_b,
+                final_state_mol=t2_b,
                 nr_of_conformations=nr_of_conformations,
                 enforceChirality=enforceChirality,
             )
@@ -307,20 +332,28 @@ def generate_tautomer_class_stereobond_aware(
         t1_smiles_kappa_0 = t2_smiles
         t1_smiles_kappa_1 = change_only_stereobond(t2_smiles)
         t2_smiles = t1_smiles
+
+        t1_a = generate_rdkit_mol(t1_smiles_kappa_0)
+        t2_a = generate_rdkit_mol(t2_smiles)
+
+        t1_b = generate_rdkit_mol(t1_smiles_kappa_1)
+        t2_b = generate_rdkit_mol(t2_smiles)
+
         tautomers.append(
             Tautomer(
                 name=name,
-                initial_state_mol=generate_rdkit_mol(t1_smiles_kappa_0),
-                final_state_mol=generate_rdkit_mol(t2_smiles),
+                initial_state_mol=t1_a,
+                final_state_mol=t2_a,
                 nr_of_conformations=nr_of_conformations,
                 enforceChirality=enforceChirality,
             )
         )
+
         tautomers.append(
             Tautomer(
                 name=name,
-                initial_state_mol=generate_rdkit_mol(t1_smiles_kappa_1),
-                final_state_mol=generate_rdkit_mol(t2_smiles),
+                initial_state_mol=t1_b,
+                final_state_mol=t2_b,
                 nr_of_conformations=nr_of_conformations,
                 enforceChirality=enforceChirality,
             )

From b1a970b2f5c7122cf5dbae6f7aec58d6ce8f86fc Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 14:49:02 -0400
Subject: [PATCH 08/11] reduce code duplication in stereobond handler

---
 neutromeratio/utils.py | 112 ++++++++---------------------------------
 1 file changed, 21 insertions(+), 91 deletions(-)

diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index 5f0b6ffe..49ad5233 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -179,6 +179,11 @@ def generate_tautomer_class_stereobond_aware(
         a list of tautomer(s)
     flipped: bool
         to indicate that t1/t2 SMILES have been exchanged
+
+
+    Notes
+    -----
+    one branch (for detecting stereobonds in heterocycles) depends on name
     """
 
     tautomers = []
@@ -186,6 +191,10 @@ def generate_tautomer_class_stereobond_aware(
     flipped = False
     stereobond_type = None
 
+    def _tautomer(t1, t2):
+        """Tautomer constructor, with name, nr_of_conformations, enforceChirality applied"""
+        return Tautomer(name, t1, t2, nr_of_conformations, enforceChirality)
+
     if flag_unspec_stereo(t1_smiles) or flag_unspec_stereo(t2_smiles):
         logger.debug("Imines present ... switching to imine generation.")
         stereobond_type = "Imine"
@@ -204,24 +213,9 @@ def generate_tautomer_class_stereobond_aware(
             t1_b = t1_kappa_1
             t2_b = generate_rdkit_mol(t2_smiles)
 
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1_a,
-                    final_state_mol= t2_a,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1_b,
-                    final_state_mol=t2_b,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
+            tautomers.append(_tautomer(t1_a, t2_a))
+            tautomers.append(_tautomer(t1_b, t2_b))
+
 
         elif flag_unspec_stereo(t2_smiles):
             flipped = True
@@ -238,24 +232,8 @@ def generate_tautomer_class_stereobond_aware(
             t1_b = t2_kappa_1
             t2_b = generate_rdkit_mol(t1_smiles)
 
