Difference between revisions of "2020 DOCK tutorial 4 with PDBID 6UZW"

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This tutorial is created by AMS 536 Spring 2020 Group 4. Group 4 members include Steven Pak, Caitlyn Cardetti, MiaoMiao He and Chuanzhou (Joey) Hu.
+
This tutorial is created by AMS 536 Spring 2020 Group 4. Group 4 members include Steven Pak, Caitlyn Cardetti, MiaoMiao He and Chuanzhou (Joey) Zhu.
  
 
How to for selecting protein: Pick a protein with few substrates and rotational bonds?
 
How to for selecting protein: Pick a protein with few substrates and rotational bonds?
Line 7: Line 7:
 
'''Dock Prep Program used: Chimera'''  
 
'''Dock Prep Program used: Chimera'''  
  
−
===I. Introduction ===
+
=I. Introduction=
  
−
=DOCK=
+
===DOCK===
 
DOCK 6.9 is a molecular modeling program that identifies interactions between chemical compounds and the receptors. DOCK has many features that can help with drug discovery( ex: Virtual Screening and Denovo). This tutorial will show us how to use Virtual Screening.  
 
DOCK 6.9 is a molecular modeling program that identifies interactions between chemical compounds and the receptors. DOCK has many features that can help with drug discovery( ex: Virtual Screening and Denovo). This tutorial will show us how to use Virtual Screening.  
  
 
===6UZW===
 
===6UZW===
−
The tutorial will be based on the PDB file 6UZE downloaded from the PDB Database.
+
The tutorial will be based on the PDB file 6UZW downloaded from the PDB Database.
−
6UZW is the crystal structure for GLUN1/GLUN2A complexed with (2S,3R)-1-[7-(2-carboxyethyl)phenanthrene-2-carbonyl]piperazine-2,3-dicarboxylic acid and glycine.  
+
6UZW is the crystal structure for GLUN1/GLUN2A complexed with (2S,3R)-1-[7-(2-carboxyethyl)phenanthrene-2-carbonyl]piperazine-2,3-dicarboxylic acid and glycine.
  
−
 
+
===Organization of Directories ===
−
== Organization of Directories ==
 
 
We set up the files in our project space as such. It will be helpful to have these folders ready ahead of time.
 
We set up the files in our project space as such. It will be helpful to have these folders ready ahead of time.
  
−
              00_files
+
      00_files
−
              01_dockprep
+
      01_dockprep
−
              02_surface_spheres
+
      02_surfacespheres
−
              03_gridbox
+
      03_gridbox
−
              04_dock
+
      04_dock
−
              05_footprint
+
      05_footprint
−
              06_virtual_screen
+
      06_virtualscreen
−
              07_virtual_screen_mpi
+
      07_virtualscreen_mpi
−
              08_cartesianmin
+
      08_cartesianmin
−
              09_rescore
+
      09_rescore
−
 
 
  
 
= II. Preparation of the ligand and receptor (preparation before the usage of DOCK6.9 =
 
= II. Preparation of the ligand and receptor (preparation before the usage of DOCK6.9 =
Line 50: Line 48:
 
===Preparation of Ligand without Hydrogens===
 
===Preparation of Ligand without Hydrogens===
 
   - Open 6UZW_complex_noH.mol2 through Chimera again
 
   - Open 6UZW_complex_noH.mol2 through Chimera again
−
   - Isolate the ligand by deleting the receptor. Select on the receptor by clicking on (Select --> Residue --> QM4 --> Select --> Inverted (selected models). The receptor should be surrounded by a green film.
+
   - Isolate the ligand by deleting the receptor. Select on the receptor by clicking on (Select --> Residue  
 +
    --> QM4 --> Select --> Inverted (selected models). The receptor should be surrounded by a green film.
 
   - Delete it (Actions -> Atoms/Bonds -> Delete).  
 
   - Delete it (Actions -> Atoms/Bonds -> Delete).  
−
   - The the ligand should be by itself with out any amino acids from the receptors and any water molecules.
+
   - The ligand should be by itself without any amino acids from the receptors and any water molecules.
 
   - Save the isolated ligand as a mol2 file (File -> Save mol2 -> 6UZW_lig_noH_mol2) into 01_dockprep
 
   - Save the isolated ligand as a mol2 file (File -> Save mol2 -> 6UZW_lig_noH_mol2) into 01_dockprep
 
This ligand has been prepared without Hydrogens.
 
This ligand has been prepared without Hydrogens.
  
 +
[[File:lig_noH.jpeg|thumb|center|500px|Figure . Lig with no Hydrogens]]
  
 
===Preparation of Receptor without Hydrogens===
 
===Preparation of Receptor without Hydrogens===
 
   - Open 6UZW_complex_noH.mol2 through Chimera again
 
   - Open 6UZW_complex_noH.mol2 through Chimera again
−
   - Isolate the ligand by deleting the receptor. Select on the receptor by clicking on (Select --> Residue --> Standard Amino acids --> Select --> Inverted (selected models). The ligand and water molecules should be surrounded by a green film.
+
   - Isolate the ligand by deleting the receptor.  
 +
  - Select on the receptor by clicking on (Select --> Residue --> Standard Amino acids --> Select -->  
 +
    Inverted (selected models).  
 +
The ligand and water molecules should be surrounded by a green film.
 
   - Delete it (Actions -> Atoms/Bonds -> Delete).  
 
   - Delete it (Actions -> Atoms/Bonds -> Delete).  
 
