Difference between revisions of "2025 Denovo tutorial 2 with PDBID 1XMU"

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=Introduction=
 
=Introduction=
  
−
This tutorial describes de novo method to refine ligand structure(s) using DOCK6. Rather than screening existing compounds, de novo refinement adds fragments based on known bioactive compounds, aiming to produce new ligands that bind more tightly to protein receptors than current options. This strategy is particularly valuable for:
+
This tutorial describes de novo method to refine ligand structure(s) using DOCK6. Rather than screening existing compounds, '''(1) de novo refinement''' adds fragments based on known bioactive compounds. '''(2) Generic de novo design''', on the other hand, produce new ligands from scratch. Such that we yield new ligand beyond the current available chemotypes allowing tighter binding protein receptors. This strategy is particularly valuable for:
  
 
*Improving suboptimal ligand binding profiles
 
*Improving suboptimal ligand binding profiles
Line 8: Line 8:
 
*Exploring novel scaffolds while maintaining critical pharmacophore features
 
*Exploring novel scaffolds while maintaining critical pharmacophore features
  
−
In this tutorial, we will utilize PDB structure 1XMU - Catalytic Domain Of Human Phosphodiesterase 4B In Complex With Roflumilast.  
+
In this tutorial, we will utilize PDB structure 1XMU - Catalytic Domain Of Human Phosphodiesterase 4B In Complex With Roflumilast.
−
 
 
  
 
=System Preparation=
 
=System Preparation=
Line 15: Line 14:
 
We first prepare a folder to contain all the input and output files for de novo refinement for 1XMU. This is the current directory structure under the upper directory dock6-1xmu.
 
We first prepare a folder to contain all the input and output files for de novo refinement for 1XMU. This is the current directory structure under the upper directory dock6-1xmu.
  
−
[[File:1xmu_file.png|center|200px]]
+
[[File:1xmu_file2.png|center|200px]]
 +
 
 +
=De Novo Refinement=
 +
 
 +
Refinement requires users to first modify the original ligand file (mol2 with hydrogen) to specify the dummy atom location. The dummy atom is used as the starting point for the refinement process.
 +
 
 +
==Modify Ligand with Dummy Atom==
 +
 
 +
Below shows the original ligand with hydrogen (mol2 file).
 +
 
 +
[[File:1xmu_lig_wH.png|center|300px]]
  
−
=Input File=
 
  
−
==Ligand with Dummy Atom==
+
Select the atoms to delete and save the mol2 file as 1xmu_lig_wDu.mol2. Hover the cursor over the atom that you intend to change to a dummy atom. Here we are changing the C21 to a dummy atom.
−
Below shows the original ligand with hydrogen (mol2 file). Open chimera and select the atoms to delete. Save the mol2 file
 
  
 +
[[File:1xmu_lig_wH_del.png|center|300px]]
 +
 +
Open the 1xmu_lig_wDu.mol2 file and edit the C21 atom type to Du as shown below.
 +
 +
[[File:1xmu_du_vi.png|center|400px]]
 +
 +
Next reopen the 1xmu_lig_wDu.mol2 file to observe if the corresponding atom is now represented as Du in ''magenta''.
 +
 +
[[File:1xmu_lig_wDu.png|center|300px]]
  
 
==DOCK6 input==
 
==DOCK6 input==
Line 27: Line 43:
  
  
 +
The directory 008_dn_refine1 contains the following files:
 +
 +
[[File: 1xmu_files.png|center|400px]]
 +
 +
 +
 +
The 01dnv.in contains the DOCK6 input parameters. Please note that the bolded lines represent customized items for this tutorial, including the input and output file names. Additionally, we intend to modify the ligand with only one layer while keeping the original pose. DOCK6 parameter files and fragment libraries have been previously prepared. Please refer to the virtual screening and genetic algorithm tutorials for further instructions if needed.
 +
 +
  conformer_search_type                                        denovo
 +
  '''dn_fraglib_scaffold_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2'''
 +
  '''dn_fraglib_linker_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2'''
 +
  '''dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2'''
 +
  dn_user_specified_anchor                                    yes
 +
  '''dn_fraglib_anchor_file                                      1xmu_lig_wDu.mol2'''
 +
  '''dn_torenv_table                                              ../006_ga_lib/unique_full_sorted_fraglib.dat'''
 +
  dn_name_identifier                                          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_remove_duplicates                                        yes
 +
  dn_max_duplicates_per_mol                                    0
 +
  dn_write_pruned_duplicates                                  no
 +
  dn_advanced_pruning                                          yes
 +
  dn_prune_initial_sample                                      yes
 +
  dn_sample_torsions                                          yes
 +
  dn_prune_individual_torsions                                yes
 +
  dn_prune_combined_torsions                                  yes
 +
  dn_random_root_selection                                    no
 +
  dn_mol_wt_cutoff_type                                        soft
 +
  dn_upper_constraint_mol_wt                                  1000
 +
  dn_lower_constraint_mol_wt                                  0.0
 +
  dn_mol_wt_std_dev                                            35.0
 +
  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                                          1'''
 +
  dn_max_root_size                                            25
 +
  dn_max_layer_size                                            25
 +
  dn_max_current_aps                                          5
 +
  dn_max_scaffolds_per_layer                                  1
 +
  dn_max_successful_att_per_root                              50000
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          yes
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        no
 +
  '''dn_output_prefix                                            1xmu_out'''
 +
  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_lig_efficiency                                          no
 +
  '''grid_score_grid_prefix                                      ../003_grid/grid'''
 +
  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_final_min                                            no
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  '''vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_AMBER_parm99.defn'''
 +
  '''flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn'''
 +
  '''flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl'''
 +
 +
 +
Then, run the bash script, which contains:
 +
 +
  #!/bin/bash
 +
 