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1_a,
-                    final_state_mol=t2_a,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1_b,
-                    final_state_mol=t2_b,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
+            tautomers.append(_tautomer(t1_a, t2_a))
+            tautomers.append(_tautomer(t1_b, t2_b))
 
         else:
             raise RuntimeError("Stereobonds present in both tautomers ... aborting!")
@@ -271,26 +249,11 @@ def generate_tautomer_class_stereobond_aware(
             # no stereobond -- normal protocol
             # generate both rdkit mol
             logger.debug("No stereobonds ...")
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1,
-                    final_state_mol=t2,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
+            tautomers.append(_tautomer(t1, t2))
+
         elif name == "molDWRow_1636":
             logger.debug("molDWRow_1636 -- stereobonds in hetereocycle ...")
-            tautomers.append(
-                Tautomer(
-                    name=name,
-                    initial_state_mol=t1,
-                    final_state_mol=t2,
-                    nr_of_conformations=nr_of_conformations,
-                    enforceChirality=enforceChirality,
-                )
-            )
+            tautomers.append(_tautomer(t1, t2))
 
         else:
             # stereobonds on both endstates
@@ -307,24 +270,8 @@ def generate_tautomer_class_stereobond_aware(
         t1_b = generate_rdkit_mol(t1_smiles_kappa_1)
         t2_b = generate_rdkit_mol(t2_smiles)
 
-        tautomers.append(
-            Tautomer(
-                name=name,
-                initial_state_mol=t1_a,
-                final_state_mol=t2_a,
-                nr_of_conformations=nr_of_conformations,
-                enforceChirality=enforceChirality,
-            )
-        )
-        tautomers.append(
-            Tautomer(
-                name=name,
-                initial_state_mol=t1_b,
-                final_state_mol=t2_b,
-                nr_of_conformations=nr_of_conformations,
-                enforceChirality=enforceChirality,
-            )
-        )
+        tautomers.append(_tautomer(t1_a, t2_a))
+        tautomers.append(_tautomer(t1_b, t2_b))
 
     elif get_nr_of_stereobonds(t1_smiles) < get_nr_of_stereobonds(t2_smiles):
         stereobond_type = "generic"
@@ -339,25 +286,8 @@ def generate_tautomer_class_stereobond_aware(
         t1_b = generate_rdkit_mol(t1_smiles_kappa_1)
         t2_b = generate_rdkit_mol(t2_smiles)
 
-        tautomers.append(
-            Tautomer(
-                name=name,
-                initial_state_mol=t1_a,
-                final_state_mol=t2_a,
-                nr_of_conformations=nr_of_conformations,
-                enforceChirality=enforceChirality,
-            )
-        )
-
-        tautomers.append(
-            Tautomer(
-                name=name,
-                initial_state_mol=t1_b,
-                final_state_mol=t2_b,
-                nr_of_conformations=nr_of_conformations,
-                enforceChirality=enforceChirality,
-            )
-        )
+        tautomers.append(_tautomer(t1_a, t2_a))
+        tautomers.append(_tautomer(t1_b, t2_b))
 
     else:
         raise RuntimeError("Stereobonds present in both tautomers ... aborting!")

From 1168b955009487093ba4b00ecf1d070c662c483e Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 16:03:55 -0400
Subject: [PATCH 09/11] revert Tautomer cyclic-import

---
 neutromeratio/utils.py | 7 ++++---
 1 file changed, 4 insertions(+), 3 deletions(-)

diff --git a/neutromeratio/utils.py b/neutromeratio/utils.py
index 49ad5233..26b31fbc 100644
--- a/neutromeratio/utils.py
+++ b/neutromeratio/utils.py
@@ -9,7 +9,6 @@
 from rdkit import Chem
 from rdkit.Chem import AllChem
 from simtk import unit
-from neutromeratio import Tautomer
 
 StereoBondType = Union[Any, str]
 
@@ -133,10 +132,11 @@ def get_stereotag_of_stereobonds(smiles: str) -> int:
     return stereo_tag
 
