Difference between revisions of "2023 Denovo tutorial 1 with PDBID 4S0V"

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=Introduction=
 
=Introduction=
−
This next section will walk you through the three options for DeNovo Design:
+
DeNovo Design is where we are attempting to design a new ligand, usually with the hope of it binding more tightly to the protein, from scratch. There are three different ways of doing this:
 
#Generic DeNovo Design
 
#Generic DeNovo Design
 
#Focused Fragment Design
 
#Focused Fragment Design
 
#DeNovo Refinement
 
#DeNovo Refinement
  
−
and will be a continuation of the Virtual Screening tutorial.  We will continue this work with #4s0v from the Protein Data Base.
+
This next tutorial will walk you through the details of each and is a continuation of the Virtual Screening tutorial.  We will continue this work with #4s0v from the Protein Data Base.
 +
 
  
 
==Setting Up Your Environment==
 
==Setting Up Your Environment==
 
For this section we will need to create some more directories following this structure:
 
For this section we will need to create some more directories following this structure:
  
−
 
+
[[File: updatedStructure.png|center]]
  
 
=DeNovo Refinement=
 
=DeNovo Refinement=
−
The DeNovo Refinement calculation/algorithm within DOCK is an interesting way to determine the effects on a ligand/protein interaction by changing part of the small molecule. For this tutorial we are going to change just a terminal ring on our ligand and see what different residues DOCK suggests we look into further.
+
The DeNovo Refinement algorithm in Dock6.10 is an interesting way to determine the effects on a ligand/protein interaction by changing only part of the small molecule. The part of the ligand we want to experiment with is deleted from the structure, replaced with a dummy atom, and then run through DOCK. The program will try to find which residue can be placed in this now open position that will bind tightly to the protein. 
  
−
The overall idea behind this refinement is to delete a portion of your ligand, replace the "stub" atom (or "anchor" atom) which is now not bonded to anything with a dummy atom, and ask DOCK to find an alternative. 
+
==Setting up the dummy atom==
  
−
*Step 1: Open the ligand minimized mol2 file we generated in the previous tutorial into Chimera.
+
For the ligand from #4s0v, we will be removing a terminal ring and looking at what DOCK suggests to replace it with. The steps to do this are: 
−
*Step 2: Open the protein into the same session
+
*Open the ligand minimized mol2 file we generated in the previous tutorial into Chimera.
−
*Step 3: Examine the binding site and choose a residue on the ligand that's pointing towards the inside of the binding site.  For our protein this detailed section looks like:
+
*Open the protein into the same session
 +
*Examine the binding site and choose a residue on the ligand that's pointing towards the inside of the binding site.  For our protein this detailed section looks like:
  
 
[[File: ligandInSite.png|thumb|center]]
 
[[File: ligandInSite.png|thumb|center]]
  
−
We see an imidazole ring pointing towards the binding site so will choose to work with that.  Select the protein and hide it from view.  
+
We see an imidazole ring pointing towards the binding site so will choose to work with that.  Select the protein and hide it from view:  
  
−
*Step 4: Place your mouse over the atom connecting the ring to the rest of the ligand and note the atom and number.  In this case it's N4.
+
*Place your mouse over the atom connecting the ring to the rest of the ligand and note the atom and number.  In this case it's N4.
 
[[File: atomnumber.png|center]]
 