   - The the receptor should be by itself without the ligand
 
   - The the receptor should be by itself without the ligand
 
   - Save the isolated receptor as a mol2 file (File -> Save mol2 -> 6UZW_rec_noH_mol2) into 01_dockprep
 
   - Save the isolated receptor as a mol2 file (File -> Save mol2 -> 6UZW_rec_noH_mol2) into 01_dockprep
 
This ligand has been prepared without Hydrogens.
 
This ligand has been prepared without Hydrogens.
−
 
−
 
−
 
  
 
===Preparation of ligand with Hydrogens===
 
===Preparation of ligand with Hydrogens===
 
-Open the 6UZW_lig_noH.mol2
 
-Open the 6UZW_lig_noH.mol2
 
-Tools -> Surface Binding Analysis
 
-Tools -> Surface Binding Analysis
−
          Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
+
    Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
−
          Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field available for standard residues. Here we used AMBER ff14SB)
+
    Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field  
−
          Do not click or edit anything while going through this process.
+
    available for standard residues. Here we used AMBER ff14SB)
−
          Save as a mol2 file. (6UZW_lig_withH.mol2) into 01_dockprep
+
    Do not click or edit anything while going through this process.
 +
    Save as a mol2 file. (6UZW_lig_withH.mol2) into 01_dockprep
 +
 
 +
- If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor.
 +
    Tools -> Structure/Binding Analysis -> DockPrep
  
−
  - If you follow the step below all the above stated steps will automatically appear one after the other to prepare the receptor.  
+
[[File:lig_withH.jpg|thumb|center|500px|Figure . Lig with Hydrogens highlighted by green]]
−
          Tools -> Structure/Binding Analysis -> DockPrep
 
  
 
===Preparation of receptor with Hydrogens===
 
===Preparation of receptor with Hydrogens===
 
-Open the 6UZW_rec_noH.mol2
 
-Open the 6UZW_rec_noH.mol2
 
-Tools -> Surface Binding Analysis
 
-Tools -> Surface Binding Analysis
−
          Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
+
    Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
−
          Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field available for standard residues. Here we used AMBER ff14SB)
+
    Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field  
−
          Do not click or edit anything while going through this process.
+
    available for standard residues. Here we used AMBER ff14SB)
−
          Save as a mol2 file. (6UZW_rec_withH.mol2) into 01_dockprep
+
    Do not click or edit anything while going through this process.
 +
    Save as a mol2 file. (6UZW_rec_withH.mol2) into 01_dockprep
  
−
  - If you follow the step below all the above stated steps will automatically appear one after the other to prepare the receptor.  
+
- If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor.  
−
          Tools -> Structure/Binding Analysis -> DockPrep
+
    Tools -> Structure/Binding Analysis -> DockPrep
  
 
=III. Generating receptor surface and spheres=
 
=III. Generating receptor surface and spheres=
Line 124: Line 128:
 
Once sphgen command is successful, 6UZW_rec.sph file will be created. Open it up using Chimera along with 6UZW_rec_noH.mol2 file. You should get a similar output like the image below.
 
Once sphgen command is successful, 6UZW_rec.sph file will be created. Open it up using Chimera along with 6UZW_rec_noH.mol2 file. You should get a similar output like the image below.
  
−
[[File:Example.jpg]]
+
[[File:spheres_6UZW.jpeg|thumb|center|500px|Figure . Spheres files of 6UZW]]
  
 
===Selecting Spheres===
 
===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.
+
Here we will be selecting the spheres which define the binding pocket of the ligand because we are trying to direct the ligand towards that binding site rather than all over the receptor. To select the spheres type the following command.
  
−
   sphere_selector 2BXF_rec.sph ../1.dockprep/2BXF_lig_withH.mol2 10.0
+
   sphere_selector 6UZW_rec.sph ../01_dockprep/6UZW_lig_withH.mol2 10.0
−
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand binding site are kept and all the other spheres are deleted in the image below.
+
This command will select all of the spheres within 10.0 angstroms of the ligand and output them to selected_spheres.sph. Visualize the selected spheres using Chimera to make sure the correct spheres are selected. Notice that, spheres around the ligand-binding site are kept and all the other spheres are deleted in the image below.
−
 
 
−
[[File:2BXF_receptor_and_selected_spheres.jpg|thumb|center|400px|2nnq receptor and selected spheres]]
 
  
 +
[[File:selected_spheres_6UZW.jpeg|thumb|center|500px|Figure . Selected spheres files of 6UZW]]
  
  
Line 150: Line 153:
 
Step 4. Add charge.
 
Step 4. Add charge.
 
*Add charges Tools > Structure Editing > Add charge Typically use AM1-BCC
 
*Add charges Tools > Structure Editing > Add charge Typically use AM1-BCC
−
*You need to be careful about selecting charge because this will affect predictions for interactions (i.e. Coulomb's Law). Read paper corresponding to your PDB stucture to determine conditions of purification and proper protonation. In the case of 6UZW our paper of reference is: https://doi.org/10.1038/s41467-020-14321-0
+
*You need to be careful about selecting charge because this will affect predictions for interactions (i.e. Coulomb's Law). Read paper corresponding to your PDB structure to determine conditions of purification and proper protonation. In the case of 6UZW our paper of reference is: https://doi.org/10.1038/s41467-020-14321-0
  
