Difference between revisions of "2019 DOCK tutorial 3 with PDBID 3JQZ"

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This tutorial contains, a step by step by approach to dock a known ligand to a known receptor.
 
This tutorial contains, a step by step by approach to dock a known ligand to a known receptor.
 +
 +
NOTE: JDB - Change all saved conformers to 50 for rigid/fad/flex and include a note on rank ordering the scores.
  
 
= I. Introduction =
 
= I. Introduction =
Line 12: Line 14:
 
# The resulting configurations are 'pruned' and energy re-minimized, yielding the docked configurations.
 
# The resulting configurations are 'pruned' and energy re-minimized, yielding the docked configurations.
  
 +
If you are interested in reading more about DOCK 6 and the background to these processes, interested readers may reference the [http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm DOCK User's Manual].
  
 
===3JQZ===
 
===3JQZ===
Line 19: Line 22:
 
Hint: for docking practice you should identify a high resolution crystal structure with a well defined ligand binding site, not situated at the interface of two chains and with minimal active site waters.
 
Hint: for docking practice you should identify a high resolution crystal structure with a well defined ligand binding site, not situated at the interface of two chains and with minimal active site waters.
  
== Organisation of Directories ==
+
== Organization of Directories ==
 
Maintaining a clearly organised set of folders will be helpful in finding specific files, calling files and most importantly keeping track of everything you do. We recommend maintaining the following directory structure throughout the tutorial.
 
Maintaining a clearly organised set of folders will be helpful in finding specific files, calling files and most importantly keeping track of everything you do. We recommend maintaining the following directory structure throughout the tutorial.
  
Line 36: Line 39:
 
= II. Preparation of the ligand and receptor =
 
= II. Preparation of the ligand and receptor =
  
Download the pdb file 3JQZ from the PDB database save it in 000_files folder.
+
Download the PDB file 3JQZ from the PDB database save it in the '''000_files folder.'''
  
 
===Checking the chosen structure===
 
===Checking the chosen structure===
It can be helpful to read through the journal article associated with the PDB file to understand protonation states, charges, environmental conditions and other important information regarding the receptor and ligand under the crystallisation conditions used to obtain the structure you are working with.  
+
It can be helpful to read through the journal article associated with the PDB file to understand protonation states, charges, environmental conditions and other important information regarding the receptor and ligand under the crystallization conditions used to obtain the structure you are working with.  
  
 
To do this;
 
To do this;
 
   - Open Chimera  
 
   - Open Chimera  
 
   - File -> Fetch by ID -> Type 3JQZ into the 'PDB' box -> Fetch
 
   - File -> Fetch by ID -> Type 3JQZ into the 'PDB' box -> Fetch
+
 
 
You can now explore the crystal structure.  
 
You can now explore the crystal structure.  
  
Hint: From the tool bar at the top of the Chimera window, click Actions -> Focus, to put your structure back in the middle of the screen.  
+
Hint: From the toolbar at the top of the Chimera window, click Actions -> Focus, to put your structure back in the middle of the screen.  
  
 
Identify the main components of the model (receptor, ligand, solvent, surfactants, metal ions)
 
Identify the main components of the model (receptor, ligand, solvent, surfactants, metal ions)
Line 55: Line 58:
 
===Preparation of receptor===
 
===Preparation of receptor===
 
   - Open the PDB file (3JQZ.pdb) using Chimera
 
   - Open the PDB file (3JQZ.pdb) using Chimera
   - Isolate the receptor using select tool and delete tool in Chimera.
+
   - Isolate the receptor using the select tool and delete tool in Chimera.
   - Save the isolated receptor as a mol2 file. (2nnq_rec_noH.mol2)
+
   - Save the isolated receptor as a mol2 file. (3jqz_rec_noH.mol2)
  
   - Open 2nnq_rec_noH.mol2 file again using Chimera and use the following instructions to prepare the receptor file to be used in DOCK.
+
   - Open 3jqz_rec_noH.mol2 file again using Chimera and use the following instructions to prepare the receptor file to be used in DOCK.
 
           Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
 
           Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
 
           Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force filed available for standard residues. Here we used AMBER ff14SB)
 
           Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force filed available for standard residues. Here we used AMBER ff14SB)
           Save as a mol2 file. (2nnq_rec_withH.mol2)
+
           Save as a mol2 file. (3jqz_rec_withH.mol2)
  
   - If you follow the step below all the above stated steps will automatically appear one after the other to prepare the receptor.  
+
   - If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor.  
 
           Tools -> Structure/Binding Analysis -> DockPrep
 
           Tools -> Structure/Binding Analysis -> DockPrep
  
Line 69: Line 72:
 
   - Open the PDB file via Chimera.
 
   - Open the PDB file via Chimera.
 
   - Using Chimera, isolate the ligand, add H atoms, add charge and save it as a mol2 file by following the same steps followed for the receptor.
 
   - Using Chimera, isolate the ligand, add H atoms, add charge and save it as a mol2 file by following the same steps followed for the receptor.
 +
  - It is ''incredibly important'' both here and in all modeling experiments that you examine the ligand's protonation state and ring saturation. For this particular experiment, if you just take Chimera's protonation output you'll find it to be incorrect. Note that 6-membered ring structure will probably be protonated incorrectly - checking [https://www.sciencedirect.com/science/article/pii/S1047847710000912?via%3Dihub#aep-section-id28 the structure in the reference] shows that it should be aromatic, not saturated. You can fix this in Chimera using Tools > Structure Editing > Build Structure.
 +
 +
[[File:3jqz_good_bad_protonation.png|thumb|none|1000px|Left: The incorrect protonation of the ligand in 3JQZ. Right: The correct protonation.]]
  
Once all the files are prepared make sure to save the files in 1.dockprep folder.
+
 
 +
Once all the files are prepared make sure to save the files in 001.dockprep folder.
  
 
=III. Generating receptor surface and spheres=
 
=III. Generating receptor surface and spheres=
Line 84: Line 91:
  
 
===Generating spheres===
 
===Generating spheres===
   - Go to 003.surface_spheres folder
+
   - Go to 003_surface_spheres folder
 
   - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file.  
 
   - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file.  
  
Line 116: Line 123:
 
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand binding site are kept and all the other spheres are deleted in the image below.
 
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand binding site are kept and all the other spheres are deleted in the image below.
  
[[File:Sphgen 3jqz bj3.png|thumb|center|1000px|2nnq receptor and selected spheres]]
+
[[File:Sphgen 3jqz bj3.png|thumb|center|1000px|3jqz receptor and selected spheres]]
  
 
=IV. Generating box and grid=
 
=IV. Generating box and grid=
  
 
===Generating box===
 
===Generating box===
Move to 003_boxgrid directory
+
'''Move to 003_gridbox directory'''
 +
 
 
Create a new file showbox.in and write the following lines in the file.
 
Create a new file showbox.in and write the following lines in the file.
  
  Y
+
Y
  8.0
+
8.0
  ../002_surface_spheres/selected_spheres.sph
+
../002_surface_spheres/selected_spheres.sph
  1
+
1
  3jqz.box.pdb
+
3jqz.box.pdb
  
 
Each of the above lines indicates that;
 
Each of the above lines indicates that;
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Use the following command to generate the box.
 
Use the following command to generate the box.
  
  showbox < showbox.in
+
showbox < showbox.in
  
If this step is successful, you should see a new file (3jqz.box.pdb) in 003_boxgrid folder.
+
If this step is successful, you should see a new file (3jqz.box.pdb) in 003_gridbox folder.
 