[[File: atomnumber.png|center]]
 +
 +
*Delete all the atoms from N4 to the end.  Your ligand should now look something like:
 +
[[File: DeletedRing.png|center]]
 +
 +
*Save a .mol2 file of your ligand in this configuration.  Make sure to give it a new filename such as 4s0v_denovoRefinement.mol2
 +
*Open the .mol2 file.  If you're on a UNIX system, you can use vi; if you're on a PC, you can use textedit.  Locate the atom that will now be changed to a dummy atom:
 +
[[File: Originalmol2.png|center]]
 +
 +
*Change the atom type to 'Du1' and the bond type to 'Du':
 +
[[File: Modifiedmol2.png|center]]
 +
and save the file.
 +
 +
*Open a new session in Chimera and load the modified mol2 file.  The "dummy" atom should now be purple:
 +
[[File: dummyatom.png|thumb|center]]
 +
 +
*scp 4s0v_denovoRefinement.mol2 over to Seawulf and into the 012.denovoRefinement directory.  From this point on we will be working on the command line.
 +
 +
==Running DeNovo Refinement==
 +
Now that we have our .mol2 file on Seawulf we can run the DeNovo Refinement tool in DOCK6.10.  We need to create an input file:
 +
  vi denovoRefinement.in
 +
 +
and type the following commands into it:
 +
  conformer_search_type                                        denovo
 +
  dn_fraglib_scaffold_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_scaffold.mol2
 +
  dn_fraglib_linker_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_linker.mol2
 +
  dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_sidechain.mol2
 +
  dn_user_specified_anchor                                    yes
 +
  dn_fraglib_anchor_file                                      4s0v_ligand_denovo.mol2
 +
  dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_torenv.dat
 +
  dn_name_identifier                                          4s0v_denovo
 +
  dn_sampling_method                                          graph
 +
  dn_graph_max_picks                                          30
 +
  dn_graph_breadth                                            3
 +
  dn_graph_depth                                              2
 +
  dn_graph_temperature                                        100.0
 +
  dn_pruning_conformer_score_cutoff                            100.0
 +
  dn_pruning_conformer_score_scaling_factor                    2.0
 +
  dn_pruning_clustering_cutoff                                100.0
 +
  dn_mol_wt_cutoff_type                                        soft
 +
  dn_upper_constraint_mol_wt                                  1000.0
 +
  dn_lower_constraint_mol_wt                                  0.0
 +
  dn_mol_wt_std_dev                                            35.0
 +
  dn_constraint_rot_bon                                        15
 +
  dn_constraint_formal_charge                                  5
 +
  dn_heur_unmatched_num                                        1
 +
  dn_heur_matched_rmsd                                        2.0
 +
  dn_unique_anchors                                            1
 +
  dn_max_grow_layers                                          1
 +
  dn_max_root_size                                            25
 +
  dn_max_layer_size                                            25
 +
  dn_max_current_aps                                          5
 +
  dn_max_scaffolds_per_layer                                  1
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          no
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        no
 +
  dn_output_prefix                                            4s0v_denovo_output
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  use_database_filter                                          no
 +
  orient_ligand                                                no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  grid_score_primary                                          yes
 +
  grid_score_rep_rad_scale                                    1
 +
  grid_score_vdw_scale                                        1
 +
  grid_score_es_scale                                          1
 +
  grid_score_grid_prefix                                      ../003.gridbox/grid
 +
  minimize_ligand                                              yes
 +
  minimize_anchor                                              no
 +
  minimize_flexible_growth                                    yes
 +
  use_advanced_simplex_parameters                              no
 +
  simplex_max_cycles                                          1
 +
  simplex_score_converge                                      0.1
 +
  simplex_cycle_converge                                      1
 +
  simplex_trans_step                                          1
 +
  simplex_rot_step                                            0.1
 +
  simplex_tors_step                                            10
 +
  simplex_grow_max_iterations                                  250
 +
  simplex_grow_tors_premin_iterations                          0
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        yes
 +
  simplex_coefficient_restraint                                10
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
 +
To run your file simply type:
 +
  dock6 -i denovoRefinement.in -o denovoRefinement.out
 +
 +
Once the job has completed you will see the following new files in your directory:
 +
 +
*4s0v_denovo_output.anchor_1.root_layer_1.mol2
 +
*4s0v_denovo_output.denovo_build.mol2
 +
*denovoRefinement.out
 +
 +
==Viewing New Molecules==
 +
scp the two .mol2 files over to your local computer.  Open a new session in Chimera.  To view the new residues that DOCK added to the dummy node go to Tools → Surface/Binding Analysis → ViewDock and open 4s0v_denovo_output.denovo_build.mol2.  As before, a dialogue box will open and you can click through the different model options. 
 +
 +
[[File: denovooptions.png|center|600px]]
 +
 +
 +
Changing the input values in the input file can be powerful and gives us flexibility in how to "grow" our new ligand.  These will be looked at more closely in the next two sections.
 +
 +
=Focused DeNovo Design=
 +
The next two types of denovo design are similar to each other.  We choose a structure, like an imidazole ring to "anchor" our ligand to the protein.  DOCK will sample this structure all over the protein to find the best binding location.  From that point a new ligand is "grown" out from this anchor point.  The difference between focused and generic denovo design comes down to what residues DOCK uses to grow this new ligand.
 +
 +
==Fragment Library Generation==
 +
In focused denovo design, a library of fragments which DOCK can use to grow the new ligand must be supplied to the program.  We are "focusing" DOCK to work with specific fragments when building the new ligand.  Very often when focused denovo design is used the fragment library is created using only the residues in the original ligand.  This will be the approach we use for the following tutorial.