 
-def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str) -> Tautomer:
+def generate_new_tautomer_pair(name: str, t1_smiles: str, t2_smiles: str):
     """Constructs and returns a Tautomer pair object, generating RDKit mols
     from t1 and t2 smiles"""
     # TOOD: should this also accept nr_of_conformations, enforceChirality?
+    from neutromeratio.tautomers import Tautomer
     return Tautomer(
         name=name,
         initial_state_mol=generate_rdkit_mol(t1_smiles),
@@ -152,7 +152,7 @@ def generate_tautomer_class_stereobond_aware(
     t2_smiles: str,
     nr_of_conformations: int = 1,
     enforceChirality=True,
-) -> Tuple[StereoBondType, List[Tautomer], bool]:
+) -> Tuple[StereoBondType, List, bool]:
     """
     If a stereobond is present in the tautomer pair we need to transform from the molecule
     with the stereobond (e.g. enol) to the tautomer without the stereobond (e.g. keto). This
@@ -185,6 +185,7 @@ def generate_tautomer_class_stereobond_aware(
     -----
     one branch (for detecting stereobonds in heterocycles) depends on name
     """
+    from neutromeratio.tautomers import Tautomer
 
     tautomers = []
 

From b2692190681288746cdfbc04f8af8649ef29c5d8 Mon Sep 17 00:00:00 2001
From: Josh Fass <josh.fass@choderalab.org>
Date: Thu, 29 Oct 2020 16:11:38 -0400
Subject: [PATCH 10/11] revert float32 --> float64

---
 neutromeratio/ani.py | 4 ++--
 1 file changed, 2 insertions(+), 2 deletions(-)

diff --git a/neutromeratio/ani.py b/neutromeratio/ani.py
index f45cb0a4..d6850f30 100644
--- a/neutromeratio/ani.py
+++ b/neutromeratio/ani.py
@@ -775,7 +775,7 @@ def calculate_force(
             raise RuntimeError(f"Shape of coordinates: {x.shape} is wrong. Aborting.")
 
         coordinates = torch.tensor(
-            x, requires_grad=True, device=self.device, dtype=torch.float64
+            x, requires_grad=True, device=self.device, dtype=torch.float32
         )
 
         energy_in_kT, restraint_energy_contribution = self._calculate_energy(
@@ -921,7 +921,7 @@ def calculate_energy(
             coordinate_list.value_in_unit(unit.nanometer),
             requires_grad=requires_grad_wrt_coordinates,
             device=self.device,
-            dtype=torch.float64,
+            dtype=torch.float32,
         )
 
         logger.debug(f"coordinates tensor: {coordinates.size()}")

From d8bc9d70e49da2df2df024343d1ae904dc0b26d4 Mon Sep 17 00:00:00 2001
From: wiederm <marcus.wieder@univie.ac.at>
Date: Fri, 30 Oct 2020 19:09:02 +0100
Subject: [PATCH 11/11] adding script to generate equilibrium samples, analyse
 dG and reweight based on optimized parameters

---
 neutromeratio/parameter_gradients.py          | 105 +++++++++++++++++-
 neutromeratio/tests/test_neutromeratio.py     |   5 +-
 .../Calculate_dG_for_new_tautomer_pairs.py    |  82 ++++++++++++++
 ...amples_in_vacuum_for_new_tautomer_pairs.sh |  23 ----
 .../generate_samples_and_analyse_results.sh   |  22 ++++
 5 files changed, 209 insertions(+), 28 deletions(-)
 create mode 100644 scripts/Calculate_dG_for_new_tautomer_pairs.py
 delete mode 100644 scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
 create mode 100644 scripts/generate_samples_and_analyse_results.sh

diff --git a/neutromeratio/parameter_gradients.py b/neutromeratio/parameter_gradients.py
index a74c3fb1..9bcfdc95 100644
--- a/neutromeratio/parameter_gradients.py
+++ b/neutromeratio/parameter_gradients.py
@@ -104,9 +104,9 @@ def get_mix(lambda0, lambda1, lam=0.0):
                         f"There are {len(snapshots)} snapshots per lambda state (max: {max_snapshots_per_window}). Aborting."
                     )
 
