Difference between revisions of "2023 DOCK tutorial 3 with PDBID 2P16"

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(→‎Grid Generation)
 
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Line 86: Line 86:
 
Save the file as 2p16_noh_ligand.mol2 using File-> Save Mol2
 
Save the file as 2p16_noh_ligand.mol2 using File-> Save Mol2
  
−
[[File:2p16_noh_ligand.png|thumb|center]]
+
[[File:2p16nohligand.png|thumb|center]]
  
 
Add hydrogens and charge using Dockprep. Save the file as 2p16_dockprep_ligand.mol2
 
Add hydrogens and charge using Dockprep. Save the file as 2p16_dockprep_ligand.mol2
Line 175: Line 175:
 
<pre>showbox < showbox.in</pre>
 
<pre>showbox < showbox.in</pre>
  
−
[[File:2p16_gridbox.jpg]]
+
[[File:2p16_gridbox.png|thumb|center]]
  
 
=== '''Grid Generation''' ===
 
=== '''Grid Generation''' ===
  
 
Open up an input file titled grid.in and type in the following:
 
Open up an input file titled grid.in and type in the following:
−
 
+
<pre>allow_non_integral_charges                no
−
<pre>compute_grids                            yes
+
compute_grids                            yes
 
grid_spacing                              0.4
 
grid_spacing                              0.4
 
output_molecule                          no
 
output_molecule                          no
Line 270: Line 270:
 
This should save a new file called 2p16_ligand.min_scored.mol2. Open it up in Chimera along with the protein and ligand mol2 files for comparison.  
 
This should save a new file called 2p16_ligand.min_scored.mol2. Open it up in Chimera along with the protein and ligand mol2 files for comparison.  
  
−
[[File:2p16_ligand_min_scored.jpg]]
+
[[File:2p16ligandminscored.png|thumb|center]]
  
 
You can also view the RMSD values in the header of the file using:  
 
You can also view the RMSD values in the header of the file using:  
Line 278: Line 278:
 
=='''Footprint Analysis'''==
 
=='''Footprint Analysis'''==
  
−
We can use footprinting to visualize the protein-ligand interactrion energies on a per-residue basis.  
+
We can use footprinting to visualize the protein-ligand interaction energies on a per-residue basis.  
  
 
In the same dock directory open up an input file called footprint.in and type in the following:
 
In the same dock directory open up an input file called footprint.in and type in the following:
Line 473: Line 473:
 
  internal_energy_rep_exp                                      12
 
  internal_energy_rep_exp                                      12
 
  internal_energy_cutoff                                      100.0
 
  internal_energy_cutoff                                      100.0
−
  ligand_atom_file                                            2p16.charge.mol2
+
  ligand_atom_file                                            2p16.ligand.min_scored.mol2
 
  limit_max_ligands                                            no
 
  limit_max_ligands                                            no
 
  skip_molecule                                                no
 
  skip_molecule                                                no
Line 479: Line 479:
 
  calculate_rmsd                                              yes
 
  calculate_rmsd                                              yes
 
  use_rmsd_reference_mol                                      yes
 
  use_rmsd_reference_mol                                      yes
−
  rmsd_reference_filename                                      2p16.charge.mol2
+
  rmsd_reference_filename                                      2p16.ligand.min_scored.mol2
 
  use_database_filter                                          no
 
  use_database_filter                                          no
 
  orient_ligand                                                no
 
  orient_ligand                                                no
Line 668: Line 668:
  
 
By looking at the comparison (green is the flex output), you can see that the pose reproduction went successfully. The output file showed the RMSD of 0.92 Å, which also indicates a success. This structure was found in the first in scored.mol2 showing that this pose was in fact the best pose after rotational bond and orientation searching.
 