 +
 +
cd into your 013a.fragLib directory and create the input file:
 +
  vi fragLib.in
 +
 +
Type the following commands into the input file:
 +
  conformer_search_type                                        flex
 +
  write_fragment_libraries                                    yes
 +
  fragment_library_prefix                                      4s0v_fragLib
 +
  fragment_library_freq_cutoff                                1
 +
  fragment_library_sort_method                                freq
 +
  fragment_library_trans_origin                                no
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  ligand_atom_file                                            ../001.structure/4s0v_ligand_hydrogens.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              no
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  bump_filter                                                  no
 +
  score_molecules                                              no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
  ligand_outfile_prefix                                        4s0v_focused
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  write_conformations                                          no
 +
  cluster_conformations                                        yes
 +
  cluster_rmsd_threshold                                      2.0
 +
  rank_ligands                                                no
 +
 +
and run the program with:
 +
  dock6 -i fragLib.in -o fragLib.out
 +
 +
Once the program has successfully run you'll see the following new files in your directory:
 +
*4s0v_focused_scored.mol2
 +
*4s0v_fragLib_linker.mol2
 +
*4s0v_fragLib_rigid.mol2
 +
*4s0v_fragLib_scaffold.mol2
 +
*4s0v_fragLib_sidechain.mol2
 +
*4s0v_fragLib_torenv.dat
 +
 +
==DeNovo Design==
 +
For this next section, cd into your 013b.focusGrowth directory.
 +
 +
There are three .mol2 files generated in the previous step that we need to give DOCK to perform the focused denovo growth.  For this next step we again need an input file:
 +
  vi deNovoFocus.in
 +
 +
and type the following commands into the input file:
 +
  conformer_search_type                                        denovo
 +
  dn_fraglib_scaffold_file                                    ../013a.fragLib/4s0v_fragLib_scaffold.mol2
 +
  dn_fraglib_linker_file                                      ../013a.fragLib/4s0v_fragLib_linker.mol2
 +
  dn_fraglib_sidechain_file                                    ../013a.fragLib/4s0v_fragLib_sidechain.mol2
 +
  dn_user_specified_anchor                                    no
 +
  dn_use_torenv_table                                          yes
 +
  dn_torenv_table                                              ../013a.fragLib/4s0v_fragLib_torenv.dat
 +
  dn_sampling_method                                          graph
 +
  dn_graph_max_picks                                          30
 +
  dn_graph_breadth                                            3
 +
  dn_graph_depth                                              2
 +
  dn_graph_temperature                                        100.0
 +
  dn_pruning_conformer_score_cutoff                            100.0
 +
  dn_pruning_conformer_score_scaling_factor                    1.0
 +
  dn_pruning_clustering_cutoff                                100.0
 +
  dn_constraint_mol_wt                                        550.0
 +
  dn_constraint_rot_bon                                        15
 +
  dn_constraint_formal_charge                                  2.0
 +
  dn_heur_unmatched_num                                        1
 +
  dn_heur_matched_rmsd                                        2.0
 +
  dn_unique_anchors                                            2
 +
  dn_max_grow_layers                                          9
 +
  dn_max_root_size                                            25
 +
  dn_max_layer_size                                            25
 +
  dn_max_current_aps                                          5
 +
  dn_max_scaffolds_per_layer                                  1
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          no
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        yes
 +
  dn_output_prefix                                            4s0v_focused
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          yes
 +
  grid_score_secondary                                        no
 +
  grid_score_rep_rad_scale                                    1
 +
  grid_score_vdw_scale                                        1
 +
  grid_score_es_scale                                          1
 +
  grid_score_grid_prefix                                      ../003.gridbox/grid
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_secondary                        no
 +
  pharmacophore_score_secondary                                no
 +
  descriptor_score_secondary                                  no
 +
  gbsa_zou_score_secondary                                    no
 +
  gbsa_hawkins_score_secondary                                no
 +
  SASA_score_secondary                                        no
 +
  amber_score_secondary                                        no
 +
  minimize_ligand                                              yes
 +
  minimize_anchor                                              yes
 +
  minimize_flexible_growth                                    yes
 +
  use_advanced_simplex_parameters                              no
 +
  simplex_max_cycles                                          1
 +
  simplex_score_converge                                      0.1
 +
  simplex_cycle_converge                                      1.0
 +
  simplex_trans_step                                          1.0
 +
  simplex_rot_step                                            0.1
 +
  simplex_tors_step                                            10.0
 +
  simplex_anchor_max_iterations                                500
 +
  simplex_grow_max_iterations                                  500
 +
  simplex_grow_tors_premin_iterations                          0
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
 +
Finally, to run this command type:
 +
  dock6 -i deNovoFocus.in -o deNovoFocus.out
 +
 +
Once this has successfully run you will see many new files in your directory.  These all contain possible new ligands which can bind to your protein.
 +
 +
==ReScoring Designed Molecules==
 +
The numerous molecules that were grown in the previous step need to be narrowed down to possible hits which can be investigated further.  One way to do this is to reScore each ligand to find those that are similar to the original ligand in terms of interactions with the protein and overall composition.  For these next steps, cd into your 013c.focusReScore directory and create your input file:
 +
  vi focusReScore.in
 +
 +
type the following commands into your input file:
 +
  conformer_search_type                                        rigid
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  ligand_atom_file                                            ../013b.focusGrowth/4s0v_focused.denovo_build.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              no
 +
  use_database_filter                                          no
 +
  orient_ligand                                                no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          no
 +
  grid_score_secondary                                        no
 +
  multigrid_score_primary                                      no
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_primary                                        no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_primary                                    no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_primary                          no
 +
  footprint_similarity_score_secondary                        no
 +
  pharmacophore_score_primary                                  no
 +
  pharmacophore_score_secondary                                no
 +
  descriptor_score_primary                                    yes
 +
  descriptor_score_secondary                                  no
 +
  descriptor_use_grid_score                                    no
 +
  descriptor_use_multigrid_score                              no
 +
  descriptor_use_continuous_score                              no
 +
  descriptor_use_footprint_similarity                          yes
 +
  descriptor_use_pharmacophore_score                          yes
 +
  descriptor_use_tanimoto                                      yes
 +
  descriptor_use_hungarian                                    yes
 +
  descriptor_use_volume_overlap                                yes
 +
  descriptor_fps_score_use_footprint_reference_mol2            yes
 +
  descriptor_fps_score_footprint_reference_mol2_filename      ../004.energy_min/4s0v.lig.min_scored.mol2
 +
  descriptor_fps_score_foot_compare_type                      Euclidean
 +
  descriptor_fps_score_normalize_foot                          no
 +
  descriptor_fps_score_foot_comp_all_residue                  yes
 +
  descriptor_fps_score_receptor_filename                      ../001.structure/4s0v_protein_hydrogens_charges.mol2
 +
  descriptor_fps_score_vdw_att_exp                            6
 +
  descriptor_fps_score_vdw_rep_exp                            12
 +
  descriptor_fps_score_vdw_rep_rad_scale                      1
 +
  descriptor_fps_score_use_distance_dependent_dielectric      yes
 +
  descriptor_fps_score_dielectric                              4.0
 +
  descriptor_fps_score_vdw_fp_scale                            1
 +
  descriptor_fps_score_es_fp_scale                            1
 +
  descriptor_fps_score_hb_fp_scale                            0
 +
  descriptor_fms_score_use_ref_mol2                            yes
 +
  descriptor_fms_score_ref_mol2_filename                      ../004.energy_min/4s0v.lig.min_scored.mol2
 +
  descriptor_fms_score_write_reference_pharmacophore_mol2      no
 +
  descriptor_fms_score_write_reference_pharmacophore_txt      no
 +
  descriptor_fms_score_write_candidate_pharmacophore          no
 +
  descriptor_fms_score_write_matched_pharmacophore            no
 +
  descriptor_fms_score_compare_type                            overlap
 +
  descriptor_fms_score_full_match                              yes
 +
  descriptor_fms_score_match_rate_weight                      5.0
 +
  descriptor_fms_score_match_dist_cutoff                      1.0
 +
  descriptor_fms_score_match_proj_cutoff                      0.7071
 +
  descriptor_fms_score_max_score                              20
 +
  descriptor_fingerprint_ref_filename                          ../004.energy_min/4s0v.lig.min_scored.mol2
 +
  descriptor_hms_score_ref_filename                            ../004.energy_min/4s0v.lig.min_scored.mol2
 +
  descriptor_hms_score_matching_coeff                          -5
 +
  descriptor_hms_score_rmsd_coeff                              1
 +
  descriptor_volume_score_reference_mol2_filename              ../004.energy_min/4s0v.lig.min_scored.mol2
 +
  descriptor_volume_score_overlap_compute_method              analytical
 +
  descriptor_weight_fps_score                                  1
 +
  descriptor_weight_pharmacophore_score                        1
 +
  descriptor_weight_fingerprint_tanimoto                      -1
 +
  descriptor_weight_hms_score                                  1
 +
  descriptor_weight_volume_overlap_score                      -1
 +
  gbsa_zou_score_secondary                                    no
 +
  gbsa_hawkins_score_secondary                                no
 +
  SASA_score_secondary                                        no
 +
  amber_score_secondary                                        no
 +
  minimize_ligand                                              no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
  chem_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
 +
  pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
 +
  ligand_outfile_prefix                                        4s0v_focus_rescore
 +
  write_footprints                                            yes
 +
  write_hbonds                                                yes
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  rank_ligands                                                no
 +
 +
Finally to run this command type:
 +
  dock6 -i focusReScore.in -o focusReScore.out
 +
 +
When the program has run successfully there will be three new files in your directory:
 +
*4s0v_focus_rescore_footprint_scored.txt
 +
*4s0v_focus_rescore_hbond_scored.txt
 +
*4s0v_focus_rescore_scored.mol2
 +
 +
To view these new ligands, scp the .mol2 file to your local computer and use ViewDock. 
 +
 +
For this ligand we get 42 viable results.  Looking at the new molecule with the lowest footprint score:
 +
 +
[[File: lowestFootprint.png|center|600px]]
 +
 +
and the highest footprint score:
 +
 +
[[File: Highest.png|center|600px]]
 +
 +
 +
If we open the newly designed molecule with the lowest descriptor score and the energized minimized ligand from the previous tutorial, we see:
 +
[[File: ligandcomparisons.png|thumb|center]]
 +
 +
and we can see the two ligands are similar to each other although not exactly the same.  This isn't too surprising since the library of fragments that DOCK had to work with originated from the residues in the original ligand.  In the next section we give DOCK more freedom is choosing which residues to use in the denovo design.
  