-                # test that we have not less than 80% of max_snapshots_per_window
+                # test that we have not less than 60% of max_snapshots_per_window
                 if max_snapshots_per_window != -1 and len(snapshots) < (
-                    int(max_snapshots_per_window * 0.8)
+                    int(max_snapshots_per_window * 0.6)
                 ):
                     raise RuntimeError(
                         f"There are only {len(snapshots)} snapshots per lambda state. Aborting."
@@ -125,7 +125,7 @@ def get_mix(lambda0, lambda1, lam=0.0):
                 snapshots.extend(ani_trajs[lam])
                 logger.debug(f"Snapshots per lambda {lam}: {len(ani_trajs[lam])}")
 
-            if len(snapshots) < 100:
+            if len(snapshots) < 300:
                 logger.critical(
                     f"Total number of snapshots is {len(snapshots)} -- is this enough?"
                 )
@@ -1065,3 +1065,102 @@ def parse_lambda_from_dcd_filename(dcd_filename):
 
     fec.flipped = flipped
     return fec
+
+
+def setup_mbar_for_new_tautomer_pairs(
+    name: str,
+    t1_smiles: str,
+    t2_smiles: str,
+    max_snapshots_per_window: int,
+    ANImodel: ANI,
+    bulk_energy_calculation: bool,
+    env: str = "vacuum",
+    checkpoint_file: str = "",
+    data_path: str = "../data/",
+    diameter: int = -1,
+):
+
+    from neutromeratio.analysis import setup_new_alchemical_system_and_energy_function
+    import os
+
+    if not (env == "vacuum" or env == "droplet"):
+        raise RuntimeError("Only keyword vacuum or droplet are allowed as environment.")
+    if env == "droplet" and diameter == -1:
+        raise RuntimeError("Something went wrong.")
+
+    def parse_lambda_from_dcd_filename(dcd_filename):
+        """parsed the dcd filename
+
+        Arguments:
+            dcd_filename {str} -- how is the dcd file called?
+
+        Returns:
+            [float] -- lambda value
+        """
+        l = dcd_filename[: dcd_filename.find(f"_energy_in_{env}")].split("_")
+        lam = l[-3]
+        return float(lam)
+
+    data_path = os.path.abspath(data_path)
+    if not os.path.exists(data_path):
+        raise RuntimeError(f"{data_path} does not exist!")
+
+    #######################
+    (energy_function, tautomer,) = setup_new_alchemical_system_and_energy_function(
+        name=name,
+        t1_smiles=t1_smiles,
+        t2_smiles=t2_smiles,
+        ANImodel=ANImodel,
+        checkpoint_file=checkpoint_file,
+        env=env,
+        diameter=diameter,
+        base_path=f"{data_path}/{name}/",
+    )
+    # and lambda values in list
+    dcds = glob(f"{data_path}/{name}/*.dcd")
+
+    lambdas = []
+    md_trajs = []
+    energies = []
+
+    # read in all the frames from the trajectories
+    if env == "vacuum":
+        top = tautomer.hybrid_topology
+    else:
+        top = f"{data_path}/{name}/{name}_in_droplet.pdb"
+
+    for dcd_filename in dcds:
+        lam = parse_lambda_from_dcd_filename(dcd_filename)
+        lambdas.append(lam)
+        traj = md.load_dcd(dcd_filename, top=top)
+        logger.debug(f"Nr of frames in trajectory: {len(traj)}")
+        md_trajs.append(traj)
+        f = open(
+            f"{data_path}/{name}/{name}_lambda_{lam:0.4f}_energy_in_{env}.csv", "r"
+        )
+        energies.append(np.array([float(e) * kT for e in f]))
+        f.close()
+
+    if len(lambdas) < 5:
+        raise RuntimeError(f"Below 5 lambda states for {name}")
+
+    assert len(lambdas) == len(energies)
+    assert len(lambdas) == len(md_trajs)
+
+    if env == "vacuum":
+        pickle_path = f"{data_path}/{name}/{name}_{ANImodel.name}_{max_snapshots_per_window}_{len(tautomer.hybrid_atoms)}_atoms.pickle"
+    else:
+        pickle_path = f"{data_path}/{name}/{name}_{ANImodel.name}_{max_snapshots_per_window}_{diameter}A_{len(tautomer.ligand_in_water_atoms)}_atoms.pickle"
+
+    # calculate free energy in kT
+    fec = FreeEnergyCalculator(
+        ani_model=energy_function,
+        md_trajs=md_trajs,
+        potential_energy_trajs=energies,
+        lambdas=lambdas,
+        pickle_path=pickle_path,
+        bulk_energy_calculation=bulk_energy_calculation,
+        max_snapshots_per_window=max_snapshots_per_window,
+    )
+
+    return fec
diff --git a/neutromeratio/tests/test_neutromeratio.py b/neutromeratio/tests/test_neutromeratio.py
index e00f75b5..2e9c405c 100644
--- a/neutromeratio/tests/test_neutromeratio.py
+++ b/neutromeratio/tests/test_neutromeratio.py
@@ -1324,7 +1324,6 @@ def test_setup_mbar_test_pickle_files():
     assert np.isclose(-3.2194223855155357, fec._end_state_free_energy_difference[0])
 
