View source for 2019 DOCK tutorial 3 with PDBID 3JQZ

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 [http://dock.compbio.ucsf.edu/ 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:

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

If you are interested in reading more about DOCK 6 and the background to these processes, interested readers may reference the [http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm DOCK User's Manual].

===3JQZ===
This tutorial is based on the PDB file 3JQZ downloaded from the PDB Database.
[https://www.rcsb.org/structure/3JQZ 3JQZ] is the 3.3 Angstrom resolution crystal structure of Human serum albumin complexed with Lidocaine.

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

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

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

= II. Preparation of the ligand and receptor =

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

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

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

You can now explore the crystal structure. 

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

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

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

===Preparation of receptor===
  - Open the PDB file (3JQZ.pdb) using Chimera
  - Isolate the receptor using the select tool and delete tool in Chimera.
  - Save the isolated receptor as a mol2 file. (3jqz_rec_noH.mol2)

  - Open 3jqz_rec_noH.mol2 file again using Chimera and use the following instructions to prepare the receptor file to be used in DOCK.
           Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
           Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force filed available for standard residues. Here we used AMBER ff14SB)
           Save as a mol2 file. (3jqz_rec_withH.mol2)

  - If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor. 
           Tools -> Structure/Binding Analysis -> DockPrep

===Preparation of ligand===
  - Open the PDB file via Chimera.
  - Using Chimera, isolate the ligand, add H atoms, add charge and save it as a mol2 file by following the same steps followed for the receptor.
  - It is ''incredibly important'' both here and in all modeling experiments that you examine the ligand's protonation state and ring saturation. For this particular experiment, if you just take Chimera's protonation output you'll find it to be incorrect. Note that 6-membered ring structure will probably be protonated incorrectly - checking [https://www.sciencedirect.com/science/article/pii/S1047847710000912?via%3Dihub#aep-section-id28 the structure in the reference] shows that it should be aromatic, not saturated. You can fix this in Chimera using Tools > Structure Editing > Build Structure.

[[File:3jqz_good_bad_protonation.png|thumb|none|1000px|Left: The incorrect protonation of the ligand in 3JQZ. Right: The correct protonation.]]


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

=III. Generating receptor surface and spheres=
===Preparation of DMS file===
  - Open 3jqz_rec_noH.mol2 using chimera.
  - Action -> Surface -> Show
  - Tools -> Structure Editing -> Write DMS
  - Save the 3jqz_rec_noH.dms into 3.surface_spheres folder

Reopen the file and make sure the surface was generated. 

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

===Generating spheres===
  - Go to 003_surface_spheres folder
  - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file. 

 3jqz_rec_noH.dms
 R
 X
 0.0
 4.0
 1.4
 3jqz_rec.sph

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

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

  sphgen -i INSPH -o OUTSPH

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

Some potential problems:
*If you are unable to run the sphgen command because the command isn't found, ensure that DOCK is in your PATH.
*If you run sphgen multiple times, be sure to delete any files generated by sphgen before running it again (ex. OUTSPH), as it isn't done automatically. You should just have '3jqz_rec_noH.dms' and 'INSPH' before running the command.
[[File:Sphgen 3jqz BJ.png|thumb|center|1000px|All the spheres generated for 3JQZ receptor]]

===Selecting Spheres===

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

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

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

[[File:Sphgen 3jqz bj3.png|thumb|center|1000px|3jqz receptor and selected spheres]]

=IV. Generating box and grid=

===Generating box===
'''Move to 003_gridbox directory'''

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

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

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

Use the following command to generate the box.

 showbox < showbox.in

If this step is successful, you should see a new file (3jqz.box.pdb) in 003_gridbox folder.
[[File:Gridbox 3jqz bj1.png|thumb|center|1000px|Box grid (blue) generated for 3JQZ receptor (gray surface) at the ligand binding site (green surface). Selected spheres are shown in pink. ]]

===Generating grid===
Create a new file (grid.in) 

Include the following lines in the grid.in file:


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

Use the following command to generate the grid.
  grid -i grid.in -o gridinfo.out

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

=V. Docking a single molecule for pose reproduction =
Under this section, the ligand for 3jqz.pdb will be re-docked into the receptor. 3 Methods will be used to achieve this.

