View source for 2024 DOCK tutorial 1 with PDBID 2ITO

= Introduction =

When the function of a protein gets involved in multiple diseases, it becomes a target protein. Pharmaceutical companies target this protein by developing small-molecule inhibitors to prevent its function. Earlier scientists used to identify these molecules by High Throughput Screening where these small molecules would be chemically synthesized and exposed to cells to determine their activity. This method was time-consuming and expensive to employ. When Virtual Screening came into the market it eased the whole process of screening the activity of thousands of molecules using computational algorithms. 

A virtual screen is set up by preparing the structure of the receptor by adding appropriate charges and hydrogens, which is also done for the ligand that we want to dock the receptor to. A footprint is then generated to identify the receptor residues where the ligand can bind. A molecular modeling program named DOCK is used here to dock our protein of interest with ligand. 

DOCK employs algorithms to bring together the ligand and the receptor together. This tutorial would involve preparing a protein and ligand for docking by downloading a protein-ligand complex from RCSB PDB database (https://www.rcsb.org/), which in this tutorial is 2ITO, which is a crystal structure of G719S mutation in EGFR Kinase domain, in complex with Iressa (an EGFR inhibitor)

These website tutorials are useful if you are unfamiliar with the following tools:
*[http://ringo.ams.stonybrook.edu/index.php/Unix UNIX]
*[http://ringo.ams.stonybrook.edu/index.php/Chimera Chimera]
*[http://ringo.ams.stonybrook.edu/index.php/Vi vi] 

This tutorial is for PDBID 2ITO. Replace any reference to 2ITO with the protein of your choice.

== Learning Objectives ==
* Download Protein of interest from RCSB PDB database 
* Step-by-step guide to use DOCK6.10 for docking protein of interest with a small-molecule ligand
* Analyse docking results to identify the most favorable binding geometry between the protein and ligand

== Setting Up Your Environment ==
Before beginning the docking process, the following directory structure should be set up in the seawulf cluster under the desginated group name:

[[File: 2ito_directory_structure.png|thumb|center]]

== Downloading a protein from the PDB database ==
Download a protein complex from [https://rcsb.org/ RCSB PDB database]. As part of this tutorial, the complex 2ITO will be used. On the main page, to the right of the top, there will be a search bar in which you will type in the PDBID and press Enter.

[[File:2itopdbsearchpage.png|center|thumb|upright=2.0]]

The resulting protein complex would be in display.

[[File:2itodisplaypage.png|center|500px]]


Towards the right hand side, click on Download Files, and select "Biological Assembly 1 (PDB - gz)"

[[File:2itodownload.png|center|200px]]

The file will be saved in the local directory.

= Preparation of the ligand and protein =
The following steps would demonstrate the preparation of the protein and the ligand which would be used by DOCK6.10. The steps in this section will be performed using Chimera. These steps are essential to perform so as to ensure accurate analysis of the data post the docking step. This section will demonstrate:
#Evaluating the structure to determine any missing loops
#Adding Missing Loops using Modeller
#Prepare the protein structure
#Prepare the ligand structure

=== Evaluating the Structure===
Use Chimera to open the downloaded PDB structure. While visualizing the structure in Chimera, note for any missing loops present, which is indicated by dashed lines in the structure. 
<gallery mode="packed">
File:2ito_missingloop.png|
File:2ito_missingloop2.png|
</gallery>



To fix the missing sections, go to Tools → Surface Editing → Model/Refine Loops.  Two dialogue boxes will appear:
[[File: 2ito_sequences.png|thumb|center]]

this box shows the sequence of amino acids in the PDB structure. 

The second box has the required inputs to give Chimera so it can re-model the missing loops: 
[[File: 2ito_db.png|thumb|center]]

in this box choose 'non-terminal missing structure' and click 'Apply'.  The progress of Modeller can be monitored in the lower left hand corner of the display.  Once it has finished, another dialogue box will appear showing the five choices of models for the missing sections.
[[File:2ito_lc.png|thumb|center]]

When clicking on each of the results, the fixed missing section will show up.  Decide which one you want to keep, highlight it and save the file by choosing File → Save PDB.  In the dialogue box, be sure to give this file a new name so as not to overwrite the original 2ito.pdb file, something similar to 2ito_loops_fixed.pdb.  In the 'Save models' section, choose the model number chosen. To confirm that everything has worked correctly, close the current session in Chimera and open 2ito_loops_fixed.pdb. Now no dashed lines should be seen and only the structure with the fixed loops.
[[File:2ito_fixed.png|thumb|center]] 
[[File:2ito_fixed2.png|thumb|right]]