 
-
 def test_change_stereobond():
     from ..utils import change_only_stereobond, get_nr_of_stereobonds
 
@@ -1748,8 +1747,10 @@ def test_load_parameters():
         )
     ):
         # set tweaked parameters
-        print(model_name)
+        model._reset_parameters()
+
         model_instance = model([0, 0])
+        model_instance._reset_parameters()
         # initial parameters
         params1 = list(model_instance.original_neural_network.parameters())[6][
             0
diff --git a/scripts/Calculate_dG_for_new_tautomer_pairs.py b/scripts/Calculate_dG_for_new_tautomer_pairs.py
new file mode 100644
index 00000000..8d4ba258
--- /dev/null
+++ b/scripts/Calculate_dG_for_new_tautomer_pairs.py
@@ -0,0 +1,82 @@
+import neutromeratio
+from simtk import unit
+import numpy as np
+import pickle
+import sys
+import torch
+from neutromeratio.parameter_gradients import setup_mbar_for_new_tautomer_pairs
+from neutromeratio.constants import (
+    kT,
+    device,
+    exclude_set_ANI,
+    mols_with_charge,
+    _get_names,
+)
+from glob import glob
+from neutromeratio.ani import AlchemicalANI1ccx, AlchemicalANI2x
+
+#######################
+#######################
+smiles1 = str(sys.argv[1])
+smiles2 = str(sys.argv[2])
+name = str(sys.argv[3])
+base_path = str(sys.argv[4])
+env = str(sys.argv[5])
+potential_name = str(sys.argv[6])
+#######################
+#######################
+torch.set_num_threads(4)
+
+print(f"Analysing samples for tautomer pair: {name}")
+print(f"With SMILES1: {smiles1}")
+print(f"With SMILES2: {smiles2}")
+print(f"Saving results in: {base_path}")
+print(f"Using potential: {potential_name}")
+
+if potential_name == "ANI1ccx":
+    AlchemicalANI = AlchemicalANI1ccx
+elif potential_name == "ANI2x":
+    AlchemicalANI = AlchemicalANI2x
+else:
+    raise RuntimeError("Potential needs to be either ANI1ccx or ANI2x")
+
+if env == "droplet":
+    print("Simulating in environment: droplet.")
+    fec = setup_mbar_for_new_tautomer_pairs(
+        name=name,
+        t1_smiles=smiles1,
+        t2_smiles=smiles2,
+        data_path=base_path,
+        ANImodel=AlchemicalANI,
+        bulk_energy_calculation=False,
+        env="droplet",
+        max_snapshots_per_window=300,
+        diameter=-1,
+    )
+elif env == "vacuum":
+    print("Simulating in environment: vacuum.")
+    fec = setup_mbar_for_new_tautomer_pairs(
+        name=name,
+        t1_smiles=smiles1,
+        t2_smiles=smiles2,
+        data_path=base_path,
+        ANImodel=AlchemicalANI,
+        bulk_energy_calculation=True,
+        env="vacuum",
+        max_snapshots_per_window=300,
+        checkpoint_file="../data/retraining/parameters_ANI1ccx_vacuum_best.pt",
+    )
+else:
+    raise RuntimeError("No env specified. Aborting.")
+
+DeltaF_ji, dDeltaF_ji = fec._end_state_free_energy_difference
+print(f"dG obtained from vacuum simulations: {DeltaF_ji} with {dDeltaF_ji} [kT]")
+