1. rigid docking 

2. fixed anchor docking 

3. flexible docking

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

Go to the directory 4.dock and a create a new file (touch min.in) and enter the command below.
  dock6 -i min.in

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

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../001.dockprep/3jqz_lig_withH.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                yes      
 rmsd_reference_filename                               ../001.dockprep/3jqz_lig_withH.mol2
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 simplex_max_iterations                                       1000
 simplex_tors_premin_iterations                               0
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_random_seed                                          0
 simplex_restraint_min                                        yes
 simplex_coefficient_restraint                                10.0
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        3jqz.lig.min
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no 

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

[[File:20190304 crystal and energy minimised ligand figure for wiki.jpg|thumb|center|1000px|3jqz with crystal pose ligand (blue) and the energy minimised ligand (magenta)]]

==Rigid Docking==
Create an input file for rigid docking
 touch rigid.in

Run dock using the created input file.
 dock6 -i rigid.in -o rigid.out

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

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 ligand_atom_file                                             3jqz.lig.min_scored.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                   yes    
 rmsd_reference_filename                             3jqz.lig.min_scored.mol2
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 simplex_max_iterations                                       1000
 simplex_tors_premin_iterations                               0
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        rigid.out
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

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

 Open Chimera
 File -> Open -> 3jqz_rec_withH.mol2
 File -> Open -> 3jqz_lig_withH.mol2
 Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (004.dock/rigid.out_scored.mol2)
Under File type, select Dock4,5 or 6

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

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

Your visualized structure should be similar to the image below.

[[File:20190304 rigid docking figure.jpg|thumb|center|1000px|3JQZ binding pocket with crystallographic ligand (blue) and rigid docked ligand (purple)]]

==Fixed Anchor Docking==
Create an input file for fixed anchor docking.
 touch fixed.in
Use the input file to perform fixed anchor docking
 dock6 -i fixed.in -o fixed.out

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

 conformer_search_type                                        flex
 user_specified_anchor                                        no
 limit_max_anchors                                            no
 min_anchor_size                                              5
 pruning_use_clustering                                       yes
 pruning_max_orients                                          1000
 pruning_clustering_cutoff                                    100
 pruning_conformer_score_cutoff                               100.0
 pruning_conformer_score_scaling_factor                       1.0
 use_clash_overlap                                            no
 write_growth_tree                                            no
 write_fragment_libraries                                     no
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../001.dockprep/3jqz_lig_withH.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       yes
 rmsd_reference_filename                                      ../001.dockprep/3jqz_lig_withH.mol2
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../002.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        fixed.out
 write_orientations                                           no
 num_scored_conformers                                        100
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no

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

[[File:20190304 true FA docking figure 2.jpg|thumb|center|1000px| 3JQZ with crystallographic ligand (blue) and fixed anchor docked ligand (red)]]

==Flexible Docking==
Create a new input file for flexible docking. (flex.in)
 touch flex.in
Use the created input file to perform flexible docking using DOCK6.
 dock6 -i flex.in -o flex.out

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

 conformer_search_type                                        flex
 user_specified_anchor                                        no
 limit_max_anchors                                            no
 min_anchor_size                                              5
 pruning_use_clustering                                       yes
 pruning_max_orients                                          1000
 pruning_clustering_cutoff                                    100
 pruning_conformer_score_cutoff                               100.0
 pruning_conformer_score_scaling_factor                       1.0
 use_clash_overlap                                            no
 write_growth_tree                                            no
 write_fragment_libraries                                     no
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             3jqz.lig.min_scored.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       yes  
 rmsd_reference_filename                             3jqz.lig.min_scored.mol2
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file         /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file          /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        flex.out
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no


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

[[File:20190304 FA docking figure 2.jpg|thumb|center|1000px| 3JQZ with crystallographic ligand (blue) and flexible docked ligand (green)]]

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

Go to directory 006.footprint

Generate an input file by typing;
 touch footprint.in
Use DOCK6 to generate footprints
 dock6 -i footprint.in

Use the following lines to answer the prompted questions.
 conformer_search_type                                        rigid
 use_internal_energy                                          no
 ligand_atom_file                                             ../004.dock/3jqz_lig_min_scored.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           yes
 footprint_similarity_score_secondary                         no
 fps_score_use_footprint_reference_mol2                       yes
 fps_score_footprint_reference_mol2_filename                  3jqz_lig_with.mol2
 fps_score_foot_compare_type                                  Euclidean
 fps_score_normalize_foot                                     no
 fps_score_foot_comp_all_residue                              yes
 fps_score_receptor_filename                                  ../1.dockprep/3jqz_rec_withH.mol2
 fps_score_vdw_att_exp                                        6
 fps_score_vdw_rep_exp                                        12
 fps_score_vdw_rep_rad_scale                                  1
 fps_score_use_distance_dependent_dielectric                  yes
 fps_score_dielectric                                         4.0
 fps_score_vdw_fp_scale                                        1
 fps_score_es_fp_scale                                        1
 fps_score_hb_fp_scale                                        0
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        3jqz_footprint_min_cryst
 write_footprints                                             yes
 write_hbonds                                                 yes
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no  

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

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

[[File:Correctly named footprint for wiki figure.jpg|thumb|center|1000px| Molecular Footprint of crystal pose ligand (blue) and the energy minimised ligand (red)]]

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

'''Move to the directory 007_virtual_screen.''' 