 +
  dock6 -i 01dnv.in -o 01dnv.out >&01dnv.log&
 +
 +
==Output Files ==
 +
 +
When completed, the directory now contains:
 +
 +
[[File: 1xmu_outfile.png|center|400px]]
 +
 +
1xmu_out.denovo_build.mol2 contains no data, which is an intermediate file genereated.
 +
 +
 +
== Results ==
 +
 +
 +
From ChimeraX > Tools> Structure/Binding Analysis > View Dock, load the 1xmu_out.completed.denovo_build.mol2 (''pink'') along with the 1xmu_rec_wH.mol2 (''blue'') and 1xmu_lig_wH.mol2 (''beige''). We can see the refinement at the dummy atom (''magenta'')
 +
 +
 +
[[File: 1xmu_out_foc2.png|300px|center]]
 +
 +
 +
We can compare the interaction energy from the View Dock panel.
 +
 +
[[File: 1xmu_output2.png|400px|center]]
 +
 +
=De Novo Generic=
 +
 +
Dock6 can also perform generic de novo design without specifying the exact location to refine with the dummy atom. Below we will illustrate performing generic de novo '''with or without a specify anchor'''. This approach will allow orientation of ligand molecules. Parameters for the input file should be carefully considered, e.g. Lipinski's rule of five. 
 +
 +
 +
==No Anchor Specification==
 +
 +
In the directory '''009_dn_generic1''', we created the dock6 input file '''without the anchor specification''':
 +
 +
  conformer_search_type                                        denovo
 +
  dn_fraglib_scaffold_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 +
  dn_fraglib_linker_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 +
  dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 +
  dn_user_specified_anchor                                    no
 +
  dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_torenv.dat
 +
  dn_name_identifier                                          generic
 +
  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_remove_duplicates                                        yes
 +
  dn_max_duplicates_per_mol                                    0
 +
  dn_write_pruned_duplicates                                  no
 +
  dn_advanced_pruning                                          yes
 +
  dn_prune_initial_sample                                      yes
 +
  dn_sample_torsions                                          yes
 +
  dn_prune_individual_torsions                                yes
 +
  dn_prune_combined_torsions                                  yes
 +
  dn_random_root_selection                                    no
 +
  dn_mol_wt_cutoff_type                                        soft
 +
  dn_upper_constraint_mol_wt                                  550.0
 +
  dn_lower_constraint_mol_wt                                  0.0
 +
  dn_mol_wt_std_dev                                            35.0
 +
  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_max_successful_att_per_root                              50000
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          yes
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        no
 +
  dn_write_growth_trees                                        no
 +
  dn_output_prefix                                            generic
 +
  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
 +
  grid_score_primary                                          yes
 +
  grid_score_rep_rad_scale                                    1
 +
  grid_score_vdw_scale                                        1
 +
  grid_score_es_scale                                          1
 +
  grid_lig_efficiency                                          no
 +
  grid_score_grid_prefix                                      ../003_grid/grid
 +
  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_final_min                                            no
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl
 +
 +
 +
We shall run this with a slurm script (01dng.slurm) similar to the one without specified anchor.
 +
 +
  #!/bin/bash
 +
  #
 +
  #SBATCH --job-name=1xmu_dng2
 +
  #SBATCH --output=generic
 +
  #SBATCH --ntasks=1
 +
  #SBATCH --nodes=1
 +
  #SBATCH --time=4:00:00
 +
  #SBATCH -p ood-short-96core
 +
  ## at least 6 nodes for medium
 +
  dock6 -i 01dng.in -o 01dng.out
 +
 +
Submit the slurm script with:
 +
 +
  sbatch 01dng.slurm
 +
 +
 +
==Specified Anchor Input==
 +
 +
We first need to create the mol2 file containing the fragment to use as anchor. You can look over the list of fragments (scaffold/linker/side chains) in the following file:
 +
 +
  /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 +
  /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 +
  /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 +
 +
 +
When you identify the fragment of your preference, you can copy or cat into a new mol2 file. File name '''frag.mol2''' is used in this tutorial. The copied content should contain the following:
 +
 +
 +
[[File: 1xmu_frag.png|center|400px]]
 +
 +
 