−
Pose Reprodution
+
Pose Reproduction
 
Flexible, Fixed-anchor, rigid
 
Flexible, Fixed-anchor, rigid
 +
 +
===Generating the box and grid===
 +
 +
For this part, we will be generating a box and grid for docking the ligand. A space-defined box will be searching efficiently, and a grid-based energy system is beneficial for a rapid and thorough process.
 +
 +
Step 1. Create a Box.
 +
 +
  - Go to 03_gridbox folder
 +
  - Create a new input file to create a box by typing vi showbox.in and type the following lines inside the file.
 +
 +
 +
Y
 +
8.0
 +
../02_surface_spheres/selected_spheres.sph
 +
1
 +
6UZW.box.pdb
 +
 +
Those are basically parameters/answers for questions specified in the program. The first line Y represents "Yes, we want to create a box around the sphere". 8.0 indicates that the box will have a length of 8 Angstroms, in all six directions.  The third line specifies the sphere file should be used.  1 is the cluster number for creating the box. The last line 6UZW_box.pdb creates a file that contains the box.
 +
 +
Once the showbox.in file is ready, type the following command to create the box.
 +
 +
  showbox < showbox.in
 +
 +
Once this command is successful, 6UZW.box.pdb file will be created. Open it up using Chimera and you should be able to visualize the structure like the image below.
 +
 +
 +
Step 2. Generating the Grid
 +
 +
  -In the same directory, create a new file by typing vi grid.in and type the following lines inside the file.
 +
 +
compute_grids                            yes
 +
grid_spacing                              0.4
 +
output_molecule                          no
 +
contact_score                            no
 +
energy_score                              yes
 +
energy_cutoff_distance                    9999
 +
atom_model                                a
 +
attractive_exponent                      6
 +
repulsive_exponent                        9
 +
distance_dielectric                      yes
 +
dielectric_factor                        4
 +
bump_filter                              yes
 +
bump_overlap                              0.75
 +
receptor_file                            ../01_dockprep/6UZW_rec_prepped.mol2
 +
box_file                                  6UZW.box.pdb
 +
vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
score_grid_prefix                        grid
 +
 +
Once the file is ready, type the following command to generate the grid. More information can be found in http://dock.compbio.ucsf.edu/DOCK_6/tutorials/grid_generation/generating_grid.html
 +
 +
  grid -i grid.in -o grid.out
 +
 +
The grid calculation might takes a while to complete. But once this command is successful, four more files will be in 03_gridbox directory:
 +
 +
6uzw.box.pdb
 +
grid.in
 +
gridinfo.out
 +
grid.bmp
 +
grid.nrg
 +
showbox.in
 +
 +
Open it up using Chimera along with the receptor and make sure everything looks reasonable. If all is good, you should be able to visualize the structure like the image below.
 +
[[File:Grid-image.jpg|thumb|center|500px|Figure . Grid image of 6UZW]]
 +
 +
 +
 +
=IV.Energy Minimization and Footprint Analysis=
 +
 +
===Energy Minimization===
 +
 +
Here before performing docking, we will do energy minimization for the ligand to remove unfavorable clashes that influence how the rigid docking goes.  Rigid docking need to have a better-prepared ligand since the ligand will be docked as a whole, not like any other docking method which will take into account of several orientations and torsion angles while adding individual fragment.
 +
 +
Step 1. Create an input file.
 +
 +
  -Go to the directory 04_dock
 +
  -Create a new file by typing “touch min.in"
 +
 +
Step 2. Write into min.in file.
 +
For this part, we recommend you to write into the min.in file by answering them one by one instead of including content directly from previous. It’s a safer way to keep the answers and questions correspond with each other since the software update the question with time. Enter the command below:
 +
 
 +
    dock6 -i min.in
 +
 +
The parameters you choose for those questions should be similar to the following:
 +
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            ../01_dockprep/6UZW_lig_prepped.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              yes
 +
use_rmsd_reference_mol                                      yes
 +
rmsd_reference_filename                                      ../01_dockprep/6UZW_lig_prepped.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                                      ../03_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                                        6UZW_lig.min
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
 +
 +
If the process went smoothly, a new file “ 6UZW_lig.min_scored.mol2 ” will be generated. You can visualize the structure along with the receptor using Chimera and see how different it is from the initial ligand.
 +
 +
[[File:6UZW_receptor with the original ligand and the minimized ligand.png|thumb|center|500px|Figure . 6UZW_receptor with the original ligand and the minimized ligand]]
 +
 +
 +
===Bash Script for Surface Spheres Generation to Energy Minimization===
 +
 +
#!/bin/bash
 +
PDB="$1"
 +
#run sphgen
 +
echo "run sphgen"
 +
mkdir 02_surfacespheres
 +
cd 02_surfacespheres
 +
touch INSPH
 +
cat << EOF > INSPH
 +
../01_dockprep/${PDB}_rec_noH.dms
 +
R
 +
X
 +
0.0
 +
4.0
 +
1.4
 +
${PDB}_rec.sph
 +
EOF
 +
sphgen -i INSPH -o OUTSPH                                                         
 +
#sphere_selector
 +
echo "run sphere_selector"
 +
sphere_selector ../02_surfacespheres/${PDB}_rec.sph ../01_dockprep/${PDB}_lig_prepped.mol2 10.0
 +
#showbox
 +
echo "showbox"
 +
cd ..
 +
mkdir 03_gridbox
 +
cd 03_gridbox
 +
#touch showbox.in
 +
cat << EOF > showbox.in
 +
Y
 +
8.0
 +
../02_surfacespheres/selected_spheres.sph
 +
1
 +
${PDB}.box.pdb
 +
EOF
 +
showbox < showbox.in
 +
8.0
 +
../02_surfacespheres/selected_spheres.sph
 +
1
 +
${PDB}.box.pdb
 +
EOF
 +
showbox < showbox.in
 +
#grid
 +
echo "grid"
 +
#touch grid.in
 +
cat << EOF > grid.in
 +
compute_grids                            yes
 +
grid_spacing                              0.4
 +
output_molecule                          no
 +
contact_score                            no
 +
energy_score                              yes
 +
energy_cutoff_distance                    9999
 +
atom_model                                a
 +
attractive_exponent                      6
 +
repulsive_exponent                        9
 +
distance_dielectric                      yes
 +
dielectric_factor                        4
 +
bump_filter                              yes
 +
bump_overlap                              0.75
 +
receptor_file                            ../01_dockprep/${PDB}_rec_prepped.mol2
 +
box_file                                  ${PDB}.box.pdb
 +
vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 +
score_grid_prefix                        grid
 +
EOF
 +
grid -i grid.in -o gridinfo.out
 +
#touch min.in
 +
cat << EOF > min.in
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            ../01_dockprep/${PDB}_lig_prepped.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              yes
 +
use_rmsd_reference_mol                                      yes
 +
rmsd_reference_filename                                      ../01_dockprep/${PDB}_lig_prepped.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                                      ../03_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_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                                        ${PDB}_lig_min
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
EOF
 +
dock6 -i min.in -o min.out
 +
 +
===Molecular Footprint===
 +
 +
The molecular footprint is a common way to present how a ligand interacts with the receptor in terms of electrostatic interactions and Van der Waals interactions. Here, we will generate a molecular footprint of the ligand that interacts with the receptor before and after minimization.
 +
 +
Step 1. Create a new input file
 +
  -Go to directory 05_footprint
 +
  -create a new file by typing “touch footprint.in"
 +
 +
 +
 +
Step 2. Write into min.in file.
 +
Enter the command below:
 +
 