 
=Generic DeNovo Design=
 
=Generic DeNovo Design=
 +
The final type of DeNovo design that we'll be looking at is Generic DeNovo Design.  This is similar to the focused design in the previous section except we're not limiting DOCK to building the new molecule using only fragments present in the original ligand.  This time we will be supplying DOCK with a library of many residues for it to use in generating the new molecule.  This section will be done on the command line, so please cd to your 014.genericDenovo directory.
 +
 +
Create the input file:
 +
  vi genericLib.in
 +
 +
Type the following commands into the input file:
 +
  conformer_search_type                                        denovo
 +
  dn_fraglib_scaffold_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_scaffold.mol2
 +
  dn_fraglib_linker_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_linker.mol2
 +
  dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_sidechain.mol2
 +
  dn_user_specified_anchor                                    no
 +
  dn_use_torenv_table                                          yes
 +
  dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_torenv.dat
 +
  dn_sampling_method                                          graph
 +
  dn_graph_max_picks                                          30
 +
  dn_graph_breadth                                            3
 +
  dn_graph_depth                                              2
 +
  dn_graph_temperature                                        100
 +
  dn_pruning_conformer_score_cutoff                            100
 +
  dn_pruning_conformer_score_scaling_factor                    1.0
 +
  dn_pruning_clustering_cutoff                                100.0
 +
  dn_constraint_mol_wt                                        550.0
 +
  dn_constraint_rot_bon                                        15
 +
  dn_constraint_formal_charge                                  2.0
 +
  dn_heur_unmatched_num                                        1
 +
  dn_heur_matched_rmsd                                        2.0
 +
  dn_unique_anchors                                            1
 +
  dn_max_grow_layers                                          9
 +
  dn_max_root_size                                            25
 +
  dn_max_layer_size                                            25
 +
  dn_max_current_aps                                          5
 +
  dn_max_scaffolds_per_layer                                  1
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          no
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        no
 +
  dn_output_prefix                                            4s0v_genericDenovo_output
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          no
 +
  grid_score_secondary                                        no
 +
  multigrid_score_primary                                      no
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_primary                                        no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_primary                                    no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_primary                          no
 +
  footprint_similarity_score_secondary                        no
 +
  pharmacophore_score_primary                                  no
 +
  pharmacophore_score_secondary                                no
 +
  descriptor_score_primary                                    yes
 +
  descriptor_score_secondary                                  no
 +
  descriptor_use_grid_score                                    yes
 +
  descriptor_use_pharmacophore_score                          no
 +
  descriptor_use_tanimoto                                      no
 +
  descriptor_use_hungarian                                    no
 +
  descriptor_use_volume_overlap                                no
 +
  descriptor_grid_score_rep_rad_scale                          1
 +
  descriptor_grid_score_vdw_scale                              1
 +
  descriptor_grid_score_es_scale                              1
 +
  descriptor_grid_score_grid_prefix                            ../003.gridbox/grid
 +
  descriptor_weight_grid_score                                1
 +
  gbsa_zou_score_secondary                                    no
 +
  gbsa_hawkins_score_secondary                                no
 +
  SASA_score_secondary                                        no
 +
  amber_score_secondary                                        no
 +
  minimize_ligand                                              yes
 +
  minimize_anchor                                              yes
 +
  minimize_flexible_growth                                    yes
 +
  use_advanced_simplex_parameters                              no
 +
  simplex_max_cycles                                          1
 +
  simplex_score_converge                                      0.1
 +
  simplex_cycle_converge                                      1.0
 +
  simplex_trans_step                                          1.0
 +
  simplex_rot_step                                            0.1
 +
  simplex_tors_step                                            10.0
 +
  simplex_anchor_max_iterations                                500
 +
  simplex_grow_max_iterations                                  500
 +
  simplex_grow_tors_premin_iterations                          0
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
 +
This calculation can be computationally expensive so it's best to run the input file with a slurm script:
 +
  #!/bin/bash
 +
  #
 +
  #SBATCH --job-name=4s0v_genericLib
 +
  #SBATCH --output=genericLib_output.txt
 +
  #SBATCH --ntasks-per-node=24
 +
  #SBATCH --nodes=1
 +
  #SBATCH --time=48:00:00
 +
  #SBATCH -p long-24core
 +
 
 +
  dock6 -i genericLib.in -o genericLib.out
 +
 +
Once the program is finished running you will see multiple new files in your directory including 4s0v_genericDenovo_output.denovo_build.mol2.  scp this file over to your local computer.  Start a new session in Chimera and open this file with ViewDock.
 +
 +
Using the command:
 +
  grep MOLECULE 4s0v_genericDenovo_output.denovo_build.mol2 | wc -l
 +
 +
you can determine how many new ligands DOCK "grew".  In this case it's 537.
 +
 +
Looking at the molecule with the lowest grid score:
 +
 +
[[File: lowestGrids.png|center|600px]]
 +
 +
and if we overlay this molecule to the original minimized ligand:
 +
 +
[[File: DenovoComparisons.png|center|600px]]
 +
 +
we see that the ligands aren't the same at all.  The original minimized ligand has a GridScore of -75.2 and this newly designed ligand has a GridScore of -75.8.
 +
 +
And finally we can look at both of these ligands interacting with the protein:
  
−
=Focused Fragment Design=
+
[[File: FinalLigand.png|center|700px]]

Latest revision as of 09:11, 6 May 2024

Introduction

DeNovo Design is where we are attempting to design a new ligand, usually with the hope of it binding more tightly to the protein, from scratch. There are three different ways of doing this:

  1. Generic DeNovo Design
  2. Focused Fragment Design
  3. DeNovo Refinement

This next tutorial will walk you through the details of each and is a continuation of the Virtual Screening tutorial. We will continue this work with #4s0v from the Protein Data Base.


Setting Up Your Environment

For this section we will need to create some more directories following this structure:

UpdatedStructure.png

DeNovo Refinement

The DeNovo Refinement algorithm in Dock6.10 is an interesting way to determine the effects on a ligand/protein interaction by changing only part of the small molecule. The part of the ligand we want to experiment with is deleted from the structure, replaced with a dummy atom, and then run through DOCK. The program will try to find which residue can be placed in this now open position that will bind tightly to the protein.