[[File:Gridbox 3jqz bj1.png|thumb|center|1000px|Box grid (blue) generated for 3JQZ receptor (gray surface) at the ligand binding site (green surface). Selected spheres are shown in pink. ]]
 
[[File:Gridbox 3jqz bj1.png|thumb|center|1000px|Box grid (blue) generated for 3JQZ receptor (gray surface) at the ligand binding site (green surface). Selected spheres are shown in pink. ]]
  
Line 157: Line 165:
 
  atom_model                                a
 
  atom_model                                a
 
  attractive_exponent                      6
 
  attractive_exponent                      6
  repulsive_exponent                        12
+
  repulsive_exponent                        9
 
  distance_dielectric                      yes
 
  distance_dielectric                      yes
 
  dielectric_factor                        4
 
  dielectric_factor                        4
 
  bump_filter                              yes
 
  bump_filter                              yes
 
  bump_overlap                              0.75
 
  bump_overlap                              0.75
  receptor_file                            ../1.dockprep/3jqz_rec_withH.mol2
+
  receptor_file                            ../001_dockprep/3jqz_rec_withH.mol2
 
  box_file                                  3jqz.box.pdb
 
  box_file                                  3jqz.box.pdb
 
  vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 
  vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
Line 173: Line 181:
  
 
=V. Docking a single molecule for pose reproduction =
 
=V. Docking a single molecule for pose reproduction =
Under this section, the ligand for 2nnq.pdb will be re-docked into the receptor. 3 Methods will be used to achieve this.
+
Under this section, the ligand for 3jqz.pdb will be re-docked into the receptor. 3 Methods will be used to achieve this.
  
 
1. rigid docking  
 
1. rigid docking  
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Before performing docking, here the ligand will be subjected to energy minimization in order to remove unfavorable clashes. These clashes will affect rigid docking because in rigid docking the ligand will be docked as the complete ligand, whereas in other docking methods the ligand will be broken into fragments and the ligand will be built step by step considering favorable orientations and torsion angles after each fragment addition.
 
Before performing docking, here the ligand will be subjected to energy minimization in order to remove unfavorable clashes. These clashes will affect rigid docking because in rigid docking the ligand will be docked as the complete ligand, whereas in other docking methods the ligand will be broken into fragments and the ligand will be built step by step considering favorable orientations and torsion angles after each fragment addition.
  
Go to the directory 4.dock and a create a new file (min.in) and enter the command below.
+
Go to the directory 4.dock and a create a new file (touch min.in) and enter the command below.
 
   dock6 -i min.in
 
   dock6 -i min.in
  
Line 193: Line 201:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
  ligand_atom_file                                            ../1.dockprep/2nnq_lig_withH.mol2
+
  ligand_atom_file                                            ../001.dockprep/3jqz_lig_withH.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
 
  read_mol_solvation                                          no
 
  read_mol_solvation                                          no
 
  calculate_rmsd                                              yes
 
  calculate_rmsd                                              yes
  use_rmsd_reference_mol                                       ../1.dockprep/2nnq_lig_withH.mol2
+
  use_rmsd_reference_mol                               yes     
 +
rmsd_reference_filename                              ../001.dockprep/3jqz_lig_withH.mol2
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                no
 
  orient_ligand                                                no
Line 210: Line 219:
 
  grid_score_vdw_scale                                        1
 
  grid_score_vdw_scale                                        1
 
  grid_score_es_scale                                          1
 
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                      ../3.boxgrid/grid
+
  grid_score_grid_prefix                                      ../003.gridbox/grid
 
  multigrid_score_secondary                                    no
 
  multigrid_score_secondary                                    no
 
  dock3.5_score_secondary                                      no
 
  dock3.5_score_secondary                                      no
Line 237: Line 246:
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        2nnq.lig.min
+
  ligand_outfile_prefix                                        3jqz.lig.min
 
  write_orientations                                          no
 
  write_orientations                                          no
 
  num_scored_conformers                                        1
 
  num_scored_conformers                                        1
 
  rank_ligands                                                no  
 
  rank_ligands                                                no  
  
If the process is successful a new file (2nnq.lig.min_scored.mol2) will be generated. You can compare how is it changed from the initial structure by analyzing the RMSD value generated in the file. Visualize the new mol2 file along with receptor and the initial ligand mol2 files using Chimera to see the differences.
+
If the process is successful a new file (3jqz.lig.min_scored.mol2) will be generated. You can compare how is it changed from the initial structure by analyzing the RMSD value generated in the file. Visualize the new mol2 file along with receptor and the initial ligand mol2 files using Chimera to see the differences.
  
[[File:2nnq min lig.png|thumb|center|1000px|2nnq_receptor with the original ligand and the minimized ligand]]
+
[[File:20190304 crystal and energy minimised ligand figure for wiki.jpg|thumb|center|1000px|3jqz with crystal pose ligand (blue) and the energy minimised ligand (magenta)]]
  
 
==Rigid Docking==
 
==Rigid Docking==
Line 251: Line 260:
  
 
Run dock using the created input file.
 
Run dock using the created input file.
  dock6 -i rigid.in
+
  dock6 -i rigid.in -o rigid.out
  
 
Follow a similar approach as we did for minimization to answer the prompted questions by either answering them manually using the answers in the lines below or by including the following lines in the input file before running dock.
 
Follow a similar approach as we did for minimization to answer the prompted questions by either answering them manually using the answers in the lines below or by including the following lines in the input file before running dock.
Line 257: Line 266:
 
  conformer_search_type                                        rigid
 
  conformer_search_type                                        rigid
 
  use_internal_energy                                          yes
 
  use_internal_energy                                          yes
  ligand_atom_file                                            2nnq.lig.min_scored.mol2
+
  ligand_atom_file                                            3jqz.lig.min_scored.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
 
  read_mol_solvation                                          no
 
  read_mol_solvation                                          no
 
  calculate_rmsd                                              yes
 
  calculate_rmsd                                              yes
  use_rmsd_reference_mol                                       2nnq.lig.min_scored.mol2
+
  use_rmsd_reference_mol                                   yes   
 +
rmsd_reference_filename                            3jqz.lig.min_scored.mol2
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                yes
 
  orient_ligand                                                yes
 
  automated_matching                                          yes
 
  automated_matching                                          yes
  receptor_site_file                                          ../2.surface_spheres/selected_spheres.sph
+
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 
  max_orientations                                            1000
 
  max_orientations                                            1000
 
  critical_points                                              no
 
  critical_points                                              no
Line 280: Line 290:
 
  grid_score_vdw_scale                                        1
 
  grid_score_vdw_scale                                        1
 
  grid_score_es_scale                                          1
 
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                      ../3.boxgrid/grid
+
  grid_score_grid_prefix                                      ../003.gridbox/grid
 
  multigrid_score_secondary                                    no
 
  multigrid_score_secondary                                    no
 
  dock3.5_score_secondary                                      no
 
  dock3.5_score_secondary                                      no
Line 314: Line 324:
  
 
  Open Chimera
 
  Open Chimera
  File -> Open -> 2nnq_rec_withH.mol2
+
  File -> Open -> 3jqz_rec_withH.mol2
  File -> Open -> 2nnq_lig_withH.mol2
+
  File -> Open -> 3jqz_lig_withH.mol2
  Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (rigid.out_scored.mol2)
+
  Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (004.dock/rigid.out_scored.mol2)
In the loaded dialog box select Dock4,5 or 6
+
Under File type, select Dock4,5 or 6
  
 
Once everything is loaded go to the ViewDock window and use it's menu to view all the calculated properties regarding the rigid docked ligand by following the steps below.
 
Once everything is loaded go to the ViewDock window and use it's menu to view all the calculated properties regarding the rigid docked ligand by following the steps below.
Line 327: Line 337:
 
Your visualized structure should be similar to the image below.
 
Your visualized structure should be similar to the image below.
  