===Preparing the Protein file===
The first step in preparing the protein is to get the protein structure alone in a .pdb file.  To do this:
#Select an atom on the protein
#Press the up arrow until the entire protein is selected
#Go to Select → Invert (all models).  This will change the selection from the protein to everything else in the structure
#Go to Actions → Atoms/Bonds → Delete
#Save the structure with a new file name (i.e. 2ito_protein_only.pdb).  Your pdb file will now look similar to this:

At this point we also need to generate a .mol2 file for the structure in this state.  Go to File → Save Mol2 and give the file a descriptive name such as 2ito_protein_no_hydrogens_no_charges.mol2

There are now two more steps necessary for its preparation:
#Adding hydrogens
#Adding charge

To add hydrogen atoms click on: Tools → Structure Editing → AddH.  This command will cause the following dialogue box to appear:


Click 'OK'.  When the program is finished, the bottom left-hand side will say "Hydrogens added" but you won't see any change in the structure.  It's good practice to make sure that the program worked properly by showing the atoms at a small area of the protein and ensuring hydrogens were added.  To do with we're going to use the 'zone' command which is quite useful:
#Click on one atom anywhere on the protein
#Click on Select → Zone.  This will cause the following dialogue box to appear:


make sure to click the same options as shown above and click on 'OK'.  Go to Actions → Atoms/Bonds → show and the atoms in the atoms in the selected area will be shown.  If the hydrogen atoms were successfully added you'll see structure similar to:

and you can see the white ends to the atoms which are the hydrogens.  The final step is to add charges to the protein.  Before you do this you should clear your selection by clicking on Select → Clear Selection.  To add charges click on Tools → Structure Editing → Add Charge and the following dialogue box will show up:


Click on 'OK' and once the program is finished the bottom left hand corner will tell you what the total charge of the structure is.  This number should be an integer.  Your protein structure is now completely prepped and ready for docking.  The final step is to save two files, a .pdb of the structure in this state and a .mol2 file.  Simply go to File → Save PDB and choose a filename such as 2ito_charges.pdb; then go to File → Save Mol2 and choose a filename such as 2ito_protein_with_charges.mol2.

===Preparing the Ligand File===


===Final Steps===

=Creating the Protein Binding Site Surface=

===Creating the Required Surface (DMS) File===

===Generating Spheres for the Binding Site===

 
===Binding Site Spheres===

=Box and Grid Generation=

The next step in the docking process is the box and grid generation. Instead of generating the energy interactions between all atoms of the protein and ligand, DOCK gets around this computationally expensive step by focusing solely on the area of interest around the protein-ligand binding pocket. Atoms that are far away from this site can be thought to have negligible impact on the energy interactions. 

===Generating the Box===

The first step is to create a box around the active site. Create a DOCK program called showbox. Do this in the 003.gridbox directory.

  vi showbox.in

This creates a new file called showbox.in and open it in vi. You will then need to type the following in the file:

  Y 
  8.0
  ../002.surface_spheres/selected_spheres.sph
  1
  2ITO.box.pdb

This will automoatically construct boxes to enclose the spheres, give 8.0 Angstroms from the spheres to draw the box, tells the file where to find the sphere file that we had previously generated, the cluster number, and the output filename.

To run this file, type the following to receive 2ITO.box.pdb in your directory

  showbox < showbox.in

After running this script, 2ITO.box.pdb should show up in your directory.

===Generating the Grid===

The next step is to generate the grid. Create a DOCK program called grid.

  vi grid.in

Use the following code:

  allow_non_integral_charges                no
  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
  allow_non_integral_charges                yes
  bump_filter                               yes
  bump_overlap                              0.75
  receptor_file                             ../001.structure/2ITO_protein_rec_final.mol2
  box_file                                  2ITO.box.pdb
  vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
  score_grid_prefix                         grid

Once this file is saved, run it with the following code:
  grid -i grid.in -o 2ITOGridInfo.out

This will take a while to run and after it finishes, you should have the following in your repository:
* grid.bmp
* grid.nrg
* 2ITOGridInfo.out

=Energy Minimization=

To allow DOCK to accurately calculate the energy interactions between atoms of the protein and ligand, it is important to make sure the ligand is in the lowest energy state before docking it into the binding site of the protein.