Setting up the dummy atom

For the ligand from #4s0v, we will be removing a terminal ring and looking at what DOCK suggests to replace it with. The steps to do this are:

  • Open the ligand minimized mol2 file we generated in the previous tutorial into Chimera.
  • Open the protein into the same session
  • Examine the binding site and choose a residue on the ligand that's pointing towards the inside of the binding site. For our protein this detailed section looks like:
LigandInSite.png

We see an imidazole ring pointing towards the binding site so will choose to work with that. Select the protein and hide it from view:

  • Place your mouse over the atom connecting the ring to the rest of the ligand and note the atom and number. In this case it's N4.
Atomnumber.png
  • Delete all the atoms from N4 to the end. Your ligand should now look something like:
DeletedRing.png
  • Save a .mol2 file of your ligand in this configuration. Make sure to give it a new filename such as 4s0v_denovoRefinement.mol2
  • Open the .mol2 file. If you're on a UNIX system, you can use vi; if you're on a PC, you can use textedit. Locate the atom that will now be changed to a dummy atom:
Originalmol2.png
  • Change the atom type to 'Du1' and the bond type to 'Du':
Modifiedmol2.png

and save the file.

  • Open a new session in Chimera and load the modified mol2 file. The "dummy" atom should now be purple:
Dummyatom.png
  • scp 4s0v_denovoRefinement.mol2 over to Seawulf and into the 012.denovoRefinement directory. From this point on we will be working on the command line.

Running DeNovo Refinement

Now that we have our .mol2 file on Seawulf we can run the DeNovo Refinement tool in DOCK6.10. We need to create an input file: 

 vi denovoRefinement.in

and type the following commands into it: 

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_sidechain.mol2
 dn_user_specified_anchor                                     yes
 dn_fraglib_anchor_file                                       4s0v_ligand_denovo.mol2
 dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_torenv.dat
 dn_name_identifier                                           4s0v_denovo
 dn_sampling_method                                           graph
 dn_graph_max_picks                                           30
 dn_graph_breadth                                             3
 dn_graph_depth                                               2
 dn_graph_temperature                                         100.0
 dn_pruning_conformer_score_cutoff                            100.0
 dn_pruning_conformer_score_scaling_factor                    2.0
 dn_pruning_clustering_cutoff                                 100.0
 dn_mol_wt_cutoff_type                                        soft
 dn_upper_constraint_mol_wt                                   1000.0
 dn_lower_constraint_mol_wt                                   0.0
 dn_mol_wt_std_dev                                            35.0
 dn_constraint_rot_bon                                        15
 dn_constraint_formal_charge                                  5
 dn_heur_unmatched_num                                        1
 dn_heur_matched_rmsd                                         2.0
 dn_unique_anchors                                            1
 dn_max_grow_layers                                           1
 dn_max_root_size                                             25 
 dn_max_layer_size                                            25
 dn_max_current_aps                                           5
 dn_max_scaffolds_per_layer                                   1
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          no
 dn_write_orients                                             no
 dn_write_growth_trees                                        no
 dn_output_prefix                                             4s0v_denovo_output
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 grid_score_primary                                           yes
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 minimize_ligand                                              yes
 minimize_anchor                                              no
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1
 simplex_trans_step                                           1
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10
 simplex_grow_max_iterations                                  250
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        yes
 simplex_coefficient_restraint                                10
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl

To run your file simply type: 

 dock6 -i denovoRefinement.in -o denovoRefinement.out

Once the job has completed you will see the following new files in your directory:

  • 4s0v_denovo_output.anchor_1.root_layer_1.mol2
  • 4s0v_denovo_output.denovo_build.mol2
  • denovoRefinement.out

Viewing New Molecules

scp the two .mol2 files over to your local computer. Open a new session in Chimera. To view the new residues that DOCK added to the dummy node go to Tools → Surface/Binding Analysis → ViewDock and open 4s0v_denovo_output.denovo_build.mol2. As before, a dialogue box will open and you can click through the different model options.

Denovooptions.png


Changing the input values in the input file can be powerful and gives us flexibility in how to "grow" our new ligand. These will be looked at more closely in the next two sections.

Focused DeNovo Design

The next two types of denovo design are similar to each other. We choose a structure, like an imidazole ring to "anchor" our ligand to the protein. DOCK will sample this structure all over the protein to find the best binding location. From that point a new ligand is "grown" out from this anchor point. The difference between focused and generic denovo design comes down to what residues DOCK uses to grow this new ligand.

Fragment Library Generation

In focused denovo design, a library of fragments which DOCK can use to grow the new ligand must be supplied to the program. We are "focusing" DOCK to work with specific fragments when building the new ligand. Very often when focused denovo design is used the fragment library is created using only the residues in the original ligand. This will be the approach we use for the following tutorial.

cd into your 013a.fragLib directory and create the input file: 

 vi fragLib.in

Type the following commands into the input file: 

 conformer_search_type                                        flex
 write_fragment_libraries                                     yes
 fragment_library_prefix                                      4s0v_fragLib
 fragment_library_freq_cutoff                                 1
 fragment_library_sort_method                                 freq
 fragment_library_trans_origin                                no
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../001.structure/4s0v_ligand_hydrogens.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        4s0v_focused
 write_orientations                                           no
 num_scored_conformers                                        1
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no

and run the program with: 

 dock6 -i fragLib.in -o fragLib.out

Once the program has successfully run you'll see the following new files in your directory:

  • 4s0v_focused_scored.mol2
  • 4s0v_fragLib_linker.mol2
  • 4s0v_fragLib_rigid.mol2
  • 4s0v_fragLib_scaffold.mol2
  • 4s0v_fragLib_sidechain.mol2
  • 4s0v_fragLib_torenv.dat

DeNovo Design

For this next section, cd into your 013b.focusGrowth directory.

