2019 DOCK tutorial 2 with PDBID 2P16

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This tutorial teaches you how to dock a drug molecule to a receptor.

/*This page is under construction*/

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.

Users of this tutorial may want to refer to a complete DOCK User Manual for more detailed instructions.

2P16

2P16 is the crystal structure of factor Xa in complex with the inhibitor APIXABAN (tradename Eliquis). You can get the pdb file from here 2P16. Click Download Files --> PDB format to download the PDB file to your directory. You may want to use a file editor like vi to check the content of a pdb file.

 vi 2p16.pdb

There are usually many useful informations other than structure data in a pdb file.

To quit vi, use

 :q

Organization of Directories

In this part, we are going to unify our nomenclature of directories. It's always helpful to maintain a good order of files.

First, cd into your working directory on SeaWulf.

Second, use mkdir command to create the following folders:

 mkdir 001.files 002.surface_spheres 003.gridbox 004.dock 005.virtual_screen 006.virtual_screen_mpi 007.cartesianmin 008.rescore

Then, you can use the following command to check the folders you just created

 ls -l

Files created in different steps will be saved to different folders accordingly in this tutorial.

II. Preparation of the ligand and receptor

Download the pdb file 2P16 from PDB database save it in 001.files folder.

Checking the structure

Read the article related to the PDB file [1] 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. You can do it by using the following command in shell

 chimera 2p16.pdb

or simply using the graphical user interface of chimera.

2p16.pdb

Identify the main components of the model (receptor, ligand, solvent, surfactants, metal ions). In our case, the protein contains two separate chains. You can also find a Mg ion shown in green sphere, and some water molecules. Because there is no hydrogen atom in the pdb file, the water molecules are only shown in oxygen atoms. By default, only 3 waters are shown. You can use Actions -> Atoms/Bonds -> Show to view all the waters.

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

In this part we are going to generate the receptor file and the ligand file that will going to be used in docking by chimera.

Clean the structure

As you have seen, the pdb file is not very clean. We need to do some modifications to make it suitable for docking studies. With the pdb file loaded by chimera, do Select -> Chain -> L to select the smaller chain. Use Actions -> Atoms/Bonds -> Delete to delete it. Then do Select -> Structure -> Ions to select the Mg ion. Use the same method to delete it.

Then do Select -> Structure -> protein to selected the protein. Hold Ctrl and Shift button, using mouse to left-click the ligand. This would include a atom or a bond of the ligand in to the selection. Then press up arrow to select to whole ligand. With Ctrl and Shift holding, left-click the water (oxygen atom) which is in the binding pocket. Now the selected structure should include the whole protein, the ligand and one oxygen atom. Do Select -> Invert (all models) to invert the selection. Now all the waters except one will be selected. Delete those molecules. Do File -> Save MOL2 to save the structure as 2p16_noh_com.mol2.

2p16_noh_com.mol2

Prepare the Ligand File

Do Select -> Structure -> ligand to select the ligand. Do Select -> Invert (all models), then delete the selected atoms. This will left the ligand molecule only. Save it as 2p16_noh_lig.mol2.

2p16_noh_lig.mol2

Prepare the Receptor File

Do Select -> Structure -> ligand to select the ligand. Then delete the selected atoms. This will left the protein and one water. Save it as 2p16_noh_rec.mol2.

2p16_noh_rec.mol2

Adding hydrogen and charge

Now we are going to add hydrogen atoms and charges to our receptor and ligand. Open 2p16_noh_rec.mol2 file again using Chimera and use the following instructions to prepare the receptor file to be used in DOCK.

 Tools -> surface/Binding Analysis -> Dock Prep

De-check the first row "Delete Solvent" (it's already done by yourself manually). Then click 'OK'. Keep pressing 'OK' in the next window. In the following window, make sure we are using AMBER ff14SB and AM1-BCC, then click 'OK'. It will add hydrogen atoms and partial charges to the receptor. Save it as 2p16_wh_rec.mol2.

2p16_wh_rec.mol2


Open 2p16_noh_lig.mol2, follow the same steps for rec file, add hydrogen and charges, and save the file as 2p16_wh_lig.mol2.

2p16_wh_lig.mol2

III. Generating receptor surface and spheres

Prepare the environment

 vi ~/.bashrc

add the following lines to the bashrc file

export DOCKHOME="/gpfs/projects/AMS536/zzz.programs/dock6/"
export PATH=$DOCKHOME/bin\:$PATH

save the file and quit. Then do

source ~/.bashrc

Preparation of DMS file

Open 2p16_noh_rec.mol2 using chimera. Do

 Action -> Surface -> Show
 Tools -> Structure Editing -> Write DMS

Save the structure as 2p16_noh_rec.dms.

Reopen the file using chimera and make sure the surface was generated.

Transfer all the files created so far to SeaWulf cluster to be used in DOCK. You can use the following command:

 scp 2p16* username@login.seawulf.stonybrook.edu:/gpfs/projects/AMS536/your_directory/

From now on, we will be working on the SeaWulf clusters instead of your own machine.