+if potential_name == "ANI1ccx":
+    DeltaF_ji = fec._compute_free_energy_difference()
+    print(
+        f"dG obtained with optimized parameters from vacuum simulations: {DeltaF_ji} [kT]"
+    )
+
+else:
+    print("We only provide optimized parameters for ANI1ccx.")
diff --git a/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh b/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
deleted file mode 100644
index 96d09aa3..00000000
--- a/scripts/generate_equilibrium_samples_in_vacuum_for_new_tautomer_pairs.sh
+++ /dev/null
@@ -1,23 +0,0 @@
-
-# User specified runtime variables
-########################################
-SMILES1='OC1=CC=C2C=CC=CC2=N1'
-SMILES2='O=C1NC2=C(C=CC=C2)C=C1'
-lambda_value=0.0   # alchemical coupling parameter (between 0. and 1.) --> we recommend using 11 lambda_values
-name='SAMP2Lmol4'
-potential_name='ANI1ccx' # which potential do you want to use? (ANI1ccx, ANI1x, ANI2x)
-n_steps=200 # nr of steps (dt = 0.5fs)
-########################################
-########################################
-
-
-env='vacuum' 
-echo 'Using potential ' ${potential_name}
-echo 'Nr of steps : ' ${n_steps}
-echo 'Simulating in : ' ${env}
-echo 'Lambda value: ' ${lambda_value}
-
-base_path="./" # where do you want to save the ouput files
-mkdir -p ${base_path}
-
-python Generate_equilibrium_samples_for_new_tautomer_pairs.py ${SMILES1} ${SMILES2} ${name} ${lambda_value} ${n_steps} ${base_path} ${env} ${potential_name}
\ No newline at end of file
diff --git a/scripts/generate_samples_and_analyse_results.sh b/scripts/generate_samples_and_analyse_results.sh
new file mode 100644
index 00000000..0137559a
--- /dev/null
+++ b/scripts/generate_samples_and_analyse_results.sh
@@ -0,0 +1,22 @@
+# This is a simple wrapper script that generates equilibrium samples for a given tautomer set 
+# and estimates the free energy difference with ANI1cccx and the tautomer optimized parameter set
+################################################
+SMILES1='OC1=CC=C2C=CC=CC2=N1'
+SMILES2='O=C1NC2=C(C=CC=C2)C=C1'
+name='test_mol' # defines where the output directory name
+base_path="./" # where do you want to save the ouput files -> the ouput will have the form ${base_path}/${name}
+potential_name='ANI1ccx' # which potential do you want to use? (ANI1ccx, ANI1x, ANI2x)
+n_steps=10000 # nr of steps (dt = 0.5fs)
+env='vacuum' 
+################################################
+echo 'Using potential ' ${potential_name}
+echo 'Nr of steps : ' ${n_steps}
+echo 'Simulating in : ' ${env}
+echo 'Lambda value: ' ${lambda_value}
+mkdir -p ${base_path}
+
+for lambda_value in $(seq 0 0.2 1); # using 6 lambda states
+do python Generate_equilibrium_samples_for_new_tautomer_pairs.py ${SMILES1} ${SMILES2} ${name} ${lambda_value} ${n_steps} ${base_path} ${env} ${potential_name};
+done
+
+python Calculate_dG_for_new_tautomer_pairs.py ${SMILES1} ${SMILES2} ${name} ${base_path} ${env} ${potential_name};