==Ligand Minimization==

Move on to the 004.energy_min directory. Create a DOCK program called min:

  vi min.in

Use the following code:

  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../001.structure/2ITO_ligand_Gefitinib_final.mol2
  limit_max_ligands                                            no
  skip_molecule                                                no
  read_mol_solvation                                           no
  calculate_rmsd                                               yes
  use_rmsd_reference_mol                                       yes
  rmsd_reference_filename                                      ../001.structure/2ITO_ligand_Gefitinib_final.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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        2ITO.lig.min
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

Run with the following:
  dock6 -i min.in -o min.out

2 new files will be  outputted to your directory:
* min.out
* 2ITO.lig.min.mol2

The mol2 file should look like this:

[[File:2ITO.lig.min.mol2.png|thumb|center]]

The 2ITO.ligand from PDB with hydrogen and charges is brown and the newly generated 2ITO.lig.min.mol2 is blue.
 

==Footprint Analysis==

Footprint analysis allows us to visualize the electrostatic and VDW interactions between the ligand and protein. This is useful when designing new ligands to compare the set of energetics.

In the 005.footprint directory, create a DOCK program called footprint.

  vi footprint.in

Use the following code: 

  conformer_search_type                                        rigid
  use_internal_energy                                          no
  ligand_atom_file                                             2ITO.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.structure/2ITO_ligand_Gefitinib_final.mol2
  fps_score_foot_compare_type                                  Euclidean
  fps_score_normalize_foot                                     no
  fps_score_foot_comp_all_residue                              yes
  fps_score_receptor_filename                                  ../001.structure/2ITO_protein_rec_final.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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        footprint.out
  write_footprints                                             yes
  write_hbonds                                                 yes
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no


Run the following to generate the footprint:

  dock6 -i footprint.in

3 files will be added to this inventory
* 2ITO_footprint.out_footprint_scored.txt
* 2ITO_footprint.out_hbond_scored.txt
* 2ITO_footprint.out_scored.mol2

Now we need to visualize the results with a python script. Copy it from the class directory to your 005.footprint directory with the following:

  cp /gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py .

Load up python with the following:

  module load py/2.7.15

Then run the script with the following:

  python plot_footprint_single_magnitude.py 2ITO_footprint.out_footprint_scored.txt  50

Once this script is complete, footprint.out_footprint_scored.txt.pdf should show up in your directory. Copy it onto your local computer to open it up. The file should look like this:


[[File:2ITO.footprint.png|thumb|center]]

=DOCK=

There are 3 types of docking that we will be covering in this tutorial. The types and key differences are displayed below:

* Rigid Docking - conformation search: rigid, orient: yes
* Fixed Anchor Docking - conformation search: flex, orient: no
* Flexible Docking - conformation search: flex, orient: yes

===Rigid Docking===

Rigid docking treats the ligand as a rigid structure and fits it the best it can into the binding site of the protein. We will be working in the 006.rigid_docking directory.

Create the rigid.in file using the command:

   vi rigid.in

The following lines need to be typed into the file: 

  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             2ITO.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                                      2ITO.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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        rigid.out
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

This will take a few minutes to complete but once it is done, you should see the following files in your directory. 

* rigid.out_scored.mol2
* rigid.out

Move the .mol2 file to your local computer and visualize it in Chimera. Compare it with the lig.min_scored,mol2 and you should see the following:

[[File:fixed.out_scored.mol2.png|thumb|center]]

The 2ITO.lig.min_scored.mol2 is blue and the newly generated fixed.out_scored.mol2 is brown.

Look at the rigid.out file. This will allow you to look at the scoring:

  Molecule: 2ito.pdb1
  Elapsed time for docking:      26 seconds
  Anchors:               1
  Orientations:          1000
  Conformations:         992
                          Grid_Score:          -72.913971
                     Grid_vdw_energy:          -63.204918
                      Grid_es_energy:           -9.709055
           Internal_energy_repulsive:           11.595561
  1 Molecules Processed

===Fixed Anchor Docking===

In this session, we will be showing the tutorial for Fixed Anchor Docking. For this mode of docking, the algorithm would first identify the largest fragment extracted from original ligand input. Noted that, the fragments are segmented based on the rotatable bonds of the ligands. Starting from this fragment pose, the algorithm will use it as an anchor to continuously add segments until the whole original ligand pose has been fully re-grown. Noted that when each fragment is added, it would be oriented for a pose that is energetically minimal. Comparing this method to the previous rigid docking mode, fixed anchor docking offer flexibility to the side chain while still being able to keep the general location of the ligand’s binding site as it fixes the anchor position.