 +
    dock6 -i footprint.in
 +
 +
Again, the file will be all set by answering a set of questions. The parameters you choose for those questions should be similar to the following:
 +
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          no
 +
ligand_atom_file                                            '''../04_dock/6UZW_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                  '''../01_dockprep/6UZW_lig_prepped.mol2'''
 +
fps_score_foot_compare_type                                  Euclidean
 +
fps_score_normalize_foot                                    no
 +
fps_score_foot_comp_all_residue                              yes
 +
fps_score_receptor_filename                                  '''../01_dockprep/6UZW_rec_prepped.mol2'''
 +
fps_score_vdw_att_exp                                        6
 +
fps_score_vdw_rep_exp                                        12
 +
fps_score_vdw_rep_rad_scale                                  1
 +
fps_score_use_distance_dependent_dielectric                  yes
 +
fps_score_dielectric                                        4.0
 +
fps_score_vdw_fp_scale                                      1
 +
fps_score_es_fp_scale                                        1
 +
fps_score_hb_fp_scale                                        0
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_secondary                                  no
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              no
 +
atom_model                                                  all                                     
 +
vdw_defn_file                                '''/gpfs/projects/AMS536/zzz.programs/dock6.9_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                                        '''6UZW_footprint_min_cryst'''
 +
write_footprints                                            yes
 +
write_hbonds                                                yes
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no                                       
 +
 +
Once everything is successfully processed, several output files should be generated:
 +
 +
footprint.out
 +
6UZW_footprint_min_cryst_footprint_scored.txt
 +
6UZW_footprint_min_cryst_hbond_scored.txt
 +
6UZW_footprint_min_cryst_scored.mol2
 +
 +
In order to visualize the result of the molecular footprint. A Python command,  which could be accessed in the previous DOCK tutorials, will be used to generate a PDF file. The PDF file will list the first 20 amino acids with large deviations in energy.
 +
 +
/gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py /PATHto/filename 20
 +
1. first input is the path to "6UZW_footprint_min_cryst_footprint_scored.txt" in this case
 +
2. the second input is the number of max residues you want to plot on the pdf below.
 +
 +
[[File:Footprintpdf.jpeg|thumb|center|500px|Footprint of 6UZW]]
 +
 +
=V.Docking=
 +
===Rigid Docking===
 +
We perform the rigid docking first. Create an input file by typing "touch rigid.in" in the directory 04_dock.
 +
Write in the rigid.in by answering the following questions manually.
 +
  touch rigid.in
 +
  dock6 -i rigid.in
 +
The corresponding parameters and answers for the questions should be like this:
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            '''6UZW_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                                      '''6UZW_lig.min_scored.mol2'''
 +
use_database_filter                                          no
 +
orient_ligand                                                yes
 +
automated_matching                                          yes
 +
receptor_site_file                                          '''../02_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                                      '''../03_gridbox/grid'''
 +
multigrid_score_secondary                                    no
 +
dock3.5_score_secondary                                      no
 +
continuous_score_secondary                                  no
 +
footprint_similarity_score_secondary                        no
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_secondary                                  no
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              yes
 +
simplex_max_iterations                                      1000
 +
simplex_tors_premin_iterations                              0
 +
simplex_max_cycles                                          1
 +
simplex_score_converge                                      0.1
 +
simplex_cycle_converge                                      1.0
 +
simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                              '''/gpfs/projects/AMS536/zzz.programs/dock6.9_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                                        1
 +
rank_ligands                                                no
 +
It is highly recommended to double check the rigid.in by typing "vi rigid in". Edit all typos and make sure all paths correct because that is the most commonly happended error.
 +
Run dock using the created input file by entering the command:
 +
dock6 -i rigid.in -o rigid.out
 +
Once the rigid docking completed, two more files (rigid.out and rigid.out_scored.mol2) will be generated. Then the mol2 file can be visualized by Chimera which should be similar to the figure below.
 +
 +
 +
[[File:Rigid_results.jpg|thumb|center|500px]]
 +
 +
 +
===Fixed Anchor Docking===
 +
The procedure of fixed anchor docking is similar to rigid docking that we just did. Create an input file of fixed anchor docking in the directory 4_dock and write in the file.
