Difference between revisions of "2020 DOCK tutorial 3 with PDBID 4F4P"

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(→‎Generating box)
(→‎IX Rescoring Docked Molecules)
 
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Line 31: Line 31:
 
===Prepare the Receptor file===
 
===Prepare the Receptor file===
  
−
With the PDB file loaded by chimera, do Select -> Residue -> SO4 to select the sulfate. Use Actions -> Atoms/Bonds -> Delete to delete it. Then do Select -> Residue -> HOH to select the water molecules. Use the same method to delete it. Then do Select -> Structure -> (0SB) to select ligand molecule. Use the same method to delete it. Save the receptor as 4f4p_rec_noh.mol2.
+
With the PDB file loaded by chimera, do  
 +
Select -> Residue -> SO4  
 +
to select the sulfate. Use  
 +
Actions -> Atoms/Bonds -> Delete  
 +
to delete it. Then do  
 +
Select -> Residue -> HOH  
 +
to select the water molecules. Use the same method to delete it. Then do  
 +
Select -> Structure -> (0SB)  
 +
to select ligand molecule. Use the same method to delete it. Save the receptor as 4f4p_rec_noh.mol2.
  
 
OR
 
OR
  
−
With the PDB file loaded by chimera, hold the control button and click on the protein chain to select a protein residue. Then click up arrow button to select the whole protein chain (Receptor). Then Select -> Invert(all models) to invert your selection (This will select all the components in your PDB file except receptor). Then do Actions->Atoms/bonds->Delete. You will remain with a protein receptor. Save the receptor as 4f4p_rec_noh.mol2.
+
With the PDB file loaded by chimera, hold the control button and click on the protein chain to select a protein residue. Then click up arrow button to select the whole protein chain (Receptor). Then  
 +
Select -> Invert(all models)
 +
to invert your selection (This will select all the components in your PDB file except receptor). Then do  
 +
Actions->Atoms/bonds->Delete.  
 +
You will remain with a protein receptor. Save the receptor as 4f4p_rec_noh.mol2.
  
  
Line 42: Line 54:
 
===Prepare the Ligand File===
 
===Prepare the Ligand File===
 
Open the pdf file again.
 
Open the pdf file again.
−
Do Select -> Structure -> ligand(0SB) to select the ligand. Do Select -> Invert (all models), then delete the selected atoms. This will left the ligand molecule only. Save it as 4f4p_lig_noh.mol2.
+
Do  
 +
Select -> Structure -> ligand(0SB)
 +
to select the ligand. Do  
 +
Select -> Invert (all models),  
 +
then delete the selected atoms. This will left the ligand molecule only. Save it as 4f4p_lig_noh.mol2.
  
 
[[File:4f4p_lig_noh.png|thumb|center|500px|Figure 3: Ligand with no Hydrogens and charges (4f4p_lig_noh.mol2)]]
 
[[File:4f4p_lig_noh.png|thumb|center|500px|Figure 3: Ligand with no Hydrogens and charges (4f4p_lig_noh.mol2)]]
Line 90: Line 106:
 
   sphgen -i INSPH -o OUTSPH
 
   sphgen -i INSPH -o OUTSPH
  
−
Once sphgen command is successful, 4f4p_rec.sph file will be created. Open it up using Chimera along with 4f4p_rec_noh.mol2 file. You should get a similar output like the image below.
+
Once sphgen command is successful, following output files will be created.  
 +
  OUTSPH
 +
  4f4p_rec.sph
 +
 
 +
Open the f4p_rec.sph using Chimera along with 4f4p_rec_noh.mol2 file. You should get a similar output like the image below.
 +
 
 
[[File:4f4precnohsph.png|thumb|center|700px|Figure 6:Chimera view of generated spheres along with the receptor(4f4p_rec_noh.mol2 + 4f4p_rec.sph)]]
 
[[File:4f4precnohsph.png|thumb|center|700px|Figure 6:Chimera view of generated spheres along with the receptor(4f4p_rec_noh.mol2 + 4f4p_rec.sph)]]
  
Line 99: Line 120:
 
   sphere_selector 4f4p_rec.sph ../001.files/4f4p_noh_lig.mol2 10.0
 
   sphere_selector 4f4p_rec.sph ../001.files/4f4p_noh_lig.mol2 10.0
  
−
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand binding site are kept and all the other spheres are deleted in the image below.
+
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph.  
 +
 
 +
Output files:
 +
  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 by following sphere selector command .
  
 
[[File: 4f4pre.png |thumb|center|700px|Figure 7: Selected spheres along with the receptor (4f4p_rec_noh.mol2 + selected_spheres.sph)]]
 
[[File: 4f4pre.png |thumb|center|700px|Figure 7: Selected spheres along with the receptor (4f4p_rec_noh.mol2 + selected_spheres.sph)]]
Line 108: Line 134:
 
Create a new file showbox.in and write the following lines in the file(# means comment ).
 
Create a new file showbox.in and write the following lines in the file(# means comment ).
  
−
   Y                                                                           #Say 'Yes' to make a box
+
   Y #Say 'Yes' to make a box
−
   8.0                                                                         #The boundary of the box is at least 8 Angstroms from any spheres
+
   8.0   #The boundary of the box is at least 8 Angstroms from any spheres
−
   ../002.surface_spheres/selected_spheres.sph   #The location and file name of the sphere file
+
   ../002.surface_spheres/selected_spheres.sph #The location and file name of the sphere file
−
   1                                                                           #Use the first cluster of spheres (only 1 cluster in this case)
+
   1   #Use the first cluster of spheres (only 1 cluster in this case)
−
   4f4p.box.pdb                                                       #The output filename
+
   4f4p.box.pdb #The output filename
 +
 
 +
Use the following command to generate the box.
 +
 
 +
  showbox < showbox.in
 +
 
 +
If this step is successful, you should see a new file (4f4p.box.pdb) in 003.gridbox folder.
 +
 