Please navigate to the directory designated for this docking mode: 007.fixed_anchor_docking. Here, we can start create the input parameters, file name – fixed.in:

  vi fixed.in

The following is the input parameter for fixed anchor docking. While you can copy and paste this into the body of your script, please make sure to change the input files directory (ligand and protein structure folder) to your personal directory. Also it should be a good practice to make sure the directories for the dock parameters should also be up-to-date.

  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                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../004.energy_min/2tio.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                                      ../004.energy_min/2tio.lig.min_scored.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 
  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
  simplex_trans_step                                           1
  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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        2tio_fixed_output
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

Now, you are ready to run dock6 implementing the above input parameters:

   dock6 -i fixed.in -o fixed.out

Dock6 should be running for a couple of minutes. If the program finished right away, it would be recommended that you should check the output file to see if there are any reported errors. Successful run should give you two additional files as followed:

*fixed.out
*2tio_fixed_output_scored.mol2

The mol2 file containing the docked ligand could be viewed in ChimeraX under ViewDock. To compare with the reference pose, this mol2 file (blue) has been overlaid on top of the original ligand pose (gold). For this result, it can be seen that although the docking score for the docked ligand is comparable with other docking modes, the RMSD computed for the docked result was larger than 2A, which has been reflected by the pose where the sidechain to the right is shifted further away than in original structure.

[[File:2tio_fixed_anchor.png|thumb|center]]

[[File:2tio fixed anchor docking vd.png|thumb|center]]


The fixed.out file summary the overall docking score of the run:

[[File:2tio_fixed_anchor_out.png|thumb|center]]

The steps for the next docking mode should also be similar.

===Fixed Anchor Docking===

In this session, we will be showing the tutorial for Flexible Docking. Among the three docking methods introduced in this tutorial, this would be the most computationally expensive method since the algorithm will be sampling all of the possible conformations of each input ligand and test their binding ability with the binding site. Thus, one might expect the computing time for this method to be exceed other introduced methods.

Please navigate to the directory designated for this docking mode: 008.flex_docking. Here, we can start create the input parameters, file name – flex.in:

  vi flex.in

The following is the input parameter for fixed anchor docking. While you can copy and paste this into the body of your script, please make sure to change the input files directory (ligand and protein structure folder) to your personal directory. Also it should be a good practice to make sure the directories for the dock parameters should also be up-to-date.

   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                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../004.energy_min/2tio.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                                      ../004.energy_min/2tio.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/dock10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        flex.out
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

Now, you are ready to run dock6 implementing the above input parameters:

   dock6 -i flex.in -o flex.out

Dock6 should be running for a couple of minutes. If the program finished right away, it would be recommended that you should check the output file to see if there are any reported errors. Successful run should give you two additional files as followed:

*flex.out
*2tio_flex.out_scored.mol2

The mol2 file containing the docked ligand could be viewed in ChimeraX under ViewDock. To compare with the reference pose, this mol2 file (blue) has been overlaid on top of the original ligand pose (gold). Under ViewDock in Chimera, it can be observed that the docked ligand showed great structural overlap with the reference pose and this has also been reflected in the reported RMSD score, which has been maintained below 2A.

[[File:2tio_flex.png|thumb|center]]

[[File:2tio_vd.png|thumb|center]]

The fixed.out file summary the overall docking score of the run:

[[File:2tio_flex_out.png|thumb|center]]

As we have finished running all the docking modes, one step could be carried out here is to compare all the three docked results with the original reference file and see if all the docking results have been suggesting a binding mode that resemble the reference pose. Attached is the overlaid docking results, where we can see the most rigid docking modes give the closest result to the reference structure, whereas fixed anchor docking has the right-side chain shifted further away. Although being the most flexible method, flexible docking gives a good scoring. (gold - original pose, blue - flex, green - rigid, magenta - fixed anchor). Here, you can also open ViewDock, navigate to columns and show grid scores or RMSD score to compare the docking efficiency between the three model, typically, a successful re-dock attempt should have the ligand less than 2A away from the original pose.