[[File:2nnq Rigid.png|thumb|center|1000px|Rigid docking results for 2nnq]]
+
[[File:20190304 rigid docking figure.jpg|thumb|center|1000px|3JQZ binding pocket with crystallographic ligand (blue) and rigid docked ligand (purple)]]
  
 
==Fixed Anchor Docking==
 
==Fixed Anchor Docking==
Line 333: Line 343:
 
  touch fixed.in
 
  touch fixed.in
 
Use the input file to perform fixed anchor docking
 
Use the input file to perform fixed anchor docking
  dock6 -i fixed.in
+
  dock6 -i fixed.in -o fixed.out
  
 
Use the following lines to answer the prompted questions as we did in rigid docking.
 
Use the following lines to answer the prompted questions as we did in rigid docking.
Line 352: Line 362:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
  ligand_atom_file                                            ../1.dockprep/2nnq_lig_withH.mol2
+
  ligand_atom_file                                            ../001.dockprep/3jqz_lig_withH.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
Line 358: Line 368:
 
  calculate_rmsd                                              yes
 
  calculate_rmsd                                              yes
 
  use_rmsd_reference_mol                                      yes
 
  use_rmsd_reference_mol                                      yes
  rmsd_reference_filename                                      ../1.dockprep/2nnq_lig_withH.mol2
+
  rmsd_reference_filename                                      ../001.dockprep/3jqz_lig_withH.mol2
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                no
 
  orient_ligand                                                no
Line 370: Line 380:
 
  grid_score_vdw_scale                                        1
 
  grid_score_vdw_scale                                        1
 
  grid_score_es_scale                                          1
 
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                      ../2.boxgrid/grid
+
  grid_score_grid_prefix                                      ../002.gridbox/grid
 
  multigrid_score_secondary                                    no
 
  multigrid_score_secondary                                    no
 
  dock3.5_score_secondary                                      no
 
  dock3.5_score_secondary                                      no
Line 400: Line 410:
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        2nnq_fad
+
  ligand_outfile_prefix                                        fixed.out
 
  write_orientations                                          no
 
  write_orientations                                          no
 
  num_scored_conformers                                        100
 
  num_scored_conformers                                        100
Line 408: Line 418:
 
  rank_ligands                                                no
 
  rank_ligands                                                no
  
Once docking is completed an output file will be generated. (2nnq_fad_scored.mol2) Follow the same method used in rigid docking to visualize the docked poses using Chimera. Once it is visualized, it should like the image below. Notice all the poses 50 generated are in the same cluster and standard RMSD is 0.75. These indicate that docking is very successful.  
+
Once docking is completed an output file will be generated. (fixed.out_scored.mol2) Follow the same method used in rigid docking to visualize the docked poses using Chimera. Once it is visualized, it should like the image below.  
  
[[File:2nnq ixed.png|thumb|center|1000px| Poses generated for fixed anchor docking]]
+
[[File:20190304 true FA docking figure 2.jpg|thumb|center|1000px| 3JQZ with crystallographic ligand (blue) and fixed anchor docked ligand (red)]]
  
 
==Flexible Docking==
 
==Flexible Docking==
Line 416: Line 426:
 
  touch flex.in
 
  touch flex.in
 
Use the created input file to perform flexible docking using DOCK6.
 
Use the created input file to perform flexible docking using DOCK6.
  dock6 -i flex.in
+
  dock6 -i flex.in -o flex.out
  
 
Answer the prompted questions using the following lines as we did in rigid and fixed anchor docking.
 
Answer the prompted questions using the following lines as we did in rigid and fixed anchor docking.
Line 435: Line 445:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
  ligand_atom_file                                            2nnq.lig.min_scored.mol2
+
  ligand_atom_file                                            3jqz.lig.min_scored.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
 
  read_mol_solvation                                          no
 
  read_mol_solvation                                          no
 
  calculate_rmsd                                              yes
 
  calculate_rmsd                                              yes
  use_rmsd_reference_mol                                      2nnq.lig.min_scored.mol2
+
  use_rmsd_reference_mol                                      yes 
 +
rmsd_reference_filename                            3jqz.lig.min_scored.mol2
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                yes
 
  orient_ligand                                                yes
 
  automated_matching                                          yes
 
  automated_matching                                          yes
  receptor_site_file                                          ../2.surface_spheres/selected_spheres.sph
+
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 
  max_orientations                                            1000
 
  max_orientations                                            1000
 
  critical_points                                              no
 
  critical_points                                              no
Line 458: Line 469:
 
  grid_score_vdw_scale                                        1
 
  grid_score_vdw_scale                                        1
 
  grid_score_es_scale                                          1
 
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                      ../3.boxgrid/grid
+
  grid_score_grid_prefix                                      ../003.gridbox/grid
 
  multigrid_score_secondary                                    no
 
  multigrid_score_secondary                                    no
 
  dock3.5_score_secondary                                      no
 
  dock3.5_score_secondary                                      no
Line 485: Line 496:
 
  simplex_restraint_min                                        no
 
  simplex_restraint_min                                        no
 
  atom_model                                                  all
 
  atom_model                                                  all
  vdw_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
+
  vdw_defn_file         /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
+
  flex_defn_file         /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
  flex_drive_file                                             /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
+
  flex_drive_file     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 
  ligand_outfile_prefix                                        flex.out
 
  ligand_outfile_prefix                                        flex.out
 
  write_orientations                                          no
 
  write_orientations                                          no
Line 496: Line 507:
 
Once flexible docking is completed an output mol2 file will be generated. (flex.out_scored.mol2). Use the visualization steps used in rigid and fixed anchor docking and study the properties of the docking results.  
 
Once flexible docking is completed an output mol2 file will be generated. (flex.out_scored.mol2). Use the visualization steps used in rigid and fixed anchor docking and study the properties of the docking results.  
  
[[File:2nnq Flex.png|thumb|center|1000px| Flexible docking results for 2nnq]]
+
[[File:20190304 FA docking figure 2.jpg|thumb|center|1000px| 3JQZ with crystallographic ligand (blue) and flexible docked ligand (green)]]
  
 
==Molecular Footprint==
 
==Molecular Footprint==
 
Molecular footprints can be used to determine how a ligand interacts with the receptor. Usually, the molecular footprint shows electrostatic interactions and Van der Waals interactions. Here, the molecular footprint will be used to determine how the ligand interacts with the receptor before and after minimization. To generate molecular footprints use following steps.
 
Molecular footprints can be used to determine how a ligand interacts with the receptor. Usually, the molecular footprint shows electrostatic interactions and Van der Waals interactions. Here, the molecular footprint will be used to determine how the ligand interacts with the receptor before and after minimization. To generate molecular footprints use following steps.
  
Go to directory 6.footprint
+
Go to directory 006.footprint
  
 
Generate an input file by typing;
 