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

Create a new input file for virtual screen.
 touch virtual.in
Use the input file to perform virtual screen using DOCK6.
 dock6 -i virtual.in

Use the following lines to answer the prompted questions.
 conformer_search_type                                        flex
 user_specified_anchor                                        no
 limit_max_anchors                                            no
 min_anchor_size                                              5
 pruning_use_clustering                                       yes
 pruning_max_orients                                          1000
 pruning_clustering_cutoff                                    100
 pruning_conformer_score_cutoff                               100.0
 pruning_conformer_score_scaling_factor                       1.0
 use_clash_overlap                                            no
 write_growth_tree                                            no
 write_fragment_libraries                                     no
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             VS_library_25K.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no 
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002_surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no 
 bump_filter                                                  no 
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003_gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no 
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0 
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        virtual.out
 write_orientations                                           no 
 num_scored_conformers                                        1
 rank_ligands                                                 no

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

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

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

'''Move to a new directory (008.virtual_screen_mpi).'''

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

To submit the job to the seawulf cluster we are using a new file. (virtual.sh)
 vim virtual.sh
Type the following lines in the new file.
 #!/bin/bash
 #PBS -l walltime=48:00:00
 #PBS -l nodes=4:ppn=28
 #PBS -q long
 #PBS -N 3jqz.virtual
 #PBS -V
 cd $PBS_O_WORKDIR
 mpirun -np 112 dock6.mpi -i virtual.in -o 3jqz.virtual.mpi.out

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

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

=VIII.Cartesian Minimization=

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

'''Move to the directory 009.cartesianmin.'''

Create a new input file for the minimization.
 touch min.in
Use the input file to perform the cartesian minimization using DOCK6.
 dock6 -i min.in

Use the following lines to answer the prompted questions.

   conformer_search_type                                        rigid
   use_internal_energy                                          yes
   internal_energy_rep_exp                                      12
   internal_energy_cutoff                                       100.0
   ligand_atom_file      ../008.virtual_screen_mpi/3jqz.virtualscreen_scored.mol2
   limit_max_ligands                                            no
   skip_molecule                                                no
   read_mol_solvation                                           no
   calculate_rmsd                                               no
   use_database_filter                                          no
   orient_ligand                                                no
   bump_filter                                                  no
   score_molecules                                              yes
   contact_score_primary                                        no
   contact_score_secondary                                      no
   grid_score_primary                                           no
   grid_score_secondary                                         no
   multigrid_score_primary                                      no
   multigrid_score_secondary                                    no
   dock3.5_score_primary                                        no
   dock3.5_score_secondary                                      no
   continuous_score_primary                                     yes
   continuous_score_secondary                                   no
   cont_score_rec_filename                                      ../001.dockprep/3jqz.rec.charged.mol2 SHOULD WE GENERATE A CHARGED STRUCTURE??
   cont_score_att_exp                                           6
   cont_score_rep_exp                                           12
   cont_score_rep_rad_scale                                     1
   cont_score_use_dist_dep_dielectric                           yes
   cont_score_dielectric                                        4.0
   cont_score_vdw_scale                                         1
   cont_score_es_scale                                          1
   footprint_similarity_score_secondary                         no
   pharmacophore_score_secondary                                no
   descriptor_score_secondary                                   no
   gbsa_zou_score_secondary                                     no
   gbsa_hawkins_score_secondary                                 no
   SASA_score_secondary                                         no
   amber_score_secondary                                        no
   minimize_ligand                                              yes
   simplex_max_iterations                                       1000
   simplex_tors_premin_iterations                               0
   simplex_max_cycles                                           1
   simplex_score_converge                                       0.1
   simplex_cycle_converge                                       1.0
   simplex_trans_step                                           1.0
   simplex_rot_step                                             0.1
   simplex_tors_step                                            10.0
   simplex_random_seed                                          0
   simplex_restraint_min                                        no
   atom_model                                                   all
   vdw_defn_file                     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
   flex_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
   flex_drive_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
   ligand_outfile_prefix                                        3jqz.virtualscreen.min
   write_orientations                                           no
   num_scored_conformers                                        1
   rank_ligands                                                 no

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

Create a new submission script for the minimization.
 vi min.sh

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


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

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

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

'''Move to the directory 010.rescore.'''