 +
 +
In the directory '''009_dn_generic2''', we created the dock6 input file '''with the anchor specification''':
 +
 +
 +
  conformer_search_type                                        denovo
 +
  dn_fraglib_scaffold_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 +
  dn_fraglib_linker_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 +
  dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 +
  '''dn_user_specified_anchor                                    yes'''
 +
  '''dn_fraglib_anchor_file                                      frag.mol2'''
 +
  dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_torenv.dat
 +
  dn_name_identifier                                          generic
 +
  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_remove_duplicates                                        yes
 +
  dn_max_duplicates_per_mol                                    0
 +
  dn_write_pruned_duplicates                                  no
 +
  dn_advanced_pruning                                          yes
 +
  dn_prune_initial_sample                                      yes
 +
  dn_sample_torsions                                          yes
 +
  dn_prune_individual_torsions                                yes
 +
  dn_prune_combined_torsions                                  yes
 +
  dn_random_root_selection                                    no
 +
  dn_mol_wt_cutoff_type                                        soft
 +
  dn_upper_constraint_mol_wt                                  550.0
 +
  dn_lower_constraint_mol_wt                                  0.0
 +
  dn_mol_wt_std_dev                                            35.0
 +
  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_max_successful_att_per_root                              50000
 +
  dn_write_checkpoints                                        yes
 +
  dn_write_prune_dump                                          yes
 +
  dn_write_orients                                            no
 +
  dn_write_growth_trees                                        no
 +
  dn_write_growth_trees                                        no
 +
  dn_output_prefix                                            generic
 +
  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
 +
  grid_score_primary                                          yes
 +
  grid_score_rep_rad_scale                                    1
 +
  grid_score_vdw_scale                                        1
 +
  grid_score_es_scale                                          1
 +
  grid_lig_efficiency                                          no
 +
  grid_score_grid_prefix                                      ../003_grid/grid
 +
  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_final_min                                            no
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_de_novo.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl
 +
 +
We shall run this with a slurm script (01dng.slurm) similar to the one without specified anchor.
 +
 +
  #!/bin/bash
 +
  #
 +
  #SBATCH --job-name=1xmu_dng2
 +
  #SBATCH --output=generic
 +
  #SBATCH --ntasks=1
 +
  #SBATCH --nodes=1
 +
  #SBATCH --time=4:00:00
 +
  #SBATCH -p ood-short-96core
 +
  ## at least 6 nodes for medium
 +
  dock6 -i 01dng.in -o 01dng.out
 +
 +
Submit the slurm script with:
 +
 +
  sbatch 01dng.slurm
 +
 +
==Output==
 +
 +
This shows the output files at the end of run for the generic de novo design with specified anchor.
 +
 +
[[File:1xmu_output3.png|center|400px]]
 +
 +
Take the last 10000 (or more) lines from the generic.completed.denovo_build.mol2 into a new mol2 file by:
 +
 +
  tail -10000 generic.completed.denovo_build.mol2 > out.mol2
 +
 +
Open the mol2 file and remove the lines until you get to the first Grid Score. For example, in our out.mol2 file for this tutorial, we need to remove 65 lines.
 +
 +
[[File:1xmu_output5.png|center|400px]]
 +
 +
==Results==
 +
 +
Open your results in chimera and you shall see the overlapped designs.
 +
 +
[[File:1xmu_output4.png|center|200px]]
 +
 +
Again, we can analyze by using View Dock. Load the out.mol2 (''blue'') along with the 1xmu_rec_wH.mol2 (''beige'').
 +
 +
 +
[[File:1xmu_output6.png|300px|center]]
 +
 +
 +
We can compare the grid score and the fragment strings from the View Dock panel.
  