There are three .mol2 files generated in the previous step that we need to give DOCK to perform the focused denovo growth. For this next step we again need an input file: 

 vi deNovoFocus.in

and type the following commands into the input file: 

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     ../013a.fragLib/4s0v_fragLib_scaffold.mol2
 dn_fraglib_linker_file                                       ../013a.fragLib/4s0v_fragLib_linker.mol2
 dn_fraglib_sidechain_file                                    ../013a.fragLib/4s0v_fragLib_sidechain.mol2
 dn_user_specified_anchor                                     no
 dn_use_torenv_table                                          yes
 dn_torenv_table                                              ../013a.fragLib/4s0v_fragLib_torenv.dat
 dn_sampling_method                                           graph
 dn_graph_max_picks                                           30
 dn_graph_breadth                                             3
 dn_graph_depth                                               2
 dn_graph_temperature                                         100.0
 dn_pruning_conformer_score_cutoff                            100.0
 dn_pruning_conformer_score_scaling_factor                    1.0
 dn_pruning_clustering_cutoff                                 100.0
 dn_constraint_mol_wt                                         550.0
 dn_constraint_rot_bon                                        15
 dn_constraint_formal_charge                                  2.0
 dn_heur_unmatched_num                                        1
 dn_heur_matched_rmsd                                         2.0
 dn_unique_anchors                                            2
 dn_max_grow_layers                                           9
 dn_max_root_size                                             25
 dn_max_layer_size                                            25
 dn_max_current_aps                                           5
 dn_max_scaffolds_per_layer                                   1
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          no
 dn_write_orients                                             no
 dn_write_growth_trees                                        yes
 dn_output_prefix                                             4s0v_focused
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl

Finally, to run this command type: 

 dock6 -i deNovoFocus.in -o deNovoFocus.out

Once this has successfully run you will see many new files in your directory. These all contain possible new ligands which can bind to your protein.

ReScoring Designed Molecules

The numerous molecules that were grown in the previous step need to be narrowed down to possible hits which can be investigated further. One way to do this is to reScore each ligand to find those that are similar to the original ligand in terms of interactions with the protein and overall composition. For these next steps, cd into your 013c.focusReScore directory and create your input file: 

 vi focusReScore.in

type the following commands into your input file: 

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../013b.focusGrowth/4s0v_focused.denovo_build.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_primary                                  no
 pharmacophore_score_secondary                                no
 descriptor_score_primary                                     yes
 descriptor_score_secondary                                   no
 descriptor_use_grid_score                                    no
 descriptor_use_multigrid_score                               no
 descriptor_use_continuous_score                              no
 descriptor_use_footprint_similarity                          yes
 descriptor_use_pharmacophore_score                           yes
 descriptor_use_tanimoto                                      yes
 descriptor_use_hungarian                                     yes
 descriptor_use_volume_overlap                                yes
 descriptor_fps_score_use_footprint_reference_mol2            yes
 descriptor_fps_score_footprint_reference_mol2_filename       ../004.energy_min/4s0v.lig.min_scored.mol2
 descriptor_fps_score_foot_compare_type                       Euclidean
 descriptor_fps_score_normalize_foot                          no
 descriptor_fps_score_foot_comp_all_residue                   yes
 descriptor_fps_score_receptor_filename                       ../001.structure/4s0v_protein_hydrogens_charges.mol2
 descriptor_fps_score_vdw_att_exp                             6
 descriptor_fps_score_vdw_rep_exp                             12
 descriptor_fps_score_vdw_rep_rad_scale                       1
 descriptor_fps_score_use_distance_dependent_dielectric       yes
 descriptor_fps_score_dielectric                              4.0
 descriptor_fps_score_vdw_fp_scale                            1
 descriptor_fps_score_es_fp_scale                             1
 descriptor_fps_score_hb_fp_scale                             0
 descriptor_fms_score_use_ref_mol2                            yes
 descriptor_fms_score_ref_mol2_filename                       ../004.energy_min/4s0v.lig.min_scored.mol2
 descriptor_fms_score_write_reference_pharmacophore_mol2      no
 descriptor_fms_score_write_reference_pharmacophore_txt       no
 descriptor_fms_score_write_candidate_pharmacophore           no
 descriptor_fms_score_write_matched_pharmacophore             no
 descriptor_fms_score_compare_type                            overlap
 descriptor_fms_score_full_match                              yes
 descriptor_fms_score_match_rate_weight                       5.0
 descriptor_fms_score_match_dist_cutoff                       1.0
 descriptor_fms_score_match_proj_cutoff                       0.7071
 descriptor_fms_score_max_score                               20
 descriptor_fingerprint_ref_filename                          ../004.energy_min/4s0v.lig.min_scored.mol2
 descriptor_hms_score_ref_filename                            ../004.energy_min/4s0v.lig.min_scored.mol2
 descriptor_hms_score_matching_coeff                          -5
 descriptor_hms_score_rmsd_coeff                              1
 descriptor_volume_score_reference_mol2_filename              ../004.energy_min/4s0v.lig.min_scored.mol2
 descriptor_volume_score_overlap_compute_method               analytical
 descriptor_weight_fps_score                                  1
 descriptor_weight_pharmacophore_score                        1
 descriptor_weight_fingerprint_tanimoto                       -1
 descriptor_weight_hms_score                                  1
 descriptor_weight_volume_overlap_score                       -1
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 chem_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
 pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
 ligand_outfile_prefix                                        4s0v_focus_rescore
 write_footprints                                             yes
 write_hbonds                                                 yes
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Finally to run this command type: 