 +
Output file:
 +
  4f4p.box.pdb
 +
 
 +
[[File:4f4p_rec_noh_selected_sphere_box.png|thumb|center|500px|Figure 8:surface of the receptor,selected spheres with box]]
 +
 
 +
===Generating Grid===
 +
Create a new input file called grid.in and write the following information there.
 +
 
 +
  compute_grids                            yes
 +
  grid_spacing                              0.4
 +
  output_molecule                          no
 +
  contact_score                            no
 +
  energy_score                              yes
 +
  energy_cutoff_distance                    9999
 +
  atom_model                                a
 +
  attractive_exponent                      6
 +
  repulsive_exponent                        9
 +
  distance_dielectric                      yes
 +
  dielectric_factor                        4
 +
  bump_filter                              yes
 +
  bump_overlap                              0.75
 +
  receptor_file                            ../001.files/4f4p_rec_h.mol2
 +
  box_file                                  4f4p.box.pdb
 +
  vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  score_grid_prefix                        grid
 +
 
 +
save the file and run the following command.
 +
 
 +
  grid -i grid.in -o grid.out
 +
 
 +
If this is successful, you will get following output files.
 +
 
 +
  grid.out
 +
  grid.nrg
 +
  grid.bmp
 +
 
 +
=V. Docking=
 +
===Energy minimization===
 +
 
 +
Before doing docking pose reproduction, energy minimization can be done in order to remove unfavorable intra-molecular clashes of the ligand. (If not it will affect the later steps of docking)
 +
 
 +
Go to 004.dock folder and open a file called min.in and type the following content there.
 +
 
 +
  conformer_search_type                                        rigid
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  internal_energy_cutoff                                      100.0
 +
  ligand_atom_file                                            ../001.files/4f4p_lig_h.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              yes
 +
  use_rmsd_reference_mol                                      ../001.files/4f4p_lig_h.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
  ligand_outfile_prefix                                        4f4p.lig.min
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  rank_ligands                                                no
 +
 
 +
Save the file and run the following command.
 +
 
 +
  dock6 -i min.in -o min.out
 +
 
 +
If you have done correctly, you will get followig out files.
 +
 
 +
  min.out
 +
  4f4p.lig.min_scored.mol2
 +
 
 +
[[File:Rec min lig complex.png |thumb|center|700px|Figure 9: Receptor with energy minimized ligand]]
 +
 
 +
=== Footprint Analysis===
 +
 
 +
In order to see the minimized ligand compared to the original ligand through footprint analysis, open a file called footprint.in and type following.
 +
 
 +
  conformer_search_type                                        rigid
 +
  use_internal_energy                                          no
 +
  ligand_atom_file                                            4f4p.lig.min_scored.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              no
 +
  use_database_filter                                          no
 +
  orient_ligand                                                no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          no
 +
  grid_score_secondary                                        no
 +
  multigrid_score_primary                                      no
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_primary                                        no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_primary                                    no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_primary                          yes
 +
  footprint_similarity_score_secondary                        no
 +
  fps_score_use_footprint_reference_mol2                      yes
 +
  fps_score_footprint_reference_mol2_filename                  ../001.files/4f4p_lig_h.mol2
 +
  fps_score_foot_compare_type                                  Euclidean
 +
  fps_score_normalize_foot                                    no
 +
  fps_score_foot_comp_all_residue                              yes
 +
  fps_score_receptor_filename                                  ../001.files/4f4p_rec_h.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
  ligand_outfile_prefix                                        fps.min.output
 +
  write_footprints                                            yes
 +
  write_hbonds                                                yes
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  rank_ligands                                                no
 +
 
 +
If footprint runs successfully, you can find the following files generated
 +
 
 +
  footprint.out
 +
  fps.min.output_footprint_scored.txt
 +
  fps.min.output_hbond_scored.txt
 +
  fps.min.output_scored.mol2
 +
 
 +
In order to visualize the footprint, first we need to copy a python script into our directory, then run it:
 +
 
 +
  cp /gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py ./
 +
  python plot_footprint_single_magnitude.py fps.min.output_footprint_scored.txt 50
 +
 
 +
A pdf document named fps.min.output_footprint_scored.txt.pdf will be generated. It looks like this:
 +
[[File:Footprint_output.jpeg|thumb|center|700px|Figure 10: Footprint of minimized ligand,red:minimized, blue:unminimized (original ligand) ]]
 +
 
 +
===Rigid Docking===
 +
Create an input file for rigid docking
 +
touch rigid.in
 +
 
 +
Write lines below into the rigid.in
 +
 
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      9
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            ../001.files/4f4p_lig_h.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              yes
 +
use_rmsd_reference_mol                                      yes
 +
rmsd_reference_filename                                      ../001.files/4f4p_lig_h.mol2
 +
use_database_filter                                          no
 +
orient_ligand                                                yes
 +
automated_matching                                          yes
 +
receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
max_orientations                                            1000
 +
critical_points                                              no
 +
chemical_matching                                            no
 +
use_ligand_spheres                                          no
 +
bump_filter                                                  no
 +
score_molecules                                              yes
 +
contact_score_primary                                        no
 +
contact_score_secondary                                      no
 +
grid_score_primary                                          yes
 +
grid_score_secondary                                        no
 +
grid_score_rep_rad_scale                                    1
 +
grid_score_vdw_scale                                        1
 +
grid_score_es_scale                                          1
 +
grid_score_grid_prefix                                      ../003.gridbox/grid
 +
multigrid_score_secondary                                    no
 +
dock3.5_score_secondary                                      no
 +
continuous_score_secondary                                  no
 +
footprint_similarity_score_secondary                        no
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_secondary                                  no
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              yes
 +
simplex_max_iterations                                      1000
 +
simplex_tors_premin_iterations                              2
 +
simplex_max_cycles                                          1
 +
simplex_score_converge                                      0.1
 +
simplex_cycle_converge                                      1.0
 +
simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        rigid.out
 +
write_orientations                                          no
 +
num_scored_conformers                                        40
 +
write_conformations                                          yes
 +
cluster_conformations                                        yes
 +
cluster_rmsd_threshold                                      2.0
 +
rank_ligands                                                no
 +
 +
Run dock using the created input file.
 +
dock6 -i rigid.in -o rigid.out
 +
 