Generate an input file by typing;
Line 511: Line 522:
 
  conformer_search_type                                        rigid
 
  conformer_search_type                                        rigid
 
  use_internal_energy                                          no
 
  use_internal_energy                                          no
  ligand_atom_file                                            2nnq_lig_min.mol2
+
  ligand_atom_file                                            ../004.dock/3jqz_lig_min_scored.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
Line 533: Line 544:
 
  footprint_similarity_score_secondary                        no
 
  footprint_similarity_score_secondary                        no
 
  fps_score_use_footprint_reference_mol2                      yes
 
  fps_score_use_footprint_reference_mol2                      yes
  fps_score_footprint_reference_mol2_filename                  2nnq_lig_with.mol2
+
  fps_score_footprint_reference_mol2_filename                  3jqz_lig_with.mol2
 
  fps_score_foot_compare_type                                  Euclidean
 
  fps_score_foot_compare_type                                  Euclidean
 
  fps_score_normalize_foot                                    no
 
  fps_score_normalize_foot                                    no
 
  fps_score_foot_comp_all_residue                              yes
 
  fps_score_foot_comp_all_residue                              yes
  fps_score_receptor_filename                                  ../1.dockprep/2nnq_rec_withH.mol2
+
  fps_score_receptor_filename                                  ../1.dockprep/3jqz_rec_withH.mol2
 
  fps_score_vdw_att_exp                                        6
 
  fps_score_vdw_att_exp                                        6
 
  fps_score_vdw_rep_exp                                        12
 
  fps_score_vdw_rep_exp                                        12
Line 557: Line 568:
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        footprint.out
+
  ligand_outfile_prefix                                        3jqz_footprint_min_cryst
 
  write_footprints                                            yes
 
  write_footprints                                            yes
 
  write_hbonds                                                yes
 
  write_hbonds                                                yes
Line 564: Line 575:
 
  rank_ligands                                                no   
 
  rank_ligands                                                no   
  
Once everything is successful these output files should be generated. (footprint.out_footprint_scored.txt, footprint.out_hbond_scored.txt, footprint.out_scored.mol2)
+
If successful, the following output files should be generated. (3jqz_footprint_min_cryst_footprint_scored.txt, 3jqz_footprint_min_cryst_hbond_scored.txt, 3jqz_footprint_min_cryst_scored.mol2)
  
Use a python script to visualize the molecular footprint. The script can be accessed in the previous DOCK tutorials. Once the script is used, the molecular footprint should be similar to the image below. Notice the large deviations in energy at different amino acid residues. Those residues contribute more towards, the interaction between the ligand and therefore can be identified as important towards the binding of new ligands which can replace the original ligand in the PDB file.
+
Use a python script to visualize the molecular footprint (read the txt files to pdf). Once the script has run, a pdf will be generated and the molecular footprint should be similar to the image below. Notice the deviations in energy at charged amino acid residues. Those residues contribute more electrostatically towards the interaction between the ligand and the receptor and therefore can be identified as important towards the binding of new ligands. In this case, the crystallographic ligand lidocaine has a +1 net charge.  
  
[[File:2nnqfootprint.png|thumb|center|1000px| Flexible docking results for 2nnq]]
+
[[File:Correctly named footprint for wiki figure.jpg|thumb|center|1000px| Molecular Footprint of crystal pose ligand (blue) and the energy minimised ligand (red)]]
  
 
= VI. Virtual Screen =  
 
= VI. Virtual Screen =  
 
Virtual screening is the method of screening a ligand library (drug-like molecules) to filter the best ligands which can bind to the binding site of a specific receptor. Here we will be using a ligand library which contains 25000 molecules to select the best ligands which can be used to replace the original ligand that the PDB file contained.
 
Virtual screening is the method of screening a ligand library (drug-like molecules) to filter the best ligands which can bind to the binding site of a specific receptor. Here we will be using a ligand library which contains 25000 molecules to select the best ligands which can be used to replace the original ligand that the PDB file contained.
  
Move to the directory 7.virtual_screen. Copy the ligand library to the same directory.  
+
'''Move to the directory 007_virtual_screen.'''
 +
 
 +
Copy the ligand library to the same directory.
 +
Copy '''VS_library_25k.mol2''' from the zzz.programs directory in project space. The VS_library_25k.mol2 file is an in house library of small molecules curated from ZINC.
  
 
Create a new input file for virtual screen.
 
Create a new input file for virtual screen.
Line 596: Line 610:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
  ligand_atom_file                                            small_ligand_library.mol2
+
  ligand_atom_file                                            VS_library_25K.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
 
  read_mol_solvation                                          no  
 
  read_mol_solvation                                          no  
  calculate_rmsd                                              yes
+
  calculate_rmsd                                              no
use_rmsd_reference_mol                                      yes
 
rmsd_reference_filename                                      ../1.dockprep/2nnq_lig_withH.mol2
 
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                yes
 
  orient_ligand                                                yes
 
  automated_matching                                          yes
 
  automated_matching                                          yes
  receptor_site_file                                          ../2.surface_spheres/selected_spheres.sph
+
  receptor_site_file                                          ../002_surface_spheres/selected_spheres.sph
 
  max_orientations                                            1000
 
  max_orientations                                            1000
 
  critical_points                                              no
 
  critical_points                                              no
Line 620: Line 632:
 
  grid_score_vdw_scale                                        1
 
  grid_score_vdw_scale                                        1
 
  grid_score_es_scale                                          1
 
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                      ../3.boxgrid/grid
+
  grid_score_grid_prefix                                      ../003_gridbox/grid
 
  multigrid_score_secondary                                    no
 
  multigrid_score_secondary                                    no
 
  dock3.5_score_secondary                                      no
 
  dock3.5_score_secondary                                      no
Line 655: Line 667:
 
  rank_ligands                                                no
 
  rank_ligands                                                no
  
Since the ligand library contains 25000 molecules, it is going to take a long time to complete the virtual screen. Therefore we are going to use the mpi version of DOCK to complete the virtual screen. Therefore you can either terminate the virtual screen which is already running by pressing ctrl+c or let it run.
+
Since the ligand library contains 25000 molecules, it is going to take a long time to complete the virtual screen. Therefore we are going to use the mpi version of DOCK to complete the virtual screen.  
 +
 
 +
You can terminate the virtual screen which is already running by pressing ctrl+c and follow the next steps to submit your job to the queue.
  
 
=VII.Virtual Screen (MPI)=
 
=VII.Virtual Screen (MPI)=
 
Until now we used the head node of the seawulf cluster. By using the mpi version of DOCK we will be using 4 processors that contain 28 nodes in each and it will complete the virtual screen quicker.  
 
Until now we used the head node of the seawulf cluster. By using the mpi version of DOCK we will be using 4 processors that contain 28 nodes in each and it will complete the virtual screen quicker.  
Move to a new directory. (8.virtual_screen_mpi)
+
 
Copy the input file and the ligand database file from 7.virtual_screen directory.
+
'''Move to a new directory (008.virtual_screen_mpi).'''
 +
 
 +
Copy the input file and the ligand database file from 007.virtual_screen directory.
  
 
To submit the job to the seawulf cluster we are using a new file. (virtual.sh)
 
To submit the job to the seawulf cluster we are using a new file. (virtual.sh)
Line 669: Line 685:
 
  #PBS -l nodes=4:ppn=28
 
  #PBS -l nodes=4:ppn=28
 
  #PBS -q long
 
  #PBS -q long
  #PBS -N 2nnq.virtual
+
  #PBS -N 3jqz.virtual
 
  #PBS -V
 
  #PBS -V
 
  cd $PBS_O_WORKDIR
 
  cd $PBS_O_WORKDIR
  mpirun -np 112 dock6.mpi -i virtual.in -o 2nnq.virtual.mpi.out
+
  mpirun -np 112 dock6.mpi -i virtual.in -o 3jqz.virtual.mpi.out
  
 
The virtual screen job can be submitted to the cluster using the following command.
 
The virtual screen job can be submitted to the cluster using the following command.
Line 684: Line 700:
 
Here we will use the docked molecules and perform a cartesian minimization of them.
 
Here we will use the docked molecules and perform a cartesian minimization of them.
  
Move to the directory 8.cartesianmin.
+
'''Move to the directory 009.cartesianmin.'''
  
 
Create a new input file for the minimization.
 