By looking at the comparison (green is the flex output), you can see that the pose reproduction went successfully. The output file showed the RMSD of 0.92 Å, which also indicates a success. This structure was found in the first in scored.mol2 showing that this pose was in fact the best pose after rotational bond and orientation searching.
 +
 +
== ''' Virtual Screening ''' ==
 +
 +
Virtual screening is a computational method of docking and ranking potential small molecule drug candidates. Leveraging the knowledge of the three-dimensional structure of the target receptor, virtual screening involves the systematic evaluation of a large library of small molecules by simulating their interactions with the target. By employing van der Waals, electrostatic, and interal energy scoring functions, virtual screening provides a way to rank and evaluate potential small molecule ligands.
 +
 +
For this tutorial, you will be using the VS_library_5K.mol2, located in /gpfs/projects/AMS536/zzz.programs/ and copied to our virtual screening directory. You can create a virtual screening directory (and then go into it) if you haven’t already:
 +
 +
<pre>
 +
mkdir 005.virtual_screen
 +
cd 005.virtual_screen
 +
</pre>
 +
 +
After that, create a new input file, vs.in:
 +
<pre>
 +
search_type                                                  flex
 +
write_fragment_libraries                                    no
 +
user_specified_anchor                                        no
 +
limit_max_anchors                                            no
 +
min_anchor_size                                              5
 +
pruning_use_clustering                                      yes
 +
pruning_max_orients                                          1000
 +
pruning_clustering_cutoff                                    100
 +
pruning_conformer_score_cutoff                              100.0
 +
pruning_conformer_score_scaling_factor                      1.0
 +
use_clash_overlap                                            no
 +
write_growth_tree                                            no
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      9
 +
internal_energy_cutoff                                      100.0
 +
ligand_atom_file                                            VS_library_5K.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              no
 +
use_database_filter                                          no
 +
orient_ligand                                                yes
 +
automated_matching                                          yes
 +
receptor_site_file                                          ../002.surface_spheres/selected_spheres.sph
 +
max_orientations                                            1000
 +
critical_points                                              no
 +
chemical_matching                                            no
 +
use_ligand_spheres                                          no
 +
bump_filter                                                  no
 +
score_molecules                                              yes
 +
contact_score_primary                                        no
 +
contact_score_secondary                                      no
 +
grid_score_primary                                          yes
 +
grid_score_secondary                                        no
 +
grid_score_rep_rad_scale                                    1
 +
grid_score_vdw_scale                                        1
 +
grid_score_es_scale                                          1
 +
grid_score_grid_prefix                                      ../003.gridbox/grid
 +
multigrid_score_secondary                                    no
 +
dock3.5_score_secondary                                      no
 +
continuous_score_secondary                                  no
 +
footprint_similarity_score_secondary                        no
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_secondary                                  no
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              yes
 +
minimize_anchor                                              yes
 +
minimize_flexible_growth                                    yes
 +
use_advanced_simplex_parameters                              no
 +
simplex_max_cycles                                          1
 +
simplex_score_converge                                      0.1
 +
simplex_cycle_converge                                      1.0
 +
simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_anchor_max_iterations                                500
 +
simplex_grow_max_iterations                                  500
 +
simplex_grow_tors_premin_iterations                          0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        2p16_virtual_5k.out
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
</pre>
 +
 +
To make sure the virtual screen finishes in a timely manner, you will be utilizing MPI parallelization, which will allow you to use multiple nodes simultaneously for more processing power. Create a new file, 2p16_VS_5K.slurm, to give SLURM instructions on carrying out the virtual screen. It should look something like this:
 +
 +
<pre>
 +
#!/bin/sh
 +
#SBATCH --partition=long-28core
 +
#SBATCH --time=2-00:00:00
 +
#SBATCH --nodes=4
 +
#SBATCH --ntasks=112
 +
#SBATCH --job-name=2p16_5k_vs
 +
#SBATCH --output=log_2p16_5k_vs.txt
 +
 +
echo "starting DOCK6.10 Simulation"
 +
module load intel_stack
 +
 +
mpirun -v -np 112 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i vs.in -o vs.out
 +
echo "done"
 +
</pre>
 +
 +
This might be a bit overkill, so you may want to reduce the number of nodes or use a queue with fewer cores. The "echo" lines are there to provide an idea of whether the job started/finished properly.
 +
 +
To submit this job to the queue, run:
 +
<pre>
 +
sbatch 2p16_VS_5K.slurm
 +
</pre>
 +
 +
Each core will produce its own .out output file, but the only one of importance will be vs.out. This file should be transferred over to your local computer for viewing within Chimera, along with 2p16_virtual_5k.out_scored.mol2, which will contain the positions of the highest-scored ligands. Shown below are the aforementioned results in Chimera, as well as the comparison of the currently best-scored ligand and the original ligand:
 +
 +
[[File:2P16_vsresult_original_withRec.png|thumb|center|500px|The original ligand (blue) compared to the best-scored ligand result from virtual screening (orange).]] [[File:2P16_VS_original_lig_structure.png|thumb|center|500px|Structural comparison between the original ligand (blue) and the best-scored ligand from virtual screening (orange) Note the various differences in ring structures between both structures.]]
 +
 +
== ''' Cartesian Minimization ''' ==
 +
 +
Following the completion of the virtual screen, Cartesian minimization plays a pivotal role in refining the ligand-receptor complexes to obtain more accurate predictions of binding affinities. This process involves rigid docking of the ligands within the binding site to minimize the potential energy of the system. To start, we must create the proper directory and enter into it:
 +
 +
<pre>
 +
mkdir 006.cartesian_min
 +
cd 006.cartesian_min
 +
</pre>
 +
 +
We will need to make a new input file for Cartesian minimization, min_scored.in:
 +
<pre>
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100
 +
ligand_atom_file                                            ../005.virtual_screen/2p16_virtual_5k.out_scored.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              no
 +
use_database_filter                                          no
 +
orient_ligand                                                no
 +
bump_filter                                                  no
 +
score_molecules                                              yes
 +
vcontact_score_primary                                      no
 +
contact_score_secondary                                      no
 +
grid_score_primary                                          no
 +
grid_score_secondary                                        no
 +
multigrid_score_primary                                      no
 +
multigrid_score_secondary                                    no
 +
dock3.5_score_primary                                        no
 +
dock3.5_score_secondary                                      no
 +
continuous_score_primary                                    yes
 +
continuous_score_secondary                                  no
 +
cont_score_rec_filename                                      ../001.files/2p16_rec_withH.mol2
 +
cont_score_att_exp                                          6
 +
cont_score_rep_exp                                          12
 +
cont_score_rep_rad_scale                                    1
 +
cont_score_use_dist_dep_dielectric                          yes
 +
cont_score_dielectric                                        4.0
 +
cont_score_vdw_scale                                        1.0
 +
cont_score_es_scale                                          1.0
 +
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                                          1.0
 +
simplex_score_converge                                      0.1
 +
simplex_cycle_converge                                      1.0
 +
simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        2p16_vsc_cart_min
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
</pre>
 +
 +
Similar to virtual screening, you will need to run this on a Seawulf queue and utilize MPI. Create a slurm input file, cart_min.slurm, and add the following:
 +
<pre>
 +
#!/bin/bash
 +
#SBATCH --time=48:00:00
 +
#SBATCH --nodes=2
 +
#SBATCH --ntasks=28
 +
#SBATCH --job-name=2p16_VS_CM
 +
#SBATCH --output=log_2p16_VS_CM.txt
 +
#SBATCH -p long-28core
 +
 +
cd $SLURM_SUBMIT_DIR
 +
echo “Starting Cartesian minimization!”
 +
mpirun -np 56 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i min_scored.in -o min_scored.out
 +
echo “Done!”
 +
</pre>
 +
 +
When you are done with the previous instructions, you can submit the job:
 +
<pre>
 +
sbatch cart_min.slurm
 +
</pre>
 +
 +
We used fewer nodes for this job compared to the virtual screen, but remember to change queue, number of cores, and number of nodes based on what works best: too many nodes or submitting to a popular queue may incur a significant wait time before the job starts. Too few cores or nodes and you may be waiting quite a while for the job to finish.
 +
 +
Once finished, you can export 2p16_vsc_cart_min_scored.mol2 and min_scored.out to view the results. As you can see, the best grid-scored VS ligand may now be docked in a different position compared to the virtual screen results:
 +
 +
[[File:Ligand_structure_cartmin_vs1.png|thumb|center|500px|The previously shown ligand shown before Cartesian minimization (orange) and after (pink).]]
 +
 +
== ''' Rescoring ''' ==
 +
 +
After Cartesian minimization, our virtually screened ligands and their conformations should now be rescored. The various conformations and ligands can be scored (and eventually sorted) by other scoring functions, such as pharmacophore score, footprint similarity score, Hungarian score, etc. To start the final step of virtual screening, you will need to create a directory for rescoring:
 +
 +
<pre>
 +
mkdir 007.rescore
 +
cd 007.rescore
 +
</pre>
 +
 +
As you can guess, we then need to create an input file:
 +
 +
<pre>
 +
vi rescore.in
 +
</pre>
 +
 +
Into this file, paste the following:
 +
<pre>
 +
conformer_search_type                                        rigid
 +
use_internal_energy                                          yes
 +
internal_energy_rep_exp                                      12
 +
internal_energy_cutoff                                      100
 +
ligand_atom_file                                            ../007.cartesianmin/2p16_vsc_min_scored.mol2
 +
limit_max_ligands                                            no
 +
skip_molecule                                                no
 +
read_mol_solvation                                          no
 +
calculate_rmsd                                              no
 +
use_database_filter                                          no
 +
orient_ligand                                                no
 +
bump_filter                                                  no
 +
score_molecules                                              yes
 +
contact_score_primary                                        no
 +
contact_score_secondary                                      no
 +
grid_score_primary                                          no
 +
grid_score_secondary                                        no
 +
multigrid_score_primary                                      no
 +
multigrid_score_secondary                                    no
 +
dock3.5_score_primary                                        no
 +
dock3.5_score_secondary                                      no
 +
continuous_score_primary                                    no
 +
continuous_score_secondary                                  no
 +
footprint_similarity_score_primary                          no
 +
footprint_similarity_score_secondary                        no
 +
pharmacophore_score_primary                                  no
 +
pharmacophore_score_secondary                                no
 +
descriptor_score_primary                                    yes
 +
descriptor_score_secondary                                  no
 +
descriptor_use_grid_score                                    no
 +
descriptor_use_multigrid_score                              no
 +
descriptor_use_continuous_score                              no
 +
descriptor_use_footprint_similarity                          yes
 +
descriptor_use_pharmacophore_score                          yes
 +
descriptor_use_hungarian                                    yes
 +
descriptor_use_volume_overlap                                yes
 +
descriptor_fps_score_use_footprint_reference_mol2            yes
 +
descriptor_fps_score_footprint_reference_mol2_filename      ../004.dock/2p16.lig.min_scored.mol2
 +
descriptor_fps_score_foot_compare_type                      Euclidean
 +
descriptor_fps_score_normalize_foot                          no
 +
descriptor_fps_score_foot_comp_all_residue                  yes
 +
descriptor_fps_score_receptor_filename                      ../001.files/2p16_rec_withH.mol2
 +
descriptor_fps_score_vdw_att_exp                            6
 +
descriptor_fps_score_vdw_rep_exp                            12
 +
descriptor_fps_score_vdw_rep_rad_scale                      1
 +
descriptor_fps_score_use_distance_dependent_dielectric      yes
 +
descriptor_fps_score_dielectric                              4.0
 +
descriptor_fps_score_vdw_fp_scale                            1
 +
descriptor_fps_score_es_fp_scale                            1
 +
descriptor_fps_score_hb_fp_scale                            0
 +
descriptor_fms_score_use_ref_mol2                            yes
 +
descriptor_fms_score_ref_mol2_filename                      ../004.dock/2p16.lig.min_scored.mol2