 +
Once rigid docking is successful, you will get following output files.
 +
 
 +
  rigid.out
 +
  rigid.out_conformers.mol2
 +
  rigid.out_scored.mol2
 +
 
 +
In order to check the rigid docking success, open rigid.out_scored.mol2 output file using Chimera by following steps.
 +
 
 +
  Open Chimera
 +
  File -> Open -> 4f4p_rec_h.mol2 (Actions -> Surface -> Show)
 +
  File -> Open -> 4f4p_lig_h.mol2
 +
  Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (rigid.out_scored.mol2)
 +
  In the loaded dialog box select Dock4,5 or 6
 +
 
 +
Once everything is loaded go to the ViewDock window and use it's menu to view all the calculated properties regarding the rigid docked ligand by following the steps below.
 +
 
 +
  Column -> Show -> Grid_Score
 +
  Column -> Show -> HA_RMSDs
 +
 
 +
Follow the same steps to get all different properties as you interested.
 +
 
 +
You can choose different conformations from the ViewDock window. If you sorted it, the top one should be the conformation with lowest energy. Your visualized structure should be similar to the image below.
 +
 
 +
[[File:rigid_out1.png|thumb|center|500px|Figure 11: rigid out file (different poses with their corresponding grid scores and RMSDs are listed in the table (left), best confirmation with lowest RMSD value (cyan) is shown compared to the original ligand)]]
 +
 
 +
[[File:rigid_all.png|thumb|center|500px|Figure 12: All possible ligand confirmations of ligand after rigid docking pose reproduction]]
 +
 
 +
===Fixed Anchor Docking===
 +
 
 +
Create an input file called fad.in and use the following command.
 +
 
 +
  dock6 -i fad.in -o fad.out
 +
 
 +
Use the following lines to answer the prompted questions as we did in rigid docking.
 +
 
 +
  conformer_search_type                                        flex
 +
  write_fragment_libraries                                    no
 +
  user_specified_anchor                                        no
 +
  limit_max_anchors                                            no
 +
  min_anchor_size                                              5
 +
  pruning_use_clustering                                      yes
 +
  pruning_max_orients                                          1000
 +
  pruning_clustering_cutoff                                    100
 +
  pruning_conformer_score_cutoff                              100.0
 +
  pruning_conformer_score_scaling_factor                      1.0
 +
  use_clash_overlap                                            no
 +
  write_growth_tree                                            no
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      9
 +
  internal_energy_cutoff                                      100.0
 +
  ligand_atom_file                                            ../001.files/4f4p_lig_h.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              yes
 +
  use_rmsd_reference_mol                                      yes
 +
  rmsd_reference_filename                                      ../001.files/4f4p_lig_h.mol2
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          yes
 +
  grid_score_secondary                                        no
 +
  grid_score_rep_rad_scale                                    1
 +
  grid_score_vdw_scale                                        1
 +
  grid_score_es_scale                                          1
 +
  grid_score_grid_prefix                                      ../003.gridbox/grid
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_secondary                        no
 +
  pharmacophore_score_secondary                                no
 +
  descriptor_score_secondary                                  no
 +
  gbsa_zou_score_secondary                                    no
 +
  gbsa_hawkins_score_secondary                                no
 +
  SASA_score_secondary                                        no
 +
  amber_score_secondary                                        no
 +
  minimize_ligand                                              yes
 +
  minimize_anchor                                              yes
 +
  minimize_flexible_growth                                    yes
 +
  use_advanced_simplex_parameters                              no
 +
  simplex_max_cycles                                          1
 +
  simplex_score_converge                                      0.1
 +
  simplex_cycle_converge                                      1.0
 +
  simplex_trans_step                                          1.0
 +
  simplex_rot_step                                            0.1
 +
  simplex_tors_step                                            10.0
 +
  simplex_anchor_max_iterations                                500
 +
  simplex_grow_max_iterations                                  500
 +
  simplex_grow_tors_premin_iterations                          0
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        yes
 +
  simplex_coefficient_restraint                                10.0
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
  ligand_outfile_prefix                                        4f4p_fad
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        40
 +
  write_conformations                                          no
 +
  cluster_conformations                                        yes
 +
  cluster_rmsd_threshold                                      2.0
 +
  rank_ligands                                                no
 +
 
 +
Once docking is completed following output files will be generated.
 +
 
 +
  fad.out
 +
  4f4p_fad_scored.mol2
 +
 
 +
Follow the same method used in rigid docking to visualize the docked poses using Chimera by opening the 4f4p_fad_scored.mol2 file. Once it is visualized, it should like the image below.
 +
 
 +
[[File:Fad_out.png|thumb|center|500px|Figure 13:  Fixed Anchor docking out file (different poses with their corresponding grid scores and RMSDs are listed in the table (left), best confirmation with lowest RMSD value (cyan) is shown compared to the original ligand)]]
 +
 
 +
[[File:Fad_all.png|thumb|center|500px|Figure 14: All possible ligand confirmations of ligand after Fixed Anchor docking pose reproduction]]
 +
 
 +
===Flexible Docking===
 +
 
 +
Create a new input file for flexible docking called flex.in and perform flexible docking using DOCK6 by following command.
 +
 
 +
  dock6 -i flex.in -o flex.out
 +
 
 +
Answer the prompted questions using the following lines as we did in rigid and fixed anchor docking (or copy the following content in to flex.in file and save).
 +
 
 +
  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                                            ../004.dock/4f4p.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.dock/4f4p.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
  ligand_outfile_prefix                                        flex.out
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  rank_ligands                                                no
 +
 
 +
Once flexible docking is completed following output files will be generated.
 +
 
 +
  flex.out
 +
  flex.out_scored.mol2
 +
 
 +
Use the visualization steps used in rigid and fixed anchor docking and study the properties of the docking results by opening flex.out_scored.mol2 file.  (You will get one confirmation since you're writing num_scored_conformers as one in the above script.)
 +
 