Create a new input file for the minimization.
Line 697: Line 713:
 
   internal_energy_rep_exp                                      12
 
   internal_energy_rep_exp                                      12
 
   internal_energy_cutoff                                      100.0
 
   internal_energy_cutoff                                      100.0
   ligand_atom_file                                             2nnq.virtualscreen_scored.mol2
+
   ligand_atom_file     ../008.virtual_screen_mpi/3jqz.virtualscreen_scored.mol2
 
   limit_max_ligands                                            no
 
   limit_max_ligands                                            no
 
   skip_molecule                                                no
 
   skip_molecule                                                no
Line 716: Line 732:
 
   continuous_score_primary                                    yes
 
   continuous_score_primary                                    yes
 
   continuous_score_secondary                                  no
 
   continuous_score_secondary                                  no
   cont_score_rec_filename                                      ../1.dockprep/2nnq.rec.charged.mol2
+
   cont_score_rec_filename                                      ../001.dockprep/3jqz.rec.charged.mol2 SHOULD WE GENERATE A CHARGED STRUCTURE??
 
   cont_score_att_exp                                          6
 
   cont_score_att_exp                                          6
 
   cont_score_rep_exp                                          12
 
   cont_score_rep_exp                                          12
Line 743: Line 759:
 
   simplex_restraint_min                                        no
 
   simplex_restraint_min                                        no
 
   atom_model                                                  all
 
   atom_model                                                  all
   vdw_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
+
   vdw_defn_file                     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
   flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
+
   flex_defn_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
   flex_drive_file                                             /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
+
   flex_drive_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
   ligand_outfile_prefix                                        2nnq.virtualscreen.minimized
+
   ligand_outfile_prefix                                        3jqz.virtualscreen.min
 
   write_orientations                                          no
 
   write_orientations                                          no
 
   num_scored_conformers                                        1
 
   num_scored_conformers                                        1
 
   rank_ligands                                                no
 
   rank_ligands                                                no
 +
 +
You can terminate the cartesian minimization which is already running by pressing ctrl+c and follow the next steps to submit your job to the queue.
  
 
Create a new submission script for the minimization.
 
Create a new submission script for the minimization.
  touch min.sh
+
  vi min.sh
  
 
   #!/bin/bash
 
   #!/bin/bash
Line 762: Line 780:
 
   cd $PBS_O_WORKDIR
 
   cd $PBS_O_WORKDIR
 
   dock6 -i min.in -o min.out
 
   dock6 -i min.in -o min.out
 +
 +
 +
The minimization can be submitted to the cluster using the following command.
 +
qsub min.sh
 +
 +
Type the following command to check the status of the job and other jobs that you submitted via your user login.
 +
qstat -u username
  
 
=IX.Rescoring Docked Molecules=
 
=IX.Rescoring Docked Molecules=
 
We would also like to rank our docked ligands and extract the 100 best ligands (which have the most negative, lowest scores). Footprint similarity, pharmacophore score, tanimoto score, the hungarian and the volume overlap score will all be used by Dock to rescore the virtual screen.
 
We would also like to rank our docked ligands and extract the 100 best ligands (which have the most negative, lowest scores). Footprint similarity, pharmacophore score, tanimoto score, the hungarian and the volume overlap score will all be used by Dock to rescore the virtual screen.
  
Move to the directory 9.rescore.
+
'''Move to the directory 010.rescore.'''
  
 
Create a new input file for the rescoring.
 
Create a new input file for the rescoring.
Line 777: Line 802:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
  ligand_atom_file                            ../8.cartesianmin/2nnq.virtualscreen.minimized_scored.mol2  
+
  ligand_atom_file                            ../009.cartesianmin/3jqz.virtualscreen.min_scored.mol2  
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
Line 783: Line 808:
 
  calculate_rmsd                                              no
 
  calculate_rmsd                                              no
 
  use_database_filter                                          no
 
  use_database_filter                                          no
  orient_ligand                                                no
+
  '''orient_ligand                                                no'''
 
  bump_filter                                                  no
 
  bump_filter                                                  no
 
  score_molecules                                              yes
 
  score_molecules                                              yes
 
  contact_score_primary                                        no
 
  contact_score_primary                                        no
 
  contact_score_secondary                                      no
 
  contact_score_secondary                                      no
  grid_score_primary                                          no
+
  '''grid_score_primary                                          no'''
 
  grid_score_secondary                                        no
 
  grid_score_secondary                                        no
 
  multigrid_score_primary                                      no
 
  multigrid_score_primary                                      no
Line 800: Line 825:
 
  pharmacophore_score_primary                                  no
 
  pharmacophore_score_primary                                  no
 
  pharmacophore_score_secondary                                no
 
  pharmacophore_score_secondary                                no
  descriptor_score_primary                                    yes
+
  '''descriptor_score_primary                                    yes'''
 
  descriptor_score_secondary                                  no
 
  descriptor_score_secondary                                  no
  descriptor_use_grid_score                                    no
+
  '''descriptor_use_grid_score                                    no'''
  descriptor_use_multigrid_score                              no
+
  '''descriptor_use_multigrid_score                              no'''
  descriptor_use_continuous_energy                            no
+
  '''descriptor_use_continuous_energy                            no'''
 
  descriptor_use_footprint_similarity                          yes
 
  descriptor_use_footprint_similarity                          yes
  descriptor_use_pharmacophore_score                          yes
+
  '''descriptor_use_pharmacophore_score                          yes'''
  descriptor_use_tanimoto                                      yes
+
  '''descriptor_use_tanimoto                                      yes'''
  descriptor_use_hungarian                                    yes
+
  '''descriptor_use_hungarian                                    yes'''
  descriptor_use_volume_overlap                                yes
+
  '''descriptor_use_volume_overlap                                yes'''
  descriptor_fps_use_footprint_reference_mol2                  yes
+
  '''descriptor_fps_use_footprint_reference_mol2                  yes'''
  descriptor_fps_footprint_reference_mol2_filename            ../4.dock/2nnq.lig.min_scored.mol2
+
  descriptor_fps_footprint_reference_mol2_filename            ../004.dock/3jqz.lig.min_scored.mol2
 
  descriptor_fps_foot_compare_type                            Euclidean
 
  descriptor_fps_foot_compare_type                            Euclidean
 
  descriptor_fps_normalize_foot                                no
 
  descriptor_fps_normalize_foot                                no
 
  descriptor_fps_foot_comp_all_residue                        yes
 
  descriptor_fps_foot_comp_all_residue                        yes
  descriptor_fps_receptor_filename                            ../1.dockprep/2nnq.rec.withH.charged.mol2
+
  '''descriptor_fps_receptor_filename                            ../001.dockprep/3jqz.rec.withH.charged.mol2'''
 
  descriptor_fps_vdw_att_exp                                  6
 
  descriptor_fps_vdw_att_exp                                  6
 
  descriptor_fps_vdw_rep_exp                                  12
 
  descriptor_fps_vdw_rep_exp                                  12
Line 824: Line 849:
 
  descriptor_fps_es_fp_scale                                  1
 
  descriptor_fps_es_fp_scale                                  1
 
  descriptor_fps_hb_fp_scale                                  0
 
  descriptor_fps_hb_fp_scale                                  0
  descriptor_fms_score_use_ref_mol2                            yes
+
  '''descriptor_fms_score_use_ref_mol2                            yes'''
  descriptor_fms_score_ref_mol2_filename                      ../4.dock/2nnq.lig.min_scored.mol2
+
  descriptor_fms_score_ref_mol2_filename                      ../004.dock/3jqz.lig.min_scored.mol2
 
  descriptor_fms_score_write_reference_pharmacophore_mol2      no
 
  descriptor_fms_score_write_reference_pharmacophore_mol2      no
 
  descriptor_fms_score_write_reference_pharmacophore_txt      no
 
  descriptor_fms_score_write_reference_pharmacophore_txt      no
Line 836: Line 861:
 
  descriptor_fms_score_match_proj_cutoff                      0.7071
 
  descriptor_fms_score_match_proj_cutoff                      0.7071
 
  descriptor_fms_score_max_score                              20
 
  descriptor_fms_score_max_score                              20
  descriptor_fingerprint_ref_filename                          ../4.dock/2nnq.lig.min_scored.mol2
+
  descriptor_fingerprint_ref_filename                          ../004.dock/3jqz.lig.min_scored.mol2
  descriptor_hungarian_ref_filename                            ../4.dock/2nnq.lig.min_scored.mol2
+
  descriptor_hungarian_ref_filename                            ../004.dock/3jqz.lig.min_scored.mol2
 
  descriptor_hungarian_matching_coeff                          -5
 
  descriptor_hungarian_matching_coeff                          -5
 
  descriptor_hungarian_rmsd_coeff                              1
 
  descriptor_hungarian_rmsd_coeff                              1
  descriptor_volume_reference_mol2_filename                    ../4.dock/2nnq.lig.min_scored.mol2
+
  descriptor_volume_reference_mol2_filename                    ../004.dock/3jqz.lig.min_scored.mol2
 
  descriptor_volume_overlap_compute_method                    analytical
 
  descriptor_volume_overlap_compute_method                    analytical
 
  descriptor_weight_fps_score                                  1
 
  descriptor_weight_fps_score                                  1
Line 851: Line 876:
 