[[File:2tio_combine.png|thumb|center]]

= Virtual Screening of a Ligand Library =

After we have tested our control case for multiple docking modes, in this section we would carry out virtual screening for a larger database of ligands. In this step, the program will attempt to dock all of the input ligands into the binding site and this should be expected to be a time-consuming and computationally exhaustive step. For the purpose of this section, I would be trying out with the 5,000 molecules database, but a safety step you can take is to try running the virtual screening with a smaller subset - containing 100 molecules, to make sure that the program works correctly. The directory to the database of both the 5,000 molecules and 1000 molecules database is available in "gpfs/AMS536/zzz.programs/VS_libraries", there is also a 25,000 molecules subset if you would want to dock to an even larger set. These libraries are extracted from the ZINC library, a commercially available database that contains purchasable chemicals.

Please navigate to the directory designated for virtual screening: 009.virtual_screening. Here, we can start create the input parameters, file name – virutal.in:

  vi virtual.in

The following is the input parameter for virtual screening. While you can copy and paste this into the body of your script, please make sure to change the input files directory (ligand and protein structure folder) to your personal directory. Also it should be a good practice to make sure the directories for the dock parameters and the molecule libraries should also be up-to-date.

    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                                             /gpfs/projects/AMS536/zzz.programs/VS_libraries/VS_library_5K.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/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/dock6.10/parameters/vdw_AMBER_parm99.defn
    flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
    flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
    ligand_outfile_prefix                                        virtual.out
    write_orientations                                           no
    num_scored_conformers                                        1
    rank_ligands                                                 no

Because the step is time-consuming and might be taking up several hours to run so we will not execute the run on command line but we would submit a job through slurm for this step. In order to do so, we would need to create a job file to submit to the slurm queue:

  vi job

The content of the job file should be as followed. In this file, we are running the task on the long-28core that should be compatible with users using login node, if you use other server to submit the task (milan), you should check which core you should be submit to on the SeaWulf website: [https://it.stonybrook.edu/help/kb/choosing-the-right-login-nodes-for-your-jobs] . We are also using mpirun with 224 partition, you should also change it so that it will fit with the core you are submitting to (if you use a different core).

  #!/bin/bash  
  #
  #SBATCH --job-name=2tio_vs_tutorial
  #SBATCH --output=vs_output.txt
  #SBATCH --ntasks-per-node=24
  #SBATCH --nodes=6
  #SBATCH --time=48:00:00
  #SBATCH -p long-28core
  
  module load intel/mpi/64/2018/18.0.3
  
  mpirun -np 224 dock6.mpi -i virtual.in -o virtual.out

Close and save the job file and submit the job to slurm with the following command:

  sbatch job

You can use the command squeue -u username to check if your job has been submitted, on queue or currently running. The job is expected to take a long while to finish so if the job is executed right away, you should check if there might be a problem in your slurm output file. The output file from this step should be including 3 types of additional files: 

- The 2tio.virtual.out_scored.mol2 docked ligand collection.

- The virtual.out file capturing the minimization process - you can check for errors in this file if you cannot run the task. This file is the overall and general output files result from your virtual screening process but you should also see many virtual.out.x files, these files represents each partition from the job that you have indicated on slurm when you are submitting the job.

- The vs_output.txt file.

After you got the result, we can move on to next step of Cartesian minimization and re-scoring steps, and we can view the result at the end.

=Cartesian Minimization of Virtually Screened Small Molecules=

In this step, we would base on the results we harvested from virtual screening and minimize the energy of the docked structure. Thus, this step would not be as time-consuming as the previous step because it the ligands have already been placed in the binding site of the protein under virtual screening.

Please navigate to the directory designated for Cartesian minimization: 010.cartesian_minimization. Here, we can start create the input parameters, file name – min.in:

  vi min.in

The following is the input parameter for virtual screening. While you can copy and paste this into the body of your script, please make sure to change the input files directory (ligand and protein structure folder) to your personal directory.Again here, we want to remind you that you would also be using output from virtual screening so please check if the output of the file is correctly implemented.