 +
[[File:Flex_out.png|thumb|center|500px|Figure 15:  Flexible docking out file (pose with corresponding grid score and RMSD are listed in the table (As we got one confirmation after flexible docking) (left), confirmation with its RMSD value (cyan) is shown compared to the original ligand)]]
 +
 
 +
= VI. Virtual Screen =
 +
Virtual screening is the method of screening a ligand library (drug-like molecules) to filter the best ligands which can bind to the binding site of a specific receptor. Here we will be using a ligand library which contains 25000 molecules to select the best ligands which can be used to replace the original ligand that the PDB file contained.
 +
 
 +
Move to the directory 005.virtual_screen.
 +
 
 +
Copy the ligand library to the same directory.
 +
 
 +
cp /gpfs/projects/AMS536/2020/536_class/VS_library_5K.mol2 ./
 +
 
 +
Create a new input file for virtual screen.
 +
 
 +
touch virtual.in
 +
 
 +
Use the input file to perform virtual screen using DOCK6.
 +
 
 +
dock6 -i virtual.in -o virtual.out
 +
 
 +
Use the following lines to answer the prompted questions.
 +
 
 +
conformer_search_type                                        flex
 +
write_fragment_libraries                                    no
 +
user_specified_anchor                                        no
 +
limit_max_anchors                                            no
 +
min_anchor_size                                              5
 +
pruning_use_clustering                                      yes
 +
pruning_max_orients                                          1000
 +
pruning_clustering_cutoff                                    100
 +
pruning_conformer_score_cutoff                              100.0
 +
pruning_conformer_score_scaling_factor                      1.0
 +
use_clash_overlap                                            no
 +
write_growth_tree                                            no
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      9
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            VS_library_5K.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        virtual.out
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
                     
 +
You can run this job on head node for a short while, just to make sure everything is fine. Then you can kill the job by Ctrl-C.
 +
 
 +
= VII Virtual Screeni mpi =
 +
 
 +
Go to 006. virtual_screen_mpi directory and copy the virtual.in file here. We will run the job on the seawulf cluster using slurm script.
 +
 
 +
Creat the virtual.sh file
 +
  vi virtual.sh
 +
 
 +
Add the following lines there
 +
 +
  #!/bin/sh
 +
  #SBATCH --partition=long-40core
 +
  #SBATCH --time=48:00:00
 +
  #SBATCH --nodes=1
 +
  #SBATCH --ntasks=1
 +
  #SBATCH --job-name=........#whatever you want
 +
  #SBATCH --output=%x-%j.o
 +
 +
  dock6 -i virtual.in -o virtual.out
 +
 
 +
Submit the job on seawulf using
 +
sbatch virtual.sh
 +
To check the status of your job
 +
  squeue -u [NETID]
 +
 
 +
===Check Virtual Screen output===
 +
 
 +
The job was terminated due to time limit. In order to calculate how many molecules were screened, we run the command:
 +
grep "Molecule: ZINC" virtual.out | wc -l
 +
 
 +
Everyone in our group has got either 1066, 1068, 1061 molecules processed.
 +
 
 +
To vizualize the output, we open the file virtual.out_scored.mol2 with Chimera. The Chimera could be slow as there are many many molecules.
 +
Go to Tools -> Surface binding Analysis -> View Dock. Also open the receptor in this session.
 +
 
 +
Column -> Show -> Grid Score
 +
 
 +
Column -> Show -> Mass weight
 +
 
 +
We see that best Grid scored molecules have higher energy than those that have lower Grid score energy.
 +
The vdw energy also have the same trend with Grid score.
 +
 
 +
=VIII.Cartesian Minimization=
 +
 
 +
Here we will use the docked molecules and perform a cartesian minimization of them.
 +
 
 +
Move to the directory 007.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 -o min.out
 +
 
 +
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                                            ../005.virtual_screen/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                                    yes
 +
continuous_score_secondary                                  no
 +
cont_score_rec_filename                                      ../001.files/4f4p_rec_h.mol2
 +
cont_score_att_exp                                          6
 +
cont_score_rep_exp                                          12
 +
cont_score_rep_rad_scale                                    1
 +
cont_score_use_dist_dep_dielectric                          yes
 +
cont_score_dielectric                                        4.0
 +
cont_score_vdw_scale                                        1
 +
cont_score_es_scale                                          1
 +
footprint_similarity_score_secondary                        no
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_secondary                                  no
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              yes
 +
simplex_max_iterations                                      1000
 +
simplex_tors_premin_iterations                              0
 +
simplex_max_cycles                                          1
 +
simplex_score_converge                                      0.1
 +
simplex_cycle_converge                                      1.0
 +
simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        4f4p.virtualscreen.minimized
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
 
 +
Then in order to submit the script in to seawulf cluster, open a new script called cartesianmin.sh and write the following.
 +
 
 +
  #!/bin/sh
 +
  #SBATCH --partition=long-40core
 +
  #SBATCH --time=48:00:00
 +
  #SBATCH --nodes=1
 +
  #SBATCH --ntasks=1
 +
  #SBATCH --job-name=4f4p_cartesian
 +
  #SBATCH --output=%x-%j.o
 +
 
 +
  echo "============================= SLURM JOB ================================="
 +
  date
 +
  echo
 +
  echo " The job will be started on the following node(s):"
 +
  echo $SLURM_JOB_NODELIST
 +
  echo
 +
  echo "Slurm user:                  $SLURM_JOB_USER"
 +
  echo "Run directory:                $(pwd)"
 +
  echo "Job ID:                      $SLURM_JOB_ID"
 +
  echo "Job name:                    $SLURM_JOB_NAME"
 +
  echo "Partition:                    $SLURM_JOB_PARTITION"
 +
  echo "Number of nodes:              $SLURM_JOB_NUM_NODES"
 +
  echo "Number of tasks:              $SLURM_NTASKS"
 +
  echo "Submitted from:              $SLURM_SUBMIT_HOST:$SLURM_SUBMIT_DIR"
 +
  echo "========================================================================="
 +
 