 +
descriptor_fms_score_write_reference_pharmacophore_mol2      no
 +
descriptor_fms_score_write_reference_pharmacophore_txt      no
 +
descriptor_fms_score_write_candidate_pharmacophore          no
 +
descriptor_fms_score_write_matched_pharmacophore            no
 +
descriptor_fms_score_compare_type                            overlap
 +
descriptor_fms_score_full_match                              yes
 +
descriptor_fms_score_match_rate_weight                      5.0
 +
descriptor_fms_score_match_dist_cutoff                      1.0
 +
descriptor_fms_score_match_proj_cutoff                      .7071
 +
descriptor_fms_score_max_score                              20
 +
descriptor_fingerprint_ref_filename                          ../004.dock/2p16.lig.min_scored.mol2
 +
descriptor_hms_score_ref_filename                            ../004.dock/2p16.lig.min_scored.mol2
 +
descriptor_hms_score_matching_coeff                          -5
 +
descriptor_hms_score_rmsd_coeff                              1
 +
descriptor_volume_score_reference_mol2_filename              ../004.dock/2p16.lig.min_scored.mol2
 +
descriptor_volume_score_overlap_compute_method              analytical
 +
descriptor_weight_fps_score                                  1
 +
descriptor_weight_pharmacophore_score                        1
 +
descriptor_weight_fingerprint_tanimoto                      -1
 +
descriptor_weight_hms_score                                  1
 +
descriptor_weight_volume_overlap_score                      -1
 +
gbsa_zou_score_secondary                                    no
 +
gbsa_hawkins_score_secondary                                no
 +
SASA_score_secondary                                        no
 +
amber_score_secondary                                        no
 +
minimize_ligand                                              no
 +
atom_model                                                  all
 +
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
 +
flex_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 +
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 +
chem_defn_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
 +
pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
 +
ligand_outfile_prefix                                        2p16_rescored.out
 +
write_footprints                                            yes
 +
write_hbonds                                                yes
 +
write_orientations                                          no
 +
num_scored_conformers                                        1
 +
rank_ligands                                                no
 +
</pre>
 +
 +
This is going to be a larger job, so you should run this parallelized on Seawulf:
 +
<pre>
 +
vi rescore.sh
 +
</pre>
 +
Here’s another example slurm batch file that should cover the running of your job:
 +
<pre>
 +
#!/bin/bash
 +
#SBATCH --time=4:00:00
 +
#SBATCH --nodes=2
 +
#SBATCH --ntasks=28
 +
#SBATCH --job-name=2p16_VS_rescore
 +
#SBATCH --output=log_2p16_rescore.txt
 +
#SBATCH -p short-28core
 +
 +
cd $SLURM_SUBMIT_DIR
 +
mpirun -np 56 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i rescore.in -o rescore.out
 +
</pre>
 +
 +
Once saved, submit it to Slurm via:
 +
<pre>
 +
sbatch rescore.sh
 +
</pre>
 +
 +
Each core will produce its own .out output file, but the only rescore.out file of importance will be the unnumbered rescore.out. However, the “xxxx_rescored.out_scored.mol2” will contain all of the scored ligands, along with all of their descriptor scores. There will also be “xxx_rescored.out_descriptor_scored.txt” files that provide more details about the ligands, including potential hydrogen bonding interactions and footprints. These files should be transferred over to your local computer for viewing within Chimera via ViewDock, along with “xxxx_rescored.out_scored.mol2”.

Latest revision as of 15:20, 12 February 2024

Introduction

Docking is a molecular modeling program useful for drug discovery. It computes optimal binding conformations and affinities of a small molecule to its target protein using sampling and scoring methods respectively. It enables the discovery of new low-energy ligand binding modes to the active site of a protein, which can help optimize existing drugs or discover new candidates.

DOCK6

DOCK6 was initially developed at UCSF and now is maintained by several groups across three universities. In addition to other features, it introduces new sampling methods such as fixed anchor, de novo design, and genetic algorithms as improvements over traditional rigid docking methods.

In this tutorial we will be exploring the following functions:

  • Docking- rigid, flexible, and fixed anchor
  • Virtual Screening- docking large libraries of compounds against a protein to generate drug lead
  • Cartesian Minimization- energy minimization of docked molecules in the active site

2P16 System

2p16 refers to the pdb code for the crystal structure of Factor Xa in complex with its inhibitor Apixaban.

Factor Xa