−
=
+
[[File:1xmu_output7.png|400px|center]]

Latest revision as of 13:28, 10 March 2025

Introduction

This tutorial describes de novo method to refine ligand structure(s) using DOCK6. Rather than screening existing compounds, (1) de novo refinement adds fragments based on known bioactive compounds. (2) Generic de novo design, on the other hand, produce new ligands from scratch. Such that we yield new ligand beyond the current available chemotypes allowing tighter binding protein receptors. This strategy is particularly valuable for:

  • Improving suboptimal ligand binding profiles
  • Overcoming resistance mechanisms
  • Exploring novel scaffolds while maintaining critical pharmacophore features

In this tutorial, we will utilize PDB structure 1XMU - Catalytic Domain Of Human Phosphodiesterase 4B In Complex With Roflumilast.

System Preparation

We first prepare a folder to contain all the input and output files for de novo refinement for 1XMU. This is the current directory structure under the upper directory dock6-1xmu.

1xmu file2.png

De Novo Refinement

Refinement requires users to first modify the original ligand file (mol2 with hydrogen) to specify the dummy atom location. The dummy atom is used as the starting point for the refinement process.

Modify Ligand with Dummy Atom

Below shows the original ligand with hydrogen (mol2 file).

1xmu lig wH.png


Select the atoms to delete and save the mol2 file as 1xmu_lig_wDu.mol2. Hover the cursor over the atom that you intend to change to a dummy atom. Here we are changing the C21 to a dummy atom.

1xmu lig wH del.png

Open the 1xmu_lig_wDu.mol2 file and edit the C21 atom type to Du as shown below.

1xmu du vi.png

Next reopen the 1xmu_lig_wDu.mol2 file to observe if the corresponding atom is now represented as Du in magenta.

1xmu lig wDu.png

DOCK6 input

The directory 008_dn_refine1 contains the following files:

1xmu files.png


The 01dnv.in contains the DOCK6 input parameters. Please note that the bolded lines represent customized items for this tutorial, including the input and output file names. Additionally, we intend to modify the ligand with only one layer while keeping the original pose. DOCK6 parameter files and fragment libraries have been previously prepared. Please refer to the virtual screening and genetic algorithm tutorials for further instructions if needed. 

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 dn_user_specified_anchor                                     yes
 dn_fraglib_anchor_file                                       1xmu_lig_wDu.mol2
 dn_torenv_table                                              ../006_ga_lib/unique_full_sorted_fraglib.dat
 dn_name_identifier                                           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_remove_duplicates                                         yes
 dn_max_duplicates_per_mol                                    0
 dn_write_pruned_duplicates                                   no
 dn_advanced_pruning                                          yes
 dn_prune_initial_sample                                      yes
 dn_sample_torsions                                           yes
 dn_prune_individual_torsions                                 yes
 dn_prune_combined_torsions                                   yes
 dn_random_root_selection                                     no
 dn_mol_wt_cutoff_type                                        soft
 dn_upper_constraint_mol_wt                                   1000
 dn_lower_constraint_mol_wt                                   0.0
 dn_mol_wt_std_dev                                            35.0
 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                                           1
 dn_max_root_size                                             25
 dn_max_layer_size                                            25
 dn_max_current_aps                                           5
 dn_max_scaffolds_per_layer                                   1
 dn_max_successful_att_per_root                               50000
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          yes
 dn_write_orients                                             no
 dn_write_growth_trees                                        no
 dn_output_prefix                                             1xmu_out
 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_lig_efficiency                                          no
 grid_score_grid_prefix                                       ../003_grid/grid
 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_final_min                                            no
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl


Then, run the bash script, which contains: 

 #!/bin/bash
 
 dock6 -i 01dnv.in -o 01dnv.out >&01dnv.log&

Output Files

When completed, the directory now contains:

1xmu outfile.png

1xmu_out.denovo_build.mol2 contains no data, which is an intermediate file genereated.


Results

From ChimeraX > Tools> Structure/Binding Analysis > View Dock, load the 1xmu_out.completed.denovo_build.mol2 (pink) along with the 1xmu_rec_wH.mol2 (blue) and 1xmu_lig_wH.mol2 (beige). We can see the refinement at the dummy atom (magenta)


1xmu out foc2.png


We can compare the interaction energy from the View Dock panel.

1xmu output2.png

De Novo Generic

Dock6 can also perform generic de novo design without specifying the exact location to refine with the dummy atom. Below we will illustrate performing generic de novo with or without a specify anchor. This approach will allow orientation of ligand molecules. Parameters for the input file should be carefully considered, e.g. Lipinski's rule of five.