 +
  dock6 -i min.in -o min.out
 +
 
 +
Then submit the job into seawulf cluster.
 +
  sbatch cartesianmin.sh
 +
 
 +
Once it has done correctly, you will get a output file called 4f4p.virtualscreen.minimized_scored.mol2. This file contains molecules with minimized energies by removing unfavorable steric clashes.
 +
 
 +
= IX Rescoring Docked Molecules =
 +
 
 +
In order to rank our docked ligands and extract the best ligands (which has the most negative, lowest scores) based on different scoring functions like Footprint similarity, pharmacophore score, tanimoto score, the hungarian and the volume overlap score, we will be using Dock6 to rescore the virtual screen minimized ligands.
 +
 
 +
Move to the directory 008.rescore and 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                                            ../007.cartesianmin/4f4p.virtualscreen.minimized_scored.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              no
 +
  use_database_filter                                          no
 +
  orient_ligand                                                no
 +
  bump_filter                                                  no
 +
  score_molecules                                              yes
 +
  contact_score_primary                                        no
 +
  contact_score_secondary                                      no
 +
  grid_score_primary                                          no
 +
  grid_score_secondary                                        no
 +
  multigrid_score_primary                                      no
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_primary                                        no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_score_primary                                    no
 +
  continuous_score_secondary                                  no
 +
  footprint_similarity_score_primary                          no
 +
  footprint_similarity_score_secondary                        no
 +
  pharmacophore_score_primary                                  no
 +
  pharmacophore_score_secondary                                no
 +
  descriptor_score_primary                                    yes
 +
  descriptor_score_secondary                                  no
 +
  descriptor_use_grid_score                                    no
 +
  descriptor_use_multigrid_score                              no
 +
  descriptor_use_continuous_score                              yes
 +
  descriptor_use_footprint_similarity                          yes
 +
  descriptor_use_pharmacophore_score                          yes
 +
  descriptor_use_tanimoto                                      yes
 +
  descriptor_use_hungarian                                    yes
 +
  descriptor_use_volume_overlap                                yes
 +
  descriptor_cont_score_rec_filename                          ../001.files/4f4p_rec_h.mol2
 +
  descriptor_cont_score_att_exp                                6
 +
  descriptor_cont_score_rep_exp                                12
 +
  descriptor_cont_score_rep_rad_scale                          1
 +
  descriptor_cont_score_use_dist_dep_dielectric                yes
 +
  descriptor_cont_score_dielectric                            4.0
 +
  descriptor_cont_score_vdw_scale                              1
 +
  descriptor_cont_score_es_scale                              1
 +
  descriptor_fps_score_use_footprint_reference_mol2            yes
 +
  descriptor_fps_score_footprint_reference_mol2_filename      ../004.dock/4f4p.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.files/4f4p_rec_h.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.dock/4f4p.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/4f4p.lig.min_scored.mol2
 +
  descriptor_hms_score_ref_filename                            ../004.dock/4f4p.lig.min_scored.mol2
 +
  descriptor_hms_score_matching_coeff                          -5
 +
  descriptor_hms_score_rmsd_coeff                              1
 +
  descriptor_volume_score_reference_mol2_filename              ../004.dock/4f4p.lig.min_scored.mol2
 +
  descriptor_volume_score_overlap_compute_method              analytical
 +
  descriptor_weight_cont_score                                1
 +
  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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 +
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 +
  chem_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/chem.defn
 +
  pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/ph4.defn
 +
  ligand_outfile_prefix                                        descriptor.output
 +
  write_footprints                                            yes
 +
  write_hbonds                                                yes
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        1
 +
  rank_ligands                                                no
 +
 
 +
Once you done the rescoring correctly, you will get several output files as below.
 +
  descriptor.output_footprint_scored.txt 
 +
  descriptor.output_scored.mol2 
 +
  descriptor.output_hbond_scored.txt
 +
  rescore.out
 +
 
 +
Open up the descriptor.output_scored.mol2 file along with original ligand and receptor and analyse the best ligands based on different scoring functions using viewdock property in Chimera.
 +
 
 +
[[File:Rescore.png|thumb|center|500px|Figure 16: Rescored ligands out file along with Receptor and original ligand (sorted according to the footprint similarity score values)]]

Latest revision as of 13:10, 9 March 2020

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

/*This page is under construction*/

I. Introduction

DOCK

DOCK is an important molecular docking program in molecular modeling and drug design. It enables you to dock a small molecule (ligand) to a binding pocket of certain protein target (receptor) and find out correct binding geometry, types and number of interactions involve in binding and energies associate with binding. For this tutorial DOCK6.9 package was used. Purpose of using DOCK6.9 in this tutorial is to re-dock a ligand to a binding pocket of the receptor protein for pose reproduction and find out lead compounds which have stronger binding affinity with our receptor binding site.

Virtual Screening

This is a protocol widely used in drug design which enable you to find out small molecules that have higher binding affinity with the binding pocket of protein of interest by screening a millions of compounds in a virtual database (such as ZINC).

Organization of directories

Log in to the seawulf cluster and go to your group directory. Use following command to create files. 

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

4F4P

4F4P is the crystal structure of Tyrosine Kinase SYK in complex with ligand LASW836. You can get the pdb file from here [1]. The resolution is 2.37 Å. Click Download Files -> PDB Format to download PDB file.

Figure 1: 4F4P.pdb which contains protein receptor, ligand, sulphate ion and water molecules. (Missing residues in the protein receptor ignored since those are located away from the binding site)

II. Preparation of the ligand and receptor

Checking the structure

Read the article related to the PDB file [2] to understand protonation states, charges and other important information regarding the receptor and the ligand.

Open the pdb file through chimera and look at the structure.

Identify the main components of the model (receptor, ligand, solvent, cofactors). In our case, the PDB file contains one protein chain, ligand(OSB), sulfate ion(SO4(2-)) as a cofactor and water molecules (H2O).

Next, we are going to generate the receptor file and the ligand file.