  SASA_score_secondary                                        no
 
  SASA_score_secondary                                        no
 
  amber_score_secondary                                        no
 
  amber_score_secondary                                        no
  minimize_ligand                                              no
+
  '''minimize_ligand                                              no'''
 
  atom_model                                                  all
 
  atom_model                                                  all
 
  vdw_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 
  vdw_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
Line 858: Line 883:
 
  chem_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/chem.defn
 
  chem_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/chem.defn
 
  pharmacophore_defn_file          /gpfs/projects/AMS536/zzz.programs/dock6/parameters/ph4.defn
 
  pharmacophore_defn_file          /gpfs/projects/AMS536/zzz.programs/dock6/parameters/ph4.defn
  ligand_outfile_prefix                                        descriptor.output
+
  ligand_outfile_prefix                                        descriptor.out
  write_footprints                                            yes
+
  '''write_footprints                                            yes'''
  write_hbonds                                                yes
+
  '''write_hbonds                                                yes'''
 
  write_orientations                                          no
 
  write_orientations                                          no
 
  num_scored_conformers                                        1
 
  num_scored_conformers                                        1
 
  rank_ligands                                                no
 
  rank_ligands                                                no

Latest revision as of 11:58, 16 January 2020

This tutorial contains, a step by step by approach to dock a known ligand to a known receptor.

NOTE: JDB - Change all saved conformers to 50 for rigid/fad/flex and include a note on rank ordering the scores.

I. Introduction

DOCK

DOCK is a molecular docking program used in drug discovery. It was developed by Irwin D. Kuntz, Jr. and colleagues at UCSF (see UCSF DOCK). This program, given a protein binding site and a small molecule, tries to predict the correct binding mode of the small molecule in the binding site, and the associated binding energy. Small molecules with highly favorable binding energies could be new drug leads. This makes DOCK a valuable drug discovery tool. DOCK is typically used to screen massive libraries of millions of compounds against a protein to isolate potential drug leads. These leads are then further studied, and could eventually result in a new, marketable drug. DOCK works well as a screening procedure for generating leads, but is not currently as useful for optimization of those leads.

DOCK 6 uses an incremental construction algorithm called anchor and grow. It is described by a three-step process:

  1. Rigid portion of ligand (anchor) is docked by geometric methods.
  2. Non-rigid segments added in layers; energy minimized.
  3. The resulting configurations are 'pruned' and energy re-minimized, yielding the docked configurations.

If you are interested in reading more about DOCK 6 and the background to these processes, interested readers may reference the DOCK User's Manual.

3JQZ

This tutorial is based on the PDB file 3JQZ downloaded from the PDB Database. 3JQZ is the 3.3 Angstrom resolution crystal structure of Human serum albumin complexed with Lidocaine.

Hint: for docking practice you should identify a high resolution crystal structure with a well defined ligand binding site, not situated at the interface of two chains and with minimal active site waters.

Organization of Directories

Maintaining a clearly organised set of folders will be helpful in finding specific files, calling files and most importantly keeping track of everything you do. We recommend maintaining the following directory structure throughout the tutorial.

             000_files
             001_dockprep
             002_surface_spheres
             003_gridbox
             004_dock
             005
             006_footprint
             007_virtual_screen
             008_virtual_screen_mpi
             009_cartesianmin
             0010_rescore

II. Preparation of the ligand and receptor

Download the PDB file 3JQZ from the PDB database save it in the 000_files folder.

Checking the chosen structure

It can be helpful to read through the journal article associated with the PDB file to understand protonation states, charges, environmental conditions and other important information regarding the receptor and ligand under the crystallization conditions used to obtain the structure you are working with.

To do this;

 - Open Chimera 
 - File -> Fetch by ID -> Type 3JQZ into the 'PDB' box -> Fetch

You can now explore the crystal structure.

Hint: From the toolbar at the top of the Chimera window, click Actions -> Focus, to put your structure back in the middle of the screen.

Identify the main components of the model (receptor, ligand, solvent, surfactants, metal ions)

 - Carefully look to identify if there are any missing residues or missing loops, this can be the case where regions of the protein were flexible. For the purposes of learning the software, we want to steer clear of any systems with missing loops. The PDB 3JQZ, chosen for this tutorial, does not contain any missing loops or residues.

Preparation of receptor

 - Open the PDB file (3JQZ.pdb) using Chimera
 - Isolate the receptor using the select tool and delete tool in Chimera.
 - Save the isolated receptor as a mol2 file. (3jqz_rec_noH.mol2)
 - Open 3jqz_rec_noH.mol2 file again using Chimera and use the following instructions to prepare the receptor file to be used in DOCK.
          Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
          Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force filed available for standard residues. Here we used AMBER ff14SB)
          Save as a mol2 file. (3jqz_rec_withH.mol2)
 - If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor. 
          Tools -> Structure/Binding Analysis -> DockPrep

Preparation of ligand

 - Open the PDB file via Chimera.
 - Using Chimera, isolate the ligand, add H atoms, add charge and save it as a mol2 file by following the same steps followed for the receptor.
 - It is incredibly important both here and in all modeling experiments that you examine the ligand's protonation state and ring saturation. For this particular experiment, if you just take Chimera's protonation output you'll find it to be incorrect. Note that 6-membered ring structure will probably be protonated incorrectly - checking the structure in the reference shows that it should be aromatic, not saturated. You can fix this in Chimera using Tools > Structure Editing > Build Structure.
Left: The incorrect protonation of the ligand in 3JQZ. Right: The correct protonation.


Once all the files are prepared make sure to save the files in 001.dockprep folder.

III. Generating receptor surface and spheres

Preparation of DMS file

 - Open 3jqz_rec_noH.mol2 using chimera.
 - Action -> Surface -> Show
 - Tools -> Structure Editing -> Write DMS
 - Save the 3jqz_rec_noH.dms into 3.surface_spheres folder

Reopen the file and make sure the surface was generated.

Transfer all the folders created so far to Seawulf Cluster to be used in DOCK.

Generating spheres

 - Go to 003_surface_spheres folder
 - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file. 
3jqz_rec_noH.dms
R
X
0.0
4.0
1.4
3jqz_rec.sph

The first line 3jqz_rec_noH.dms specifies the input file. R indicates that spheres generated will be outside of the receptor surface. X specifies all the points will be used. 0.0 is the distance in angstroms and it will avoid steric clashes. 4.0 is the maximum surface radius of the spheres and 1.4 is the minimum radius in angstroms. The last line 3jqz_spheres.sph creates the sph file that contains clustered spheres.

Once the INSPH file is ready, type the following command to generate the spheres.

 sphgen -i INSPH -o OUTSPH

Once sphgen command is successful, 3jqz_spheres.sph file will be created. Open it up using Chimera along with 3jqz_rec_noH.mol2 file. You should get a similar output like the image below.