No Anchor Specification

In the directory 009_dn_generic1, we created the dock6 input file without the anchor specification: 

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 dn_user_specified_anchor                                     no
 dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_torenv.dat
 dn_name_identifier                                           generic
 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_remove_duplicates                                         yes
 dn_max_duplicates_per_mol                                    0
 dn_write_pruned_duplicates                                   no
 dn_advanced_pruning                                          yes
 dn_prune_initial_sample                                      yes
 dn_sample_torsions                                           yes
 dn_prune_individual_torsions                                 yes
 dn_prune_combined_torsions                                   yes
 dn_random_root_selection                                     no
 dn_mol_wt_cutoff_type                                        soft
 dn_upper_constraint_mol_wt                                   550.0
 dn_lower_constraint_mol_wt                                   0.0
 dn_mol_wt_std_dev                                            35.0
 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_max_successful_att_per_root                               50000
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          yes
 dn_write_orients                                             no
 dn_write_growth_trees                                        no
 dn_write_growth_trees                                        no
 dn_output_prefix                                             generic
 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
 grid_score_primary                                           yes
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_lig_efficiency                                          no
 grid_score_grid_prefix                                       ../003_grid/grid
 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_final_min                                            no
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl


We shall run this with a slurm script (01dng.slurm) similar to the one without specified anchor. 

 #!/bin/bash
 #
 #SBATCH --job-name=1xmu_dng2
 #SBATCH --output=generic
 #SBATCH --ntasks=1
 #SBATCH --nodes=1
 #SBATCH --time=4:00:00
 #SBATCH -p ood-short-96core
 ## at least 6 nodes for medium
 dock6 -i 01dng.in -o 01dng.out

Submit the slurm script with: 

 sbatch 01dng.slurm


Specified Anchor Input

We first need to create the mol2 file containing the fragment to use as anchor. You can look over the list of fragments (scaffold/linker/side chains) in the following file: 

 /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2


When you identify the fragment of your preference, you can copy or cat into a new mol2 file. File name frag.mol2 is used in this tutorial. The copied content should contain the following:


1xmu frag.png

 

In the directory 009_dn_generic2, we created the dock6 input file with the anchor specification: 


 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_sidechain.mol2
 dn_user_specified_anchor                                     yes
 dn_fraglib_anchor_file                                       frag.mol2
 dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/fraglib_torenv.dat
 dn_name_identifier                                           generic
 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_remove_duplicates                                         yes
 dn_max_duplicates_per_mol                                    0
 dn_write_pruned_duplicates                                   no
 dn_advanced_pruning                                          yes
 dn_prune_initial_sample                                      yes
 dn_sample_torsions                                           yes
 dn_prune_individual_torsions                                 yes
 dn_prune_combined_torsions                                   yes
 dn_random_root_selection                                     no
 dn_mol_wt_cutoff_type                                        soft
 dn_upper_constraint_mol_wt                                   550.0
 dn_lower_constraint_mol_wt                                   0.0
 dn_mol_wt_std_dev                                            35.0
 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_max_successful_att_per_root                               50000
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          yes
 dn_write_orients                                             no
 dn_write_growth_trees                                        no
 dn_write_growth_trees                                        no
 dn_output_prefix                                             generic
 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
 grid_score_primary                                           yes
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_lig_efficiency                                          no
 grid_score_grid_prefix                                       ../003_grid/grid
 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_final_min                                            no
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.12_ams536/parameters/flex_drive.tbl

We shall run this with a slurm script (01dng.slurm) similar to the one without specified anchor. 

 #!/bin/bash
 #
 #SBATCH --job-name=1xmu_dng2
 #SBATCH --output=generic
 #SBATCH --ntasks=1
 #SBATCH --nodes=1
 #SBATCH --time=4:00:00
 #SBATCH -p ood-short-96core
 ## at least 6 nodes for medium
 dock6 -i 01dng.in -o 01dng.out

Submit the slurm script with: 

 sbatch 01dng.slurm

Output

This shows the output files at the end of run for the generic de novo design with specified anchor.

1xmu output3.png

Take the last 10000 (or more) lines from the generic.completed.denovo_build.mol2 into a new mol2 file by: 

 tail -10000 generic.completed.denovo_build.mol2 > out.mol2

Open the mol2 file and remove the lines until you get to the first Grid Score. For example, in our out.mol2 file for this tutorial, we need to remove 65 lines.

1xmu output5.png

Results

Open your results in chimera and you shall see the overlapped designs.

1xmu output4.png

Again, we can analyze by using View Dock. Load the out.mol2 (blue) along with the 1xmu_rec_wH.mol2 (beige).


1xmu output6.png


We can compare the grid score and the fragment strings from the View Dock panel.

1xmu output7.png
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