Prepare the Receptor file

With the PDB file loaded by chimera, do 

Select -> Residue -> SO4

to select the sulfate. Use 

Actions -> Atoms/Bonds -> Delete

to delete it. Then do 

Select -> Residue -> HOH

to select the water molecules. Use the same method to delete it. Then do 

Select -> Structure -> (0SB)

to select ligand molecule. Use the same method to delete it. Save the receptor as 4f4p_rec_noh.mol2.

OR

With the PDB file loaded by chimera, hold the control button and click on the protein chain to select a protein residue. Then click up arrow button to select the whole protein chain (Receptor). Then 

Select -> Invert(all models)

to invert your selection (This will select all the components in your PDB file except receptor). Then do 

Actions->Atoms/bonds->Delete.

You will remain with a protein receptor. Save the receptor as 4f4p_rec_noh.mol2.


Figure 2: Receptor with no Hydrogens and charges (4f4p_rec_noh.mol2)

Prepare the Ligand File

Open the pdf file again. Do 

Select -> Structure -> ligand(0SB)

to select the ligand. Do 

Select -> Invert (all models),

then delete the selected atoms. This will left the ligand molecule only. Save it as 4f4p_lig_noh.mol2.

Figure 3: Ligand with no Hydrogens and charges (4f4p_lig_noh.mol2)

Adding hydrogen and charge

Now we are going to add hydrogen atoms and charges to our receptor and ligand. Open 4f4p_rec_noh.mol2 file using Chimera and use the following instructions to prepare the receptor file to be used in DOCK. Tools -> Structure editing -> AddH. This command will add hydrogen to the receptor. Tools -> Structure editing -> Add Charge. This command will add charge to atoms to make the receptor neutral. Save the file as 4f4p_rec_h.mol2


Open 4f4p_lig_noh.mol2, follow the same steps for ligand file, add hydrogen. The Chimera added 2 hydrogens to the nitrogen where it is supposed to be only one, as referred in the pdb report. So remove one hydrogen. (Hold Ctrl and select extra H, then Actions -> Atoms/Bonds -> delete.) (Therefore, Be really concern when adding Hydrogens using chimera since it is automatically adding unwanted hydrogen atoms. Compare your protonation state with PDB report and make sure you have correct structure after adding hydrogens.) After removing hydrogen add charges to the ligand Tools -> Structure editing -> Add Charge. After this save the file as 4f4p_lig_h.mol2.

Figure 4: Ligand with Hydrogen and charges (4f4p_lig_h.mol2)

copy the 4f4p_rec_h.mol2, 4f4p_rec_noh.mol2, 4f4p_lig_noh.mol2, 4f4p_lig_h.mol2 files in to 001.files directory

III. Generating receptor surface and spheres

Creating a surface (DMS) file

Open the 4f4p_rec_noh.mol2 file in chimera. Then Action -> surface -> show -> Shows the surface of the receptor without H.

Tools -> Structure editing -> write DMS -> name: 4f4p_rec_noh.dms -> This command saves the surface of the receptor in dms format.

Figure 5: Surface of the receptor (4f4p_rec_noh.dms)

copy the 4f4p_rec.dms file in to 002.surface_spheres directory.

Generating spheres

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

 vi INSPH

then type the following lines inside the file. 

 4f4p_rec_noh.dms
 R
 X
 0.0
 4.0
 1.4
 4f4p_rec.sph

The first line 4f4p_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 4f4p_rec.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, following output files will be created. 

 OUTSPH
 4f4p_rec.sph

Open the f4p_rec.sph using Chimera along with 4f4p_rec_noh.mol2 file. You should get a similar output like the image below.

Figure 6:Chimera view of generated spheres along with the receptor(4f4p_rec_noh.mol2 + 4f4p_rec.sph)

Selecting Spheres

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

 sphere_selector 4f4p_rec.sph ../001.files/4f4p_noh_lig.mol2 10.0

This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph.

Output files: 

 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 by following sphere selector command .

Figure 7: Selected spheres along with the receptor (4f4p_rec_noh.mol2 + selected_spheres.sph)

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(# means comment ). 

 Y  #Say 'Yes' to make a box
 8.0   #The boundary of the box is at least 8 Angstroms from any spheres
 ../002.surface_spheres/selected_spheres.sph  #The location and file name of the sphere file
 1    #Use the first cluster of spheres (only 1 cluster in this case)
 4f4p.box.pdb  #The output filename

Use the following command to generate the box. 

 showbox < showbox.in

If this step is successful, you should see a new file (4f4p.box.pdb) in 003.gridbox folder.

Output file: 

 4f4p.box.pdb
Figure 8:surface of the receptor,selected spheres with box

Generating Grid

Create a new input file called grid.in and write the following information there. 

 compute_grids                             yes
 grid_spacing                              0.4
 output_molecule                           no
 contact_score                             no
 energy_score                              yes
 energy_cutoff_distance                    9999
 atom_model                                a
 attractive_exponent                       6
 repulsive_exponent                        9
 distance_dielectric                       yes
 dielectric_factor                         4
 bump_filter                               yes
 bump_overlap                              0.75
 receptor_file                             ../001.files/4f4p_rec_h.mol2
 box_file                                  4f4p.box.pdb
 vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 score_grid_prefix                         grid

save the file and run the following command. 

  grid -i grid.in -o grid.out

If this is successful, you will get following output files. 

 grid.out
 grid.nrg
 grid.bmp

V. Docking

Energy minimization

Before doing docking pose reproduction, energy minimization can be done in order to remove unfavorable intra-molecular clashes of the ligand. (If not it will affect the later steps of docking)

Go to 004.dock folder and open a file called min.in and type the following content there. 