 dock6 -i focusReScore.in -o focusReScore.out

When the program has run successfully there will be three new files in your directory:

  • 4s0v_focus_rescore_footprint_scored.txt
  • 4s0v_focus_rescore_hbond_scored.txt
  • 4s0v_focus_rescore_scored.mol2

To view these new ligands, scp the .mol2 file to your local computer and use ViewDock.

For this ligand we get 42 viable results. Looking at the new molecule with the lowest footprint score:

LowestFootprint.png

and the highest footprint score:

Highest.png


If we open the newly designed molecule with the lowest descriptor score and the energized minimized ligand from the previous tutorial, we see:

Ligandcomparisons.png

and we can see the two ligands are similar to each other although not exactly the same. This isn't too surprising since the library of fragments that DOCK had to work with originated from the residues in the original ligand. In the next section we give DOCK more freedom is choosing which residues to use in the denovo design.

Generic DeNovo Design

The final type of DeNovo design that we'll be looking at is Generic DeNovo Design. This is similar to the focused design in the previous section except we're not limiting DOCK to building the new molecule using only fragments present in the original ligand. This time we will be supplying DOCK with a library of many residues for it to use in generating the new molecule. This section will be done on the command line, so please cd to your 014.genericDenovo directory.

Create the input file: 

 vi genericLib.in

Type the following commands into the input file: 

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_sidechain.mol2
 dn_user_specified_anchor                                     no
 dn_use_torenv_table                                          yes
 dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_torenv.dat
 dn_sampling_method                                           graph
 dn_graph_max_picks                                           30
 dn_graph_breadth                                             3
 dn_graph_depth                                               2
 dn_graph_temperature                                         100
 dn_pruning_conformer_score_cutoff                            100
 dn_pruning_conformer_score_scaling_factor                    1.0
 dn_pruning_clustering_cutoff                                 100.0
 dn_constraint_mol_wt                                         550.0
 dn_constraint_rot_bon                                        15
 dn_constraint_formal_charge                                  2.0
 dn_heur_unmatched_num                                        1
 dn_heur_matched_rmsd                                         2.0
 dn_unique_anchors                                            1
 dn_max_grow_layers                                           9
 dn_max_root_size                                             25
 dn_max_layer_size                                            25
 dn_max_current_aps                                           5
 dn_max_scaffolds_per_layer                                   1
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          no
 dn_write_orients                                             no
 dn_write_growth_trees                                        no
 dn_output_prefix                                             4s0v_genericDenovo_output
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_primary                                  no
 pharmacophore_score_secondary                                no
 descriptor_score_primary                                     yes
 descriptor_score_secondary                                   no
 descriptor_use_grid_score                                    yes
 descriptor_use_pharmacophore_score                           no
 descriptor_use_tanimoto                                      no
 descriptor_use_hungarian                                     no
 descriptor_use_volume_overlap                                no
 descriptor_grid_score_rep_rad_scale                          1
 descriptor_grid_score_vdw_scale                              1
 descriptor_grid_score_es_scale                               1
 descriptor_grid_score_grid_prefix                            ../003.gridbox/grid
 descriptor_weight_grid_score                                 1
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl

This calculation can be computationally expensive so it's best to run the input file with a slurm script: 

 #!/bin/bash
 #
 #SBATCH --job-name=4s0v_genericLib
 #SBATCH --output=genericLib_output.txt
 #SBATCH --ntasks-per-node=24
 #SBATCH --nodes=1
 #SBATCH --time=48:00:00
 #SBATCH -p long-24core
 
 dock6 -i genericLib.in -o genericLib.out

Once the program is finished running you will see multiple new files in your directory including 4s0v_genericDenovo_output.denovo_build.mol2. scp this file over to your local computer. Start a new session in Chimera and open this file with ViewDock.

Using the command: 

 grep MOLECULE 4s0v_genericDenovo_output.denovo_build.mol2 | wc -l

you can determine how many new ligands DOCK "grew". In this case it's 537.

Looking at the molecule with the lowest grid score:

LowestGrids.png

and if we overlay this molecule to the original minimized ligand:

DenovoComparisons.png

we see that the ligands aren't the same at all. The original minimized ligand has a GridScore of -75.2 and this newly designed ligand has a GridScore of -75.8.

And finally we can look at both of these ligands interacting with the protein:

FinalLigand.png
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