Create a new input file for the rescoring.
 touch rescore.in
Use the input file to perform the cartesian minimization using DOCK6.
 dock6 -i rescore.in

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                            ../009.cartesianmin/3jqz.virtualscreen.min_scored.mol2 
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 '''orient_ligand                                                no'''
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 '''grid_score_primary                                           no'''
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_primary                                  no
 pharmacophore_score_secondary                                no
 '''descriptor_score_primary                                     yes'''
 descriptor_score_secondary                                   no
 '''descriptor_use_grid_score                                    no'''
 '''descriptor_use_multigrid_score                               no'''
 '''descriptor_use_continuous_energy                             no'''
 descriptor_use_footprint_similarity                          yes
 '''descriptor_use_pharmacophore_score                           yes'''
 '''descriptor_use_tanimoto                                      yes'''
 '''descriptor_use_hungarian                                     yes'''
 '''descriptor_use_volume_overlap                                yes'''
 '''descriptor_fps_use_footprint_reference_mol2                  yes'''
 descriptor_fps_footprint_reference_mol2_filename             ../004.dock/3jqz.lig.min_scored.mol2
 descriptor_fps_foot_compare_type                             Euclidean
 descriptor_fps_normalize_foot                                no
 descriptor_fps_foot_comp_all_residue                         yes
 '''descriptor_fps_receptor_filename                             ../001.dockprep/3jqz.rec.withH.charged.mol2'''
 descriptor_fps_vdw_att_exp                                   6
 descriptor_fps_vdw_rep_exp                                   12
 descriptor_fps_vdw_rep_rad_scale                             1
 descriptor_fps_use_distance_dependent_dielectric             yes
 descriptor_fps_dielectric                                    4.0
 descriptor_fps_vdw_fp_scale                                  1
 descriptor_fps_es_fp_scale                                   1
 descriptor_fps_hb_fp_scale                                   0
 '''descriptor_fms_score_use_ref_mol2                            yes'''
 descriptor_fms_score_ref_mol2_filename                       ../004.dock/3jqz.lig.min_scored.mol2
 descriptor_fms_score_write_reference_pharmacophore_mol2      no
 descriptor_fms_score_write_reference_pharmacophore_txt       no
 descriptor_fms_score_write_candidate_pharmacophore           no
 descriptor_fms_score_write_matched_pharmacophore             no
 descriptor_fms_score_compare_type                            overlap
 descriptor_fms_score_full_match                              yes
 descriptor_fms_score_match_rate_weight                       5.0
 descriptor_fms_score_match_dist_cutoff                       1.0
 descriptor_fms_score_match_proj_cutoff                       0.7071
 descriptor_fms_score_max_score                               20
 descriptor_fingerprint_ref_filename                          ../004.dock/3jqz.lig.min_scored.mol2
 descriptor_hungarian_ref_filename                            ../004.dock/3jqz.lig.min_scored.mol2
 descriptor_hungarian_matching_coeff                          -5
 descriptor_hungarian_rmsd_coeff                              1
 descriptor_volume_reference_mol2_filename                    ../004.dock/3jqz.lig.min_scored.mol2
 descriptor_volume_overlap_compute_method                     analytical
 descriptor_weight_fps_score                                  1
 descriptor_weight_pharmacophore_score                        1
 descriptor_weight_fingerprint_tanimoto                       -1
 descriptor_weight_hungarian                                  1
 descriptor_weight_volume_overlap_score                       -1
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 '''minimize_ligand                                              no'''
 atom_model                                                   all
 vdw_defn_file                     /gpfs/projects/AMS536/zzz.programs/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex.defn
 flex_drive_file                   /gpfs/projects/AMS536/zzz.programs/dock6/parameters/flex_drive.tbl
 chem_defn_file                    /gpfs/projects/AMS536/zzz.programs/dock6/parameters/chem.defn
 pharmacophore_defn_file           /gpfs/projects/AMS536/zzz.programs/dock6/parameters/ph4.defn
 ligand_outfile_prefix                                        descriptor.out
 '''write_footprints                                             yes'''
 '''write_hbonds                                                 yes'''
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no