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../001.files/4f4p_lig_h.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       ../001.files/4f4p_lig_h.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        4f4p.lig.min
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Save the file and run the following command. 

 dock6 -i min.in -o min.out

If you have done correctly, you will get followig out files. 

 min.out
 4f4p.lig.min_scored.mol2
Figure 9: Receptor with energy minimized ligand

Footprint Analysis

In order to see the minimized ligand compared to the original ligand through footprint analysis, open a file called footprint.in and type following. 

 conformer_search_type                                        rigid
 use_internal_energy                                          no
 ligand_atom_file                                             4f4p.lig.min_scored.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           yes
 footprint_similarity_score_secondary                         no
 fps_score_use_footprint_reference_mol2                       yes
 fps_score_footprint_reference_mol2_filename                  ../001.files/4f4p_lig_h.mol2
 fps_score_foot_compare_type                                  Euclidean
 fps_score_normalize_foot                                     no
 fps_score_foot_comp_all_residue                              yes
 fps_score_receptor_filename                                  ../001.files/4f4p_rec_h.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        fps.min.output
 write_footprints                                             yes
 write_hbonds                                                 yes
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

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

 footprint.out
 fps.min.output_footprint_scored.txt
 fps.min.output_hbond_scored.txt
 fps.min.output_scored.mol2

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

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

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

Figure 10: Footprint of minimized ligand,red:minimized, blue:unminimized (original ligand)

Rigid Docking

Create an input file for rigid docking 

touch rigid.in

Write lines below into the rigid.in 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.files/4f4p_lig_h.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../001.files/4f4p_lig_h.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
max_orientations                                             1000 
critical_points                                              no
chemical_matching                                            no
use_ligand_spheres                                           no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           yes
grid_score_secondary                                         no
grid_score_rep_rad_scale                                     1
grid_score_vdw_scale                                         1
grid_score_es_scale                                          1
grid_score_grid_prefix                                       ../003.gridbox/grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               2
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
ligand_outfile_prefix                                        rigid.out
write_orientations                                           no
num_scored_conformers                                        40
write_conformations                                          yes
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Run dock using the created input file. 

dock6 -i rigid.in -o rigid.out

Once rigid docking is successful, you will get following output files. 

 rigid.out
 rigid.out_conformers.mol2
 rigid.out_scored.mol2

In order to check the rigid docking success, open rigid.out_scored.mol2 output file using Chimera by following steps. 

 Open Chimera
 File -> Open -> 4f4p_rec_h.mol2 (Actions -> Surface -> Show)
 File -> Open -> 4f4p_lig_h.mol2
 Tools -> Surface/binding Analysis -> ViewDock -> Select the Rigid Dock output file. (rigid.out_scored.mol2)
 In the loaded dialog box select Dock4,5 or 6

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

 Column -> Show -> Grid_Score
 Column -> Show -> HA_RMSDs

Follow the same steps to get all different properties as you interested.

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

Figure 11: rigid out file (different poses with their corresponding grid scores and RMSDs are listed in the table (left), best confirmation with lowest RMSD value (cyan) is shown compared to the original ligand)
Figure 12: All possible ligand confirmations of ligand after rigid docking pose reproduction

Fixed Anchor Docking

Create an input file called fad.in and use the following command. 

 dock6 -i fad.in -o fad.out

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

 conformer_search_type                                        flex
 write_fragment_libraries                                     no
 user_specified_anchor                                        no
 limit_max_anchors                                            no
 min_anchor_size                                              5
 pruning_use_clustering                                       yes
 pruning_max_orients                                          1000
 pruning_clustering_cutoff                                    100
 pruning_conformer_score_cutoff                               100.0
 pruning_conformer_score_scaling_factor                       1.0
 use_clash_overlap                                            no
 write_growth_tree                                            no
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      9
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../001.files/4f4p_lig_h.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       yes
 rmsd_reference_filename                                      ../001.files/4f4p_lig_h.mol2
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes 
 receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        yes
 simplex_coefficient_restraint                                10.0
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        4f4p_fad
 write_orientations                                           no
 num_scored_conformers                                        40
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no

Once docking is completed following output files will be generated. 

 fad.out
 4f4p_fad_scored.mol2

Follow the same method used in rigid docking to visualize the docked poses using Chimera by opening the 4f4p_fad_scored.mol2 file. Once it is visualized, it should like the image below.

Figure 13: Fixed Anchor docking out file (different poses with their corresponding grid scores and RMSDs are listed in the table (left), best confirmation with lowest RMSD value (cyan) is shown compared to the original ligand)
Figure 14: All possible ligand confirmations of ligand after Fixed Anchor docking pose reproduction

Flexible Docking

Create a new input file for flexible docking called flex.in and perform flexible docking using DOCK6 by following command. 

 dock6 -i flex.in -o flex.out

Answer the prompted questions using the following lines as we did in rigid and fixed anchor docking (or copy the following content in to flex.in file and save). 

 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                                             ../004.dock/4f4p.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.dock/4f4p.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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        flex.out
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Once flexible docking is completed following output files will be generated. 

 flex.out
 flex.out_scored.mol2

Use the visualization steps used in rigid and fixed anchor docking and study the properties of the docking results by opening flex.out_scored.mol2 file. (You will get one confirmation since you're writing num_scored_conformers as one in the above script.)

Figure 15: Flexible docking out file (pose with corresponding grid score and RMSD are listed in the table (As we got one confirmation after flexible docking) (left), confirmation with its RMSD value (cyan) is shown compared to the original ligand)

VI. Virtual Screen

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

Move to the directory 005.virtual_screen.

Copy the ligand library to the same directory. 

cp /gpfs/projects/AMS536/2020/536_class/VS_library_5K.mol2 ./

Create a new input file for virtual screen. 

touch virtual.in

Use the input file to perform virtual screen using DOCK6. 

dock6 -i virtual.in -o virtual.out

Use the following lines to answer the prompted questions. 

conformer_search_type                                        flex
write_fragment_libraries                                     no
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
use_internal_energy                                          yes
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             VS_library_5K.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
ligand_outfile_prefix                                        virtual.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

You can run this job on head node for a short while, just to make sure everything is fine. Then you can kill the job by Ctrl-C.

VII Virtual Screeni mpi

Go to 006. virtual_screen_mpi directory and copy the virtual.in file here. We will run the job on the seawulf cluster using slurm script.