 +
  touch fixed.in
 +
  dock6 -i fixed.in
 +
Answering a set of questions manually with the following parameters.
 +
conformer_search_type                                        flex
 +
write_fragment_libraries                                    no
 +
user_specified_anchor                                        no
 +
limit_max_anchors                                            no
 +
min_anchor_size                                              5
 +
pruning_use_clustering                                      yes
 +
pruning_max_orients                                          1000
 +
pruning_clustering_cutoff                                    100
 +
pruning_conformer_score_cutoff                              100.0
 +
pruning_conformer_score_scaling_factor                      1.0
 +
use_clash_overlap                                            no
 +
write_growth_tree                                            no
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            '''../01_dockprep/6UZW_lig_prepped.mol2'''
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              yes
 +
use_rmsd_reference_mol                                      yes
 +
rmsd_reference_filename                                      '''../01_dockprep/6UZW_lig_prepped.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                                      '''../03_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                                        fixed.out
 +
write_orientations                                          no
 +
num_scored_conformers                                        100
 +
write_conformations                                          no
 +
cluster_conformations                                        yes
 +
cluster_rmsd_threshold                                      2.0
 +
rank_ligands                                                no                                         
 +
Run dock6 with the created input file. Once the fixed anchor docking completed, two more output files (fixed.out and fixed.out_scored.mol2) will be generated.
 +
  dock6 -i fixed.in -o fixed.out
 +
Visualize the docked pose with Chimera in the same way used in rigid docking. It should look like the figure below.
 +
 +
[[File:Fixed_results.jpg|thumb|center|500px]]
 +
===Flexible Docking===
 +
Create an input file of flexible docking in the directory 4_dock and complete the file.
 +
  touch flex.in
 +
  dock6 -i flex.in
 +
Answering a set of questions manually with the following parameters.
 +
conformer_search_type                                        flex
 +
write_fragment_libraries                                    no
 +
user_specified_anchor                                        no
 +
limit_max_anchors                                            no
 +
min_anchor_size                                              5
 +
pruning_use_clustering                                      yes
 +
pruning_max_orients                                          1000
 +
pruning_clustering_cutoff                                    100
 +
pruning_conformer_score_cutoff                              100.0
 +
pruning_conformer_score_scaling_factor                      1.0
 +
use_clash_overlap                                            no
 +
write_growth_tree                                            no
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            '''6UZW_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                                      '''6UZW_lig.min_scored.mol2'''
 +
use_database_filter                                          no
 +
orient_ligand                                                yes
 +
automated_matching                                          yes
 +
receptor_site_file                                          '''../02_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                                      '''../03_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                                         
 +
Run dock6 with the created input file. Once the flexible docking completed, two more files (flex.out and flex.out_scored.mol2) will be generated.
 +
  dock6 -i flex.in -o flex.out
 +
Visualize the docked pose with Chimera. It should look like the figure below.
 +
[[File:Flex_results.jpg|thumb|center|500px]]
 +
 +
 +
===Virtual Screen===
 +
Move into directory 06_virtualscreen
 +
cd ../06_virtualscreen
 +
 +
Copy the library into directory 06_virtualscreen
 +
cp /gpfs/projects/AMS536/zzz.programs/VS_library_5K.mol2 ./
 +
 +
Create an input file in the directory 06_virtualscreen and complete the file.
 +
touch virtual.in
 +
dock6 -i virtual.in
 +
 +
Answering a set of questions manually with the following parameters.
 +
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                                          ../02_surfacespheres/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                                      ../03_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
 +
 +
Run dock6 with the created input file in SLURM.
 +
To run in SLURM, create a bash script.
 +
Touch bashvirtualscreen.sh
 +
 +
vi bashvirtualscreen.sh
 +
 +
#!/bin/bash
 +
#SBATCH --time=48:00:00
 +
#SBATCH --nodes=1
 +
#SBATCH --ntasks=24
 +
#SBATCH --job-name=6UZW_VS
 +
#SBATCH --output=6UZW_VS_out
 +
#SBATCH -p long-40core
 +
 +
cd $SLURM_SUBMIT_DIR
 +
 +
/gpfs/projects/AMS536/zzz.programs/dock6.9_release/bin/dock6 -i virtual.in -o virtual.out
 +
 +
Queue your bash script in SLURM
 +
qsub bashvirtualscreen.sh
 +
 +
You will be given a reference number.
 +
To check status of your job:
 +
qstat -u NETID