Some potential problems:

  • If you are unable to run the sphgen command because the command isn't found, ensure that DOCK is in your PATH.
  • If you run sphgen multiple times, be sure to delete any files generated by sphgen before running it again (ex. OUTSPH), as it isn't done automatically. You should just have '3jqz_rec_noH.dms' and 'INSPH' before running the command.
All the spheres generated for 3JQZ receptor

Selecting Spheres

Here we will be selecting the spheres which define the binding pocket of the ligand because we are trying to direct the ligand towards that binding site rather than all over the receptor. To select the spheres execute the following command.

 sphere_selector 3jqz_rec.sph ../001_dockprep/3jqz_lig_withH.mol2 10.0

This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand binding site are kept and all the other spheres are deleted in the image below.

3jqz receptor and selected spheres

IV. Generating box and grid

Generating box

Move to 003_gridbox directory

Create a new file showbox.in and write the following lines in the file.

Y
8.0
../002_surface_spheres/selected_spheres.sph
1
3jqz.box.pdb

Each of the above lines indicates that;

 We intend to generate a box
 The box length should be 8 Angstroms
 Use the selected_spheres file in the designated location
 The name of the file that contains the generated box.

Use the following command to generate the box.

showbox < showbox.in

If this step is successful, you should see a new file (3jqz.box.pdb) in 003_gridbox folder.

Box grid (blue) generated for 3JQZ receptor (gray surface) at the ligand binding site (green surface). Selected spheres are shown in pink.

Generating grid

Create a new file (grid.in)

Include the following lines in the grid.in file:


compute_grids                             yes
grid_spacing                              0.4
output_molecule                           no
contact_score                             no
energy_score                              yes
energy_cutoff_distance                    9999
atom_model                                a
attractive_exponent                       6
repulsive_exponent                        9
distance_dielectric                       yes
dielectric_factor                         4
bump_filter                               yes
bump_overlap                              0.75
receptor_file                             ../001_dockprep/3jqz_rec_withH.mol2
box_file                                  3jqz.box.pdb
vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
score_grid_prefix                         grid

Use the following command to generate the grid.

 grid -i grid.in -o gridinfo.out

If the command is successful, three new files will be generated. (gridinfo.out, grid.nrg, grid.bmp). Go through gridinfo.out file to make sure all the information about the receptor in the file matches with the original information of the receptor. (Eg:- Total charge, residues and their charges) If the information doesn't match, that means you have made an error in one of the steps that you followed so far.

V. Docking a single molecule for pose reproduction

Under this section, the ligand for 3jqz.pdb will be re-docked into the receptor. 3 Methods will be used to achieve this.

1. rigid docking

2. fixed anchor docking

3. flexible docking

Energy minimization

Before performing docking, here the ligand will be subjected to energy minimization in order to remove unfavorable clashes. These clashes will affect rigid docking because in rigid docking the ligand will be docked as the complete ligand, whereas in other docking methods the ligand will be broken into fragments and the ligand will be built step by step considering favorable orientations and torsion angles after each fragment addition.

Go to the directory 4.dock and a create a new file (touch min.in) and enter the command below.

 dock6 -i min.in

Answer the prompted questions using the answers given below or include the following lines in the min.in file at before entering the above command to avoid answering the questions manually.

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.dockprep/3jqz_lig_withH.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                yes      
rmsd_reference_filename                               ../001.dockprep/3jqz_lig_withH.mol2
use_database_filter                                          no
orient_ligand                                                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
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
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_random_seed                                          0
simplex_restraint_min                                        yes
simplex_coefficient_restraint                                10.0
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        3jqz.lig.min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no 

If the process is successful a new file (3jqz.lig.min_scored.mol2) will be generated. You can compare how is it changed from the initial structure by analyzing the RMSD value generated in the file. Visualize the new mol2 file along with receptor and the initial ligand mol2 files using Chimera to see the differences.

3jqz with crystal pose ligand (blue) and the energy minimised ligand (magenta)

Rigid Docking

Create an input file for rigid docking

touch rigid.in

Run dock using the created input file.

dock6 -i rigid.in -o rigid.out

Follow a similar approach as we did for minimization to answer the prompted questions by either answering them manually using the answers in the lines below or by including the following lines in the input file before running dock.

conformer_search_type                                        rigid
use_internal_energy                                          yes
ligand_atom_file                                             3jqz.lig.min_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                   yes    
rmsd_reference_filename                             3jqz.lig.min_scored.mol2
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
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
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_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        rigid.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Once rigid docking is successful, you will get an output file. (rigid.out_scored.mol2) Visualize the output file using Chimera by following steps to check the rigid docking success.

Open Chimera
File -> Open -> 3jqz_rec_withH.mol2
File -> Open -> 3jqz_lig_withH.mol2
Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (004.dock/rigid.out_scored.mol2)

Under File type, select Dock4,5 or 6

Once everything is loaded go to the ViewDock window and use it's menu to view all the calculated properties regarding the rigid docked ligand by following the steps below.

Column -> Show -> gridscore
Column -> Show -> HA_RMSDs
Follow the same steps to get all the properties

Your visualized structure should be similar to the image below.

3JQZ binding pocket with crystallographic ligand (blue) and rigid docked ligand (purple)

Fixed Anchor Docking

Create an input file for fixed anchor docking.

touch fixed.in

Use the input file to perform fixed anchor docking

dock6 -i fixed.in -o fixed.out

Use the following lines to answer the prompted questions as we did in rigid docking.

conformer_search_type                                        flex
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
write_fragment_libraries                                     no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.dockprep/3jqz_lig_withH.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../001.dockprep/3jqz_lig_withH.mol2
use_database_filter                                          no
orient_ligand                                                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                                       ../002.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/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        fixed.out
write_orientations                                           no
num_scored_conformers                                        100
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Once docking is completed an output file will be generated. (fixed.out_scored.mol2) Follow the same method used in rigid docking to visualize the docked poses using Chimera. Once it is visualized, it should like the image below.

3JQZ with crystallographic ligand (blue) and fixed anchor docked ligand (red)

Flexible Docking

Create a new input file for flexible docking. (flex.in)

touch flex.in

Use the created input file to perform flexible docking using DOCK6.

dock6 -i flex.in -o flex.out

Answer the prompted questions using the following lines as we did in rigid and fixed anchor docking.

conformer_search_type                                        flex
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
write_fragment_libraries                                     no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             3jqz.lig.min_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes  
rmsd_reference_filename                             3jqz.lig.min_scored.mol2
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/parameters/vdw_AMBER_parm99.defn
flex_defn_file          /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        flex.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


Once flexible docking is completed an output mol2 file will be generated. (flex.out_scored.mol2). Use the visualization steps used in rigid and fixed anchor docking and study the properties of the docking results.

3JQZ with crystallographic ligand (blue) and flexible docked ligand (green)

Molecular Footprint

Molecular footprints can be used to determine how a ligand interacts with the receptor. Usually, the molecular footprint shows electrostatic interactions and Van der Waals interactions. Here, the molecular footprint will be used to determine how the ligand interacts with the receptor before and after minimization. To generate molecular footprints use following steps.