Creat the virtual.sh file 

 vi virtual.sh

Add the following lines there 

 #!/bin/sh 
 #SBATCH --partition=long-40core 
 #SBATCH --time=48:00:00
 #SBATCH --nodes=1 
 #SBATCH --ntasks=1 
 #SBATCH --job-name=........#whatever you want
 #SBATCH --output=%x-%j.o

 dock6 -i virtual.in -o virtual.out

Submit the job on seawulf using 

sbatch virtual.sh

To check the status of your job 

  squeue -u [NETID]

Check Virtual Screen output

The job was terminated due to time limit. In order to calculate how many molecules were screened, we run the command: 

grep "Molecule: ZINC" virtual.out | wc -l

Everyone in our group has got either 1066, 1068, 1061 molecules processed.

To vizualize the output, we open the file virtual.out_scored.mol2 with Chimera. The Chimera could be slow as there are many many molecules. Go to Tools -> Surface binding Analysis -> View Dock. Also open the receptor in this session.

Column -> Show -> Grid Score

Column -> Show -> Mass weight

We see that best Grid scored molecules have higher energy than those that have lower Grid score energy. The vdw energy also have the same trend with Grid score.

VIII.Cartesian Minimization

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

Move to the directory 007.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 -o min.out

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                                             ../005.virtual_screen/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                                     yes
continuous_score_secondary                                   no
cont_score_rec_filename                                      ../001.files/4f4p_rec_h.mol2
cont_score_att_exp                                           6
cont_score_rep_exp                                           12
cont_score_rep_rad_scale                                     1
cont_score_use_dist_dep_dielectric                           yes
cont_score_dielectric                                        4.0
cont_score_vdw_scale                                         1
cont_score_es_scale                                          1
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
ligand_outfile_prefix                                        4f4p.virtualscreen.minimized
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Then in order to submit the script in to seawulf cluster, open a new script called cartesianmin.sh and write the following. 

 #!/bin/sh
 #SBATCH --partition=long-40core
 #SBATCH --time=48:00:00
 #SBATCH --nodes=1
 #SBATCH --ntasks=1
 #SBATCH --job-name=4f4p_cartesian
 #SBATCH --output=%x-%j.o

 echo "============================= SLURM JOB ================================="
 date
 echo
 echo " The job will be started on the following node(s):"
 echo $SLURM_JOB_NODELIST
 echo
 echo "Slurm user:                   $SLURM_JOB_USER"
 echo "Run directory:                $(pwd)"
 echo "Job ID:                       $SLURM_JOB_ID"
 echo "Job name:                     $SLURM_JOB_NAME"
 echo "Partition:                    $SLURM_JOB_PARTITION"
 echo "Number of nodes:              $SLURM_JOB_NUM_NODES"
 echo "Number of tasks:              $SLURM_NTASKS"
 echo "Submitted from:               $SLURM_SUBMIT_HOST:$SLURM_SUBMIT_DIR"
 echo "=========================================================================" 

 dock6 -i min.in -o min.out

Then submit the job into seawulf cluster. 

 sbatch cartesianmin.sh

Once it has done correctly, you will get a output file called 4f4p.virtualscreen.minimized_scored.mol2. This file contains molecules with minimized energies by removing unfavorable steric clashes.

IX Rescoring Docked Molecules

In order to rank our docked ligands and extract the best ligands (which has the most negative, lowest scores) based on different scoring functions like Footprint similarity, pharmacophore score, tanimoto score, the hungarian and the volume overlap score, we will be using Dock6 to rescore the virtual screen minimized ligands.

Move to the directory 008.rescore and 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                                             ../007.cartesianmin/4f4p.virtualscreen.minimized_scored.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_primary                                  no
 pharmacophore_score_secondary                                no
 descriptor_score_primary                                     yes
 descriptor_score_secondary                                   no
 descriptor_use_grid_score                                    no
 descriptor_use_multigrid_score                               no
 descriptor_use_continuous_score                              yes
 descriptor_use_footprint_similarity                          yes
 descriptor_use_pharmacophore_score                           yes
 descriptor_use_tanimoto                                      yes
 descriptor_use_hungarian                                     yes
 descriptor_use_volume_overlap                                yes
 descriptor_cont_score_rec_filename                           ../001.files/4f4p_rec_h.mol2
 descriptor_cont_score_att_exp                                6
 descriptor_cont_score_rep_exp                                12
 descriptor_cont_score_rep_rad_scale                          1
 descriptor_cont_score_use_dist_dep_dielectric                yes
 descriptor_cont_score_dielectric                             4.0
 descriptor_cont_score_vdw_scale                              1
 descriptor_cont_score_es_scale                               1
 descriptor_fps_score_use_footprint_reference_mol2            yes
 descriptor_fps_score_footprint_reference_mol2_filename       ../004.dock/4f4p.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.files/4f4p_rec_h.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.dock/4f4p.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/4f4p.lig.min_scored.mol2
 descriptor_hms_score_ref_filename                            ../004.dock/4f4p.lig.min_scored.mol2
 descriptor_hms_score_matching_coeff                          -5
 descriptor_hms_score_rmsd_coeff                              1
 descriptor_volume_score_reference_mol2_filename              ../004.dock/4f4p.lig.min_scored.mol2
 descriptor_volume_score_overlap_compute_method               analytical
 descriptor_weight_cont_score                                 1
 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/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 chem_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/chem.defn
 pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/ph4.defn
 ligand_outfile_prefix                                        descriptor.output
 write_footprints                                             yes
 write_hbonds                                                 yes
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Once you done the rescoring correctly, you will get several output files as below. 

 descriptor.output_footprint_scored.txt  
 descriptor.output_scored.mol2  
 descriptor.output_hbond_scored.txt 
 rescore.out

Open up the descriptor.output_scored.mol2 file along with original ligand and receptor and analyse the best ligands based on different scoring functions using viewdock property in Chimera.

Figure 16: Rescored ligands out file along with Receptor and original ligand (sorted according to the footprint similarity score values)
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