  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100
  ligand_atom_file                                             ../009.virtual_screening/virtual.out_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
  grid_score_primary                                           no
  multigrid_score_primary                                      no
  dock3.5_score_primary                                        no
  continuous_score_primary                                     yes
  cont_score_rec_filename                                      ../001.structure/2tio_protein_hydrogens_charges.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                                         1.0
  cont_score_es_scale                                          1.0
  minimize_ligand                                              yes
  simplex_max_iterations                                       1000
  simplex_tors_premin_iterations                               0
  simplex_max_cycles                                           1.0
  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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        2tio.virtual_screen.min
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no


Although this step is not as exhaustive as the previous step that we have worked on, we would still be submitting the task to slurm because the task is still bulky as we are dealing with 5,000 molecules. In order to do so, we would need to create a job file to submit to the slurm queue:

  vi job

The content of the job file should be as followed. As mentioned above, we are running the task on the long-28core that should be compatible with users using login node, if you use other server to submit the task (milan), you should check which core you should be submit to on the SeaWulf website: [https://it.stonybrook.edu/help/kb/choosing-the-right-login-nodes-for-your-jobs] . We would not be using mpirun to partition like we did with virtual screening because this step is not as bulky.

  #!/bin/bash
  #
  #SBATCH --job-name=2tio_minimization_tutorial
  #SBATCH --output=min_output.txt
  #SBATCH --ntasks-per-node=24
  #SBATCH --nodes=8
  #SBATCH --time=48:00:00
  #SBATCH -p long-28core
  
  dock6 -i min.in -o min.out

Close and save the job file and submit the job to slurm with the following command:

  sbatch job

You can use the command squeue -u username to check if your job has been submitted, on queue or currently running. Similarly if the job finished right away, you should check if there might be a problem in your slurm output file. The output file from this step should be including 3 additional files: 

- The 2tio.virtual_screen.min_scored.mol2 docked and minimized ligand collection.
- The min.out file capturing the minimization process - you can check for errors in this file if you cannot run the task.
- The min_output.txt file.

From here, you can move on the the last step of rescoring the ligand database and viewing your results in Chimera.

=Rescoring and Ranking Virtually Screened Molecules=

In this step, we would base on the resulting poses of the ligand derived from the previous step where all of the ligands have already been docked and. For this step, not only will we try to re-dock the molecule, but we would also re-rank the molecules based on Footprint Scoring and thus, by comparing these molecules with one another, we would be able to rank which one has the highest potential to be a potential lead.

Please navigate to the directory designated for rescoring the docked ligands: 011.reScore. Here, we can start create the input parameters, file name – rescore.in:

  vi rescore.in

The following is the input parameter for virtual screening. While you can copy and paste this into the body of your script, please make sure to change the input files directory (ligand and protein structure folder) to your personal directory. Just like in last step, we would be using output from virtual screening so please check if the output of the file is correctly implemented. Additionally, in order to calculate the footprint similarity score, we would be using the energy minimized structure of the original reference ligand from the 004.energy_min folder, so please make sure the code is pointing to the correct input files.

  conformer_search_type                                         rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100
  ligand_atom_file                                             ../009.virtual_screening/virtual.out_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_score                              no
  descriptor_use_footprint_similarity                          yes
  descriptor_use_pharmacophore_score                           yes
  descriptor_use_hungarian                                     yes
  descriptor_use_volume_overlap                                yes
  descriptor_fps_score_use_footprint_reference_mol2            yes
  descriptor_fps_score_footprint_reference_mol2_filename       ../004.energy_min/2tio.lig.min_scored.mol2
  descriptor_fps_score_foot_compare_type                       Euclidean
  descriptor_fps_score_normalize_foot                          no
  descriptor_fps_score_foot_comp_all_residue                   yes
  descriptor_fps_score_receptor_filename                       ../001.structure/2tio_protein_hydrogens_charges.mol2
  descriptor_fps_score_vdw_att_exp                             6
  descriptor_fps_score_vdw_rep_exp                             12
  descriptor_fps_score_vdw_rep_rad_scale                       1
  descriptor_fps_score_use_distance_dependent_dielectric       yes
  descriptor_fps_score_dielectric                              4.0
  descriptor_fps_score_vdw_fp_scale                            1
  descriptor_fps_score_es_fp_scale                             1
  descriptor_fps_score_hb_fp_scale                             0
  descriptor_fms_score_use_ref_mol2                            yes
  descriptor_fms_score_ref_mol2_filename                       ../004.energy_min/2tio.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                       .7071
  descriptor_fms_score_max_score                               20
  descriptor_fingerprint_ref_filename                          ../004.energy_min/2tio.lig.min_scored.mol2
  descriptor_hms_score_ref_filename                            ../004.energy_min/2tio.lig.min_scored.mol2
  descriptor_hms_score_matching_coeff                          -5
  descriptor_hms_score_rmsd_coeff                              1
  descriptor_volume_score_reference_mol2_filename              ../004.energy_min/2tio.lig.min_scored.mol2
  descriptor_volume_score_overlap_compute_method               analytical
  descriptor_weight_fps_score                                  1
  descriptor_weight_pharmacophore_score                        1
  descriptor_weight_fingerprint_tanimoto                       -1
  descriptor_weight_hms_score                                  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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  chem_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
  pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
  ligand_outfile_prefix                                        2tio.output
  write_footprints                                             yes
  write_hbonds                                                 yes
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no