Generating spheres

Go to 002.surface_spheres folder Create a new input file to create spheres by

 vi INSPH

then type the following lines inside the file.

 ../001.files/2p16_noh_rec.dms
 R
 X
 0.0
 4.0
 1.4
 2p16_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, 2p16_rec.sph file will be created. Open it up using Chimera along with 2p16_noh_rec.mol2 file. You should get a similar output like the image below.

2p16 sphere

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 2p16_rec.sph ../001.files/2p16_noh_lig.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.

2p16 selected sphere

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
 2p16.box.pdb

Each of the above lines means:

Say 'Yes' to make a box
The boundary of the box is at least 8 Angstroms from any spheres
The location and file name of the sphere file
Use the first cluster of spheres (Only 1 cluster in this case)
The output filename


Use the following command to generate the box.

 showbox < showbox.in

If this step is successful, you should see a new file (2p16.box.pdb) in 003.gridbox folder.

2p16 selected sphere with box

Generating grid

In the same folder, create a new file

 vi grid.in

Copy the following lines in to the 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.files/2p16_wh_rec.mol2
box_file                                  2p16.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 grid.out

If the command is successful, three new files will be generated. (grid.out, grid.nrg, grid.bmp). Go through grid.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.

All the generated grid files are binary files. It's hard for us to visualize them.

Please note that we use 9 instead of 12 as the repulsive exponent in Lennard-Jones potential. This gives a 'softer' repulsion.

V. Docking a single molecule for pose reproduction

Under this section, the ligand for 2p16.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 004.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.files/2p16_wh_lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       ../001.files/2p16_wh_lig.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                                        2p16.lig.min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no 

If you made any errors during the entering of the above lines, just keep doing. After you have finished every questions, you can use vi to correct your min.in file. Then you are ready for the minimization. Do the following:

 dock6 -i min.in -o min.out

If the process is successful a new file (2p16.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.

2p16_receptor with the crystalized ligand(green) and the minimized ligand(pink).

We can also use footprint to visualize the changes after minimization. Under the same directory, create a new file

 touch footprint.in

then

dock6 -i footprint.in

Answer the following questions interactively

conformer_search_type                                        rigid
use_internal_energy                                          no
ligand_atom_file                                             ./2p16.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                  ../001.files/2p16_wh_lig.mol2
fps_score_foot_compare_type                                  Euclidean
fps_score_normalize_foot                                     no
fps_score_foot_comp_all_residue                              yes
fps_score_receptor_filename                                  ../001.files/2p16_wh_rec.mol2
fps_score_vdw_att_exp                                        6
fps_score_vdw_rep_exp                                        9
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                                        fps.min.output
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

If dock runs successfully, you can find the following files generated

fps.min.output_footprint_scored.txt
fps.min.output_hbond_scored.txt

In order to visualize the footprint, first we need to copy a python script into our directory, then run it:

cp /gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py ./
python plot_footprint_single_magnitude.py fps.min.output_footprint_scored.txt 50

A pdf document named fps.min.output_footprint_scored.txt.pdf will be generated. It looks like this:

footprint comparison with energy minimized (red) and the un-minimized ligand(blue).

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
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.files/2p16_wh_lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       ../001.files/2p16_wh_lig.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                               2
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                                        40
write_conformations                                          yes
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
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 -> 2p16_rec_withH.mol2
File -> Open -> 2p16_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

You can choose different conformations from the ViewDock window. The top one should be the conformation with lowest energy. Your visualized structure should be similar to the image below.

Rigid docking results for 2p16, Reference and docked ligand is colored with sky blue and pink, respectively
Rigid docking results for 2p16

Fixed Anchor Docking

Create an input file for fixed anchor docking.

touch fad.in

Use the input file to perform fixed anchor docking

dock6 -i fad.in

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

conformer_search_type                                        flex
write_fragment_libraries                                     no
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
use_internal_energy                                          yes
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.files/2p16_wh_lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../001.files/2p16_wh_lig.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                                        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                                        2p16_fad
write_orientations                                           no
num_scored_conformers                                        40
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Once docking is completed an output file will be generated. (2p16_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.

Poses generated for fixed anchor docking, Reference and docked ligand is colored with sky blue and pink, respectively
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 2p16, Reference and docked ligand is colored with sky blue and pink, respectively
Flexible docking results for 2p16

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 005.virtual_screen.

Copy the ligand library to the same directory.

cp /gpfs/projects/AMS536/zzz.programs/VS_library_5K.mol2 ./

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
write_fragment_libraries                                     no
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
use_internal_energy                                          yes
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             VS_library_5K.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../001.files/2p16_wh_lig.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                                        virtual.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no
                             

You can run this job on head node for a short while, just to make sure everything is fine. Then you can kill the job by Ctrl-C. We will use the mpi version to finish this calculation. See next section.

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)

vi 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 2p16.virtual
#PBS -V
cd $PBS_O_WORKDIR
mpirun -v -np 112 dock6.mpi -i virtual.in -o 2p16.virtual.mpi.out

Before submitting the job, please check the mpirun you are using

which mpirun

make sure you are using an intel mpi.

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.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