Latest revision as of 14:45, 3 March 2020

This tutorial is created by AMS 536 Spring 2020 Group 4. Group 4 members include Steven Pak, Caitlyn Cardetti, MiaoMiao He and Chuanzhou (Joey) Zhu.

How to for selecting protein: Pick a protein with few substrates and rotational bonds?

The PDB code chosen is 6UZW which is for the crystal structure of GLUN1/GLUN2A ligand-binding domain in complex with glycine and UBP791.

Dock Prep Program used: Chimera

I. Introduction

DOCK

DOCK 6.9 is a molecular modeling program that identifies interactions between chemical compounds and the receptors. DOCK has many features that can help with drug discovery( ex: Virtual Screening and Denovo). This tutorial will show us how to use Virtual Screening.

6UZW

The tutorial will be based on the PDB file 6UZW downloaded from the PDB Database. 6UZW is the crystal structure for GLUN1/GLUN2A complexed with (2S,3R)-1-[7-(2-carboxyethyl)phenanthrene-2-carbonyl]piperazine-2,3-dicarboxylic acid and glycine.

Organization of Directories

We set up the files in our project space as such. It will be helpful to have these folders ready ahead of time. 

     00_files
     01_dockprep
     02_surfacespheres
     03_gridbox
     04_dock
     05_footprint
     06_virtualscreen
     07_virtualscreen_mpi
     08_cartesianmin
     09_rescore

II. Preparation of the ligand and receptor (preparation before the usage of DOCK6.9

- Download 6UZW to local computer off of the PDB database
- Save the .pdb file into your 00_files directory.

Checking the Structure and Preparing the Complex without Hydrogens

- Open Chimera, open the downloaded .pdb file, and check the structure for missing residues, gaps, heme groups, missing loops, and size. We recommend using proteins with no heme groups and proteins of a relatively smaller size.
- Save the .pdb file (the ligand and the receptor) as a mol2 file (6UZW_complex_noH.mol2)
- When preparing both the ligand the receptor, you can open this complex mol2 file and delete the component that you won't need (eg. for preparing the receptor, open the complex, delete the ligand, and save).

This complex (receptor and ligand) has been prepared without Hydrogens.



Preparation of Ligand without Hydrogens

 - Open 6UZW_complex_noH.mol2 through Chimera again
 - Isolate the ligand by deleting the receptor. Select on the receptor by clicking on (Select --> Residue 
   --> QM4 --> Select --> Inverted (selected models). The receptor should be surrounded by a green film.
 - Delete it (Actions -> Atoms/Bonds -> Delete). 
 - The ligand should be by itself without any amino acids from the receptors and any water molecules.
 - Save the isolated ligand as a mol2 file (File -> Save mol2 -> 6UZW_lig_noH_mol2) into 01_dockprep

This ligand has been prepared without Hydrogens.

Figure . Lig with no Hydrogens

Preparation of Receptor without Hydrogens

 - Open 6UZW_complex_noH.mol2 through Chimera again
 - Isolate the ligand by deleting the receptor. 
 - Select on the receptor by clicking on (Select --> Residue --> Standard Amino acids --> Select --> 
   Inverted (selected models).

The ligand and water molecules should be surrounded by a green film. 

 - Delete it (Actions -> Atoms/Bonds -> Delete). 
 - The the receptor should be by itself without the ligand
 - Save the isolated receptor as a mol2 file (File -> Save mol2 -> 6UZW_rec_noH_mol2) into 01_dockprep

This ligand has been prepared without Hydrogens.

Preparation of ligand with Hydrogens

-Open the 6UZW_lig_noH.mol2 -Tools -> Surface Binding Analysis 

   Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
   Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field 
   available for standard residues. Here we used AMBER ff14SB)
   Do not click or edit anything while going through this process.
   Save as a mol2 file. (6UZW_lig_withH.mol2) into 01_dockprep

- If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor. 

   Tools -> Structure/Binding Analysis -> DockPrep
Figure . Lig with Hydrogens highlighted by green

Preparation of receptor with Hydrogens

-Open the 6UZW_rec_noH.mol2 -Tools -> Surface Binding Analysis 

   Tools -> Structure Editing -> Add H (To add Hydrogen atoms)
   Tools -> Structure Editing -> Add Charge (To add the charge use the latest AMBER force field 
   available for standard residues. Here we used AMBER ff14SB)
   Do not click or edit anything while going through this process.
   Save as a mol2 file. (6UZW_rec_withH.mol2) into 01_dockprep

- If you follow the step below all the above-stated steps will automatically appear one after the other to prepare the receptor. 

    Tools -> Structure/Binding Analysis -> DockPrep

III. Generating receptor surface and spheres

Preparation of DMS file

 - Open 6UZW_rec_noH.mol2 using chimera.
 - Action -> Surface -> Show
 - Tools -> Structure Editing -> Write DMS
 - Save the 6UZW_rec_noH.dms into 02_surface_spheres folder

Reopen the file and make sure the surface was generated.

Transfer all the folders created so far to seawulf cluster to be used in DOCK using the Secure Copy command on unix from the local terminal that you have saved your files. NOT from the seawulf terminal 

scp -r [FROM] [TO]
scp -r PATH/folder/ PATH/seawulf_folder

Generating spheres

 - Go to 02_surface_spheres folder
 - Create a new input file to create spheres by typing vim INSPH and type the following lines inside the file. 

6UZW_rec_noH.dms
R
X
0.0
4.0
1.4
6UZW_rec.sph

The first line 6UZW_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 6UZW_spheres.sph creates a sphere 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, 6UZW_rec.sph file will be created. Open it up using Chimera along with 6UZW_rec_noH.mol2 file. You should get a similar output like the image below.

Figure . Spheres files of 6UZW

Selecting Spheres

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

 sphere_selector 6UZW_rec.sph ../01_dockprep/6UZW_lig_withH.mol2 10.0

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

Figure . Selected spheres files of 6UZW


Step 1. Separate protein and ligand

Step 2. Delete H2O molecules in Chimera to reduce computing time.

  • To delete: Select > Residues > H O H - they should be highlighted Click Actions > Atoms/Bonds > delete H2O molecules should now be deleted.

Step 3. Add H's.

  • PDB codes do not contain H's due to low electron density.
  • To add H's Tools > Structure Editing > AddH
  • H's MUST be added before you add charge or they will not be accounted for and will affect predictions for interactions.

Step 4. Add charge.