For this step, we would also be sending the task to slurm for running. Thus, we would need to create a job file:

  vi job

The content of the job file should be as followed. As mentioned above, we are running the task on the long-28core that should be compatible with users using login node, if you use other server to submit the task (milan), you should check which core you should be submit to on the SeaWulf website: [https://it.stonybrook.edu/help/kb/choosing-the-right-login-nodes-for-your-jobs] . Again, for this step, we would be using 224 partitions with mpirun and you should also re-calculate how many cores would you be using to maximize the runtime efficiency when you put the number down here.

  #!/bin/bash
  #
  #SBATCH --job-name=2tio_rescoring
  #SBATCH --output=rescoring.txt
  #SBATCH --ntasks-per-node=28
  #SBATCH --mail-user=an.p.nguyen@stonybrook.edu
  #SBATCH --nodes=8
  #SBATCH --time=48:00:00
  #SBATCH -p long-28core
  
  module load intel/mpi/64/2018/18.0.3
  
  mpirun -np 224 dock6.mpi -i rescore.in -o rescore.out

Close and save the job file and submit the job to slurm with the following command:

  sbatch job

You can use the command squeue -u username to check if your job has been submitted, on queue or currently running. Similarly if the job finished right away, you should check if there might be a problem in your slurm output file. The output file from this step should be including 3 additional types of file files: 

- The 2tio.output_scored.mol2 docked ligand collection.

- The rescore.out file capturing the rescoring process - you can check for errors in this file if you cannot run the task. This file is the overall and general output files result from your virtual screening process but you should also see many rescore.out.x files, these files represents each partition from the job that you have indicated on slurm so there should in total be 224 rescore.out.x files generated as we have specified that many cores to be using.

- The 2tio.output_footprint_scored.txt, 2tio.output_hbond_scored.txt, and rescoring.txt. The two first .txt files should be showing the scores based on the footprint similarity comparison and the Hydrogen bonds scoring accordingly.

The .mol2 file should be the re-scored and docked structures of the ligands whereas in next step, we would download this file to your local computer and view them in Chimera. To view the results, first, you should open the structure of the protein in Chimera and then open the docked ligands library using ViewDock. Since we are opening a set of 5,000 molecules, the step should take some time to load all the file on to the platform. After the ligands are loaded, in order to view all the ligands on Chimera, you would need to select all the molecules listed on the ViewDock ligands list, you can choose that by selecting the first ligand and scroll down to the bottom of the list and Shift-select the last ligand. Additionally, on the ViewDock window, you can also click on Show > Columns and open as many columns as you want. These columns will then list out the score accordingly, for example, in the attached image, we have shown the number of Hydrogen bonds (combined, ligands', and receptors') and scores (combined score, electrostatic score, and Van der Waals score).

[[File:2tio_vs.png|thumb|center]]

Another viewing process can be done is to go to HBond in ViewDock, and use a bright color to highlight them (in this case we are showing in magenta, with a thicker line option). Next you can go to Action > Inspect and adjust the atom model to show showing the ligand structure itself, along with that set the background to white color and hide the protein's ribbon. The output should be as followed where it would highlight the binding site that the ligands are making contact with.

[[File:2tio_vs_site.png|thumb|center]]