Go to directory 006.footprint

Generate an input file by typing;

touch footprint.in

Use DOCK6 to generate footprints

dock6 -i footprint.in

Use the following lines to answer the prompted questions.

conformer_search_type                                        rigid
use_internal_energy                                          no
ligand_atom_file                                             ../004.dock/3jqz_lig_min_scored.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                           yes
footprint_similarity_score_secondary                         no
fps_score_use_footprint_reference_mol2                       yes
fps_score_footprint_reference_mol2_filename                  3jqz_lig_with.mol2
fps_score_foot_compare_type                                  Euclidean
fps_score_normalize_foot                                     no
fps_score_foot_comp_all_residue                              yes
fps_score_receptor_filename                                  ../1.dockprep/3jqz_rec_withH.mol2
fps_score_vdw_att_exp                                        6
fps_score_vdw_rep_exp                                        12
fps_score_vdw_rep_rad_scale                                  1
fps_score_use_distance_dependent_dielectric                  yes
fps_score_dielectric                                         4.0
fps_score_vdw_fp_scale                                        1
fps_score_es_fp_scale                                        1
fps_score_hb_fp_scale                                        0
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                                              no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        3jqz_footprint_min_cryst
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no  

If successful, the following output files should be generated. (3jqz_footprint_min_cryst_footprint_scored.txt, 3jqz_footprint_min_cryst_hbond_scored.txt, 3jqz_footprint_min_cryst_scored.mol2)

Use a python script to visualize the molecular footprint (read the txt files to pdf). Once the script has run, a pdf will be generated and the molecular footprint should be similar to the image below. Notice the deviations in energy at charged amino acid residues. Those residues contribute more electrostatically towards the interaction between the ligand and the receptor and therefore can be identified as important towards the binding of new ligands. In this case, the crystallographic ligand lidocaine has a +1 net charge.

Molecular Footprint of crystal pose ligand (blue) and the energy minimised ligand (red)

VI. Virtual Screen

Virtual screening is the method of screening a ligand library (drug-like molecules) to filter the best ligands which can bind to the binding site of a specific receptor. Here we will be using a ligand library which contains 25000 molecules to select the best ligands which can be used to replace the original ligand that the PDB file contained.

Move to the directory 007_virtual_screen.

Copy the ligand library to the same directory. Copy VS_library_25k.mol2 from the zzz.programs directory in project space. The VS_library_25k.mol2 file is an in house library of small molecules curated from ZINC.

Create a new input file for virtual screen.

touch virtual.in

Use the input file to perform virtual screen using DOCK6.

dock6 -i virtual.in

Use the following lines to answer the prompted questions.

conformer_search_type                                        flex
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
write_fragment_libraries                                     no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             VS_library_25K.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                                              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/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
ligand_outfile_prefix                                        virtual.out
write_orientations                                           no 
num_scored_conformers                                        1
rank_ligands                                                 no

Since the ligand library contains 25000 molecules, it is going to take a long time to complete the virtual screen. Therefore we are going to use the mpi version of DOCK to complete the virtual screen.

You can terminate the virtual screen which is already running by pressing ctrl+c and follow the next steps to submit your job to the queue.

VII.Virtual Screen (MPI)

Until now we used the head node of the seawulf cluster. By using the mpi version of DOCK we will be using 4 processors that contain 28 nodes in each and it will complete the virtual screen quicker.

Move to a new directory (008.virtual_screen_mpi).

Copy the input file and the ligand database file from 007.virtual_screen directory.

To submit the job to the seawulf cluster we are using a new file. (virtual.sh)

vim virtual.sh

Type the following lines in the new file.

#!/bin/bash
#PBS -l walltime=48:00:00
#PBS -l nodes=4:ppn=28
#PBS -q long
#PBS -N 3jqz.virtual
#PBS -V
cd $PBS_O_WORKDIR
mpirun -np 112 dock6.mpi -i virtual.in -o 3jqz.virtual.mpi.out

The virtual screen job can be submitted to the cluster using the following command.

qsub virtual.sh

Type the following command to check the status of the job and other jobs that you submitted via your user login.

qstat -u username

VIII.Cartesian Minimization

Here we will use the docked molecules and perform a cartesian minimization of them.

Move to the directory 009.cartesianmin.

Create a new input file for the minimization.

touch min.in

Use the input file to perform the cartesian minimization using DOCK6.

dock6 -i min.in

Use the following lines to answer the prompted questions.

  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file      ../008.virtual_screen_mpi/3jqz.virtualscreen_scored.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                                     yes
  continuous_score_secondary                                   no
  cont_score_rec_filename                                      ../001.dockprep/3jqz.rec.charged.mol2 SHOULD WE GENERATE A CHARGED STRUCTURE??
  cont_score_att_exp                                           6
  cont_score_rep_exp                                           12
  cont_score_rep_rad_scale                                     1
  cont_score_use_dist_dep_dielectric                           yes
  cont_score_dielectric                                        4.0
  cont_score_vdw_scale                                         1
  cont_score_es_scale                                          1
  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
  simplex_max_iterations                                       1000
  simplex_tors_premin_iterations                               0
  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_random_seed                                          0
  simplex_restraint_min                                        no
  atom_model                                                   all
  vdw_defn_file                     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
  flex_drive_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        3jqz.virtualscreen.min
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

You can terminate the cartesian minimization which is already running by pressing ctrl+c and follow the next steps to submit your job to the queue.

Create a new submission script for the minimization.

vi min.sh
  #!/bin/bash
  #PBS -l walltime=48:00:00
  #PBS -l nodes=1:ppn=28
  #PBS -q long
  #PBS -N cartesian min
  #PBS -V 
  cd $PBS_O_WORKDIR
  dock6 -i min.in -o min.out


The minimization can be submitted to the cluster using the following command.

qsub min.sh

Type the following command to check the status of the job and other jobs that you submitted via your user login.

qstat -u username

IX.Rescoring Docked Molecules

We would also like to rank our docked ligands and extract the 100 best ligands (which have the most negative, lowest scores). Footprint similarity, pharmacophore score, tanimoto score, the hungarian and the volume overlap score will all be used by Dock to rescore the virtual screen.

Move to the directory 010.rescore.

Create a new input file for the rescoring.

touch rescore.in

Use the input file to perform the cartesian minimization using DOCK6.

dock6 -i rescore.in
conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                            ../009.cartesianmin/3jqz.virtualscreen.min_scored.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_energy                             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_use_footprint_reference_mol2                  yes
descriptor_fps_footprint_reference_mol2_filename             ../004.dock/3jqz.lig.min_scored.mol2
descriptor_fps_foot_compare_type                             Euclidean
descriptor_fps_normalize_foot                                no
descriptor_fps_foot_comp_all_residue                         yes
descriptor_fps_receptor_filename                             ../001.dockprep/3jqz.rec.withH.charged.mol2
descriptor_fps_vdw_att_exp                                   6
descriptor_fps_vdw_rep_exp                                   12
descriptor_fps_vdw_rep_rad_scale                             1
descriptor_fps_use_distance_dependent_dielectric             yes
descriptor_fps_dielectric                                    4.0
descriptor_fps_vdw_fp_scale                                  1
descriptor_fps_es_fp_scale                                   1
descriptor_fps_hb_fp_scale                                   0
descriptor_fms_score_use_ref_mol2                            yes
descriptor_fms_score_ref_mol2_filename                       ../004.dock/3jqz.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.dock/3jqz.lig.min_scored.mol2
descriptor_hungarian_ref_filename                            ../004.dock/3jqz.lig.min_scored.mol2
descriptor_hungarian_matching_coeff                          -5
descriptor_hungarian_rmsd_coeff                              1
descriptor_volume_reference_mol2_filename                    ../004.dock/3jqz.lig.min_scored.mol2
descriptor_volume_overlap_compute_method                     analytical
descriptor_weight_fps_score                                  1
descriptor_weight_pharmacophore_score                        1
descriptor_weight_fingerprint_tanimoto                       -1
descriptor_weight_hungarian                                  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/parameters/vdw_AMBER_parm99.defn
flex_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
flex_drive_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
chem_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/chem.defn
pharmacophore_defn_file           /gpfs/projects/AMS536/zzz.programs/dock6/parameters/ph4.defn
ligand_outfile_prefix                                        descriptor.out
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no