  • Add charges Tools > Structure Editing > Add charge Typically use AM1-BCC
  • You need to be careful about selecting charge because this will affect predictions for interactions (i.e. Coulomb's Law). Read paper corresponding to your PDB structure to determine conditions of purification and proper protonation. In the case of 6UZW our paper of reference is: https://doi.org/10.1038/s41467-020-14321-0

Pose Reproduction Flexible, Fixed-anchor, rigid

Generating the box and grid

For this part, we will be generating a box and grid for docking the ligand. A space-defined box will be searching efficiently, and a grid-based energy system is beneficial for a rapid and thorough process.

Step 1. Create a Box. 

 - Go to 03_gridbox folder
 - Create a new input file to create a box by typing vi showbox.in and type the following lines inside the file. 



Y
8.0
../02_surface_spheres/selected_spheres.sph
1
6UZW.box.pdb

Those are basically parameters/answers for questions specified in the program. The first line Y represents "Yes, we want to create a box around the sphere". 8.0 indicates that the box will have a length of 8 Angstroms, in all six directions. The third line specifies the sphere file should be used. 1 is the cluster number for creating the box. The last line 6UZW_box.pdb creates a file that contains the box.

Once the showbox.in file is ready, type the following command to create the box. 

  showbox < showbox.in

Once this command is successful, 6UZW.box.pdb file will be created. Open it up using Chimera and you should be able to visualize the structure like the image below.


Step 2. Generating the Grid 

 -In the same directory, create a new file by typing vi grid.in and type the following lines inside the file.

compute_grids                             yes
grid_spacing                              0.4
output_molecule                           no
contact_score                             no
energy_score                              yes
energy_cutoff_distance                    9999
atom_model                                a
attractive_exponent                       6
repulsive_exponent                        9
distance_dielectric                       yes
dielectric_factor                         4
bump_filter                               yes
bump_overlap                              0.75
receptor_file                             ../01_dockprep/6UZW_rec_prepped.mol2
box_file                                  6UZW.box.pdb
vdw_definition_file                      /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
score_grid_prefix                        grid

Once the file is ready, type the following command to generate the grid. More information can be found in http://dock.compbio.ucsf.edu/DOCK_6/tutorials/grid_generation/generating_grid.html 

 grid -i grid.in -o grid.out

The grid calculation might takes a while to complete. But once this command is successful, four more files will be in 03_gridbox directory: 

6uzw.box.pdb	
grid.in		
gridinfo.out
grid.bmp	
grid.nrg	
showbox.in

Open it up using Chimera along with the receptor and make sure everything looks reasonable. If all is good, you should be able to visualize the structure like the image below.

Figure . Grid image of 6UZW


IV.Energy Minimization and Footprint Analysis

Energy Minimization

Here before performing docking, we will do energy minimization for the ligand to remove unfavorable clashes that influence how the rigid docking goes. Rigid docking need to have a better-prepared ligand since the ligand will be docked as a whole, not like any other docking method which will take into account of several orientations and torsion angles while adding individual fragment.

Step 1. Create an input file. 

 -Go to the directory 04_dock 
 -Create a new file by typing “touch min.in"

Step 2. Write into min.in file. For this part, we recommend you to write into the min.in file by answering them one by one instead of including content directly from previous. It’s a safer way to keep the answers and questions correspond with each other since the software update the question with time. Enter the command below: 

   dock6 -i min.in

The parameters you choose for those questions should be similar to the following: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../01_dockprep/6UZW_lig_prepped.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01_dockprep/6UZW_lig_prepped.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                                       ../03_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                                        6UZW_lig.min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


If the process went smoothly, a new file “ 6UZW_lig.min_scored.mol2 ” will be generated. You can visualize the structure along with the receptor using Chimera and see how different it is from the initial ligand.

Figure . 6UZW_receptor with the original ligand and the minimized ligand


Bash Script for Surface Spheres Generation to Energy Minimization

#!/bin/bash
PDB="$1"
#run sphgen
echo "run sphgen"
mkdir 02_surfacespheres
cd 02_surfacespheres
touch INSPH
cat << EOF > INSPH
../01_dockprep/${PDB}_rec_noH.dms
R
X
0.0
4.0
1.4
${PDB}_rec.sph
EOF
sphgen -i INSPH -o OUTSPH                                                          
#sphere_selector
echo "run sphere_selector"
sphere_selector ../02_surfacespheres/${PDB}_rec.sph ../01_dockprep/${PDB}_lig_prepped.mol2 10.0
#showbox
echo "showbox"
cd ..
mkdir 03_gridbox
cd 03_gridbox
#touch showbox.in
cat << EOF > showbox.in
Y
8.0
../02_surfacespheres/selected_spheres.sph
1
${PDB}.box.pdb
EOF
showbox < showbox.in
8.0
../02_surfacespheres/selected_spheres.sph
1
${PDB}.box.pdb
EOF
showbox < showbox.in
#grid
echo "grid"
#touch grid.in
cat << EOF > grid.in
compute_grids                             yes
grid_spacing                              0.4
output_molecule                           no
contact_score                             no
energy_score                              yes
energy_cutoff_distance                    9999
atom_model                                a
attractive_exponent                       6
repulsive_exponent                        9
distance_dielectric                       yes
dielectric_factor                         4
bump_filter                               yes
bump_overlap                              0.75
receptor_file                             ../01_dockprep/${PDB}_rec_prepped.mol2
box_file                                  ${PDB}.box.pdb
vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
score_grid_prefix                         grid
EOF
grid -i grid.in -o gridinfo.out
#touch min.in
cat << EOF > min.in
conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../01_dockprep/${PDB}_lig_prepped.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01_dockprep/${PDB}_lig_prepped.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                                       ../03_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_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                                        ${PDB}_lig_min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no 
EOF
dock6 -i min.in -o min.out

Molecular Footprint

The molecular footprint is a common way to present how a ligand interacts with the receptor in terms of electrostatic interactions and Van der Waals interactions. Here, we will generate a molecular footprint of the ligand that interacts with the receptor before and after minimization.

