2018 DOCK tutorial 1 with PDBID 2NNQ

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

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.


2NNQ

The tutorial will be based on the PDB file 2NNQ downloaded from the PDB Database. 2NNQ is the crystal structure for a human adipocyte fatty acid binding protein in complex with ((2'-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-3-biphenylyl)oxy)acetic acid.

Organization of Directories

Maintaining a clearly organized set of folders will be helpful in finding specific files, calling different files in input files and most importantly keeping track of everything you do. We would like to recommend to maintain the following set of files throughout the tutorial.

             0.files
             1.dockprep
             2.surface_spheres
             3.gridbox
             4.dock
             5.
             6.footprint
             7.virtual_screen
             8.virtual_screen_mpi
             9.cartesianmin
             10.rescore

II. Preparation of the ligand and receptor

Download the pdb file 2NNQ from PDB database save it in 0.files folder.

Checking the structure

 - Read the article related to the PDB file to understand protonation states, charges, environmental conditions and other important information regarding the receptor and the ligand.
 - Open the pdb file through chimera and look at the structure. 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 particular PDB file didn't contain any missing loops or missing residues)

Preparation of receptor

 - Open the PDB file (2NNQ.pdb) via Chimera
 - Isolate the receptor using select tool and delete tool in Chimera.
 - Save the isolated receptor as a mol2 file. (2nnq_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.
          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. (22nq_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.

Once all the files are prepared make sure to save the files in 1.dockprep folder.

III. Generating receptor surface and spheres

Preparation of DMS file

 - Open 2nnq_rec_noH.mol2 using chimera.
 - Action -> Surface -> Show
 - Tools -> Structure Editing -> Write DMS
 - Save the 2nnq_rec_withH.dms into 3.surface_spheres folder

Transfer all the folders created so far to seawulf cluster to be used in DOCK.

Generating spheres

 - Go to 2.surface_spheres folder
 - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file. 
2nnq_rec_noH.dms
R
X
0.0
4.0
1.4
2nnq_rec.sph

The first line 2nnq_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 2nnq_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, 2nnq_spheres.sph file will be created. Open it up using Chimera along with 2nnq_rec_noH.mol2 file. You should get a similar output like the image below.

All the spheres generated for 2nnq receptor

Selecting Spheres

Here we will be selecting the spheres which defines 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 type the following command.

 sphere_selector 2nnq_rec.sph ../1.dockprep/2nnq_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.

2nnq receptor and selected spheres

IV. Generating box and grid

Generating box

Move to 3.boxgrid directory Create a new file showbox.in and write the following lines in the file.

 Y
 8.0
 ../2.surface_spheres/selected_spheres.sph
 1
 2nnq.box.pdb

Each of the above lines indicate 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 generated box.

Use the following command to generate the box.

 showbox < showbox.in

If this step is successful, you should see a new file (2nnq.box.pdb) in 3.boxgrid folder.

Generating grid

Create a new file (grid.in)

Use the following command to generate the grid.

 grid -i grid.in -o gridinfo.out

Answer the prompted questions with the answers given below. (or you can use the following lines and include them in the grid.in file before entering the above command. If you do that these questions won't be prompted again. They will be automatically answered by grid.in file created)

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                        12
distance_dielectric                       yes
dielectric_factor                         4
bump_filter                               yes
bump_overlap                              0.75
receptor_file                             ../1.dockprep/2nnq_rec_withH.mol2
box_file                                  2nnq.box.pdb
vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
score_grid_prefix                         grid

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 2nnq.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 (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                                             ../1.dockprep/2nnq_lig_withH.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       ../1.dockprep/2nnq_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                                       ../3.boxgrid/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                                        2nnq.lig.min
write_orientations                                           no
num_scored_conformers                                        1
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.

2nnq_receptor with the original ligand and the minimized ligand

Rigid Docking

Create an input file for rigid docking

touch rigid.in

Run dock using the created input file.

dock6 -i rigid.in

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                                             2nnq.lig.min_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       2nnq.lig.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../2.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                                       ../3.boxgrid/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 -> 2nnq_rec_withH.mol2
File -> Open -> 2nnq_lig_withH.mol2
Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (rigid.out_scored.mol2)
In the loaded dialog box 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.

Rigid docking results for 2nnq

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

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                                             ../1.dockprep/2nnq_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                                      ../1.dockprep/2nnq_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                                       ../2.boxgrid/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                                        2nnq_fad
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. (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.

Poses generated for fixed anchor docking

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

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                                             2nnq.lig.min_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       2nnq.lig.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../2.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                                       ../3.boxgrid/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.

Flexible docking results for 2nnq

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 6.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                                             2nnq_lig_min.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                  2nnq_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/2nnq_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                                        footprint.out
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
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)

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.

Flexible docking results for 2nnq

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 7.virtual_screen. Copy the ligand library to the same directory.

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                                             small_ligand_library.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no 
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../1.dockprep/2nnq_lig_withH.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../2.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                                       ../3.boxgrid/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. Therefore you can either terminate the virtual screen which is already running by pressing ctrl+c or let it run.

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. (8.virtual_screen_mpi) Copy the input file and the ligand database file from 7.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 2nnq.virtual
#PBS -V
cd $PBS_O_WORKDIR
mpirun -np 112 dock6.mpi -i virtual.in -o 2nnq.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 8.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                                             2nnq.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                                      ../1.dockprep/2nnq.rec.noH.charged.mol2
  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                                        2nnq.virtualscreen.minimized
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

Create a new submission script for the minimization.

touch 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

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 9.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                            ../8.cartesianmin/2nnq.virtualscreen.minimized_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             ../4.dock/2nnq.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                             ../1.dockprep/2nnq.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                       ../4.dock/2nnq.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                          ../4.dock/2nnq.lig.min_scored.mol2
descriptor_hungarian_ref_filename                            ../4.dock/2nnq.lig.min_scored.mol2
descriptor_hungarian_matching_coeff                          -5
descriptor_hungarian_rmsd_coeff                              1
descriptor_volume_reference_mol2_filename                    ../4.dock/2nnq.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.output
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no