Step 1. Create a new input file 

 -Go to directory 05_footprint
 -create a new file by typing “touch footprint.in"


Step 2. Write into min.in file. Enter the command below: 

   dock6 -i footprint.in

Again, the file will be all set by answering a set of questions. The parameters you choose for those questions should be similar to the following: 

conformer_search_type                                        rigid
use_internal_energy                                          no
ligand_atom_file                                             ../04_dock/6UZW_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                  ../01_dockprep/6UZW_lig_prepped.mol2
fps_score_foot_compare_type                                  Euclidean
fps_score_normalize_foot                                     no
fps_score_foot_comp_all_residue                              yes
fps_score_receptor_filename                                  ../01_dockprep/6UZW_rec_prepped.mol2
fps_score_vdw_att_exp                                        6
fps_score_vdw_rep_exp                                        12
fps_score_vdw_rep_rad_scale                                  1
fps_score_use_distance_dependent_dielectric                  yes
fps_score_dielectric                                         4.0
fps_score_vdw_fp_scale                                       1
fps_score_es_fp_scale                                        1
fps_score_hb_fp_scale                                        0
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              no
atom_model                                                   all                                       
vdw_defn_file                                 /gpfs/projects/AMS536/zzz.programs/dock6.9_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                                        6UZW_footprint_min_cryst
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Once everything is successfully processed, several output files should be generated: 

footprint.out
6UZW_footprint_min_cryst_footprint_scored.txt
6UZW_footprint_min_cryst_hbond_scored.txt
6UZW_footprint_min_cryst_scored.mol2

In order to visualize the result of the molecular footprint. A Python command, which could be accessed in the previous DOCK tutorials, will be used to generate a PDF file. The PDF file will list the first 20 amino acids with large deviations in energy. 

/gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py /PATHto/filename 20
1. first input is the path to "6UZW_footprint_min_cryst_footprint_scored.txt" in this case
2. the second input is the number of max residues you want to plot on the pdf below.
Footprint of 6UZW

V.Docking

Rigid Docking

We perform the rigid docking first. Create an input file by typing "touch rigid.in" in the directory 04_dock. Write in the rigid.in by answering the following questions manually. 

 touch rigid.in
 dock6 -i rigid.in

The corresponding parameters and answers for the questions should be like this: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             6UZW_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                                      6UZW_lig.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../02_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                                       ../03_gridbox/grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                              /gpfs/projects/AMS536/zzz.programs/dock6.9_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                                        1
rank_ligands                                                 no

It is highly recommended to double check the rigid.in by typing "vi rigid in". Edit all typos and make sure all paths correct because that is the most commonly happended error. Run dock using the created input file by entering the command: 

dock6 -i rigid.in -o rigid.out

Once the rigid docking completed, two more files (rigid.out and rigid.out_scored.mol2) will be generated. Then the mol2 file can be visualized by Chimera which should be similar to the figure below.


Rigid results.jpg


Fixed Anchor Docking

The procedure of fixed anchor docking is similar to rigid docking that we just did. Create an input file of fixed anchor docking in the directory 4_dock and write in the file. 

 touch fixed.in
 dock6 -i fixed.in

Answering a set of questions manually with the following parameters. 

conformer_search_type                                        flex
write_fragment_libraries                                     no
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../01_dockprep/6UZW_lig_prepped.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01_dockprep/6UZW_lig_prepped.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                                       ../03_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                                        fixed.out
write_orientations                                           no
num_scored_conformers                                        100
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Run dock6 with the created input file. Once the fixed anchor docking completed, two more output files (fixed.out and fixed.out_scored.mol2) will be generated. 

 dock6 -i fixed.in -o fixed.out

Visualize the docked pose with Chimera in the same way used in rigid docking. It should look like the figure below.

Fixed results.jpg

Flexible Docking

Create an input file of flexible docking in the directory 4_dock and complete the file. 

 touch flex.in
 dock6 -i flex.in

Answering a set of questions manually with the following parameters. 

conformer_search_type                                        flex
write_fragment_libraries                                     no
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             6UZW_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                                      6UZW_lig.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../02_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                                       ../03_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

Run dock6 with the created input file. Once the flexible docking completed, two more files (flex.out and flex.out_scored.mol2) will be generated. 

 dock6 -i flex.in -o flex.out

Visualize the docked pose with Chimera. It should look like the figure below.

Flex results.jpg


Virtual Screen

Move into directory 06_virtualscreen 

cd ../06_virtualscreen

Copy the library into directory 06_virtualscreen 

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

Create an input file in the directory 06_virtualscreen and complete the file. 

touch virtual.in
dock6 -i virtual.in

Answering a set of questions manually with the following parameters. 

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                                           ../02_surfacespheres/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                                       ../03_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

Run dock6 with the created input file in SLURM. To run in SLURM, create a bash script. 

Touch bashvirtualscreen.sh

vi bashvirtualscreen.sh

#!/bin/bash
#SBATCH --time=48:00:00
#SBATCH --nodes=1
#SBATCH --ntasks=24
#SBATCH --job-name=6UZW_VS
#SBATCH --output=6UZW_VS_out
#SBATCH -p long-40core

cd $SLURM_SUBMIT_DIR

/gpfs/projects/AMS536/zzz.programs/dock6.9_release/bin/dock6 -i virtual.in -o virtual.out

Queue your bash script in SLURM 

qsub bashvirtualscreen.sh

You will be given a reference number. To check status of your job: 

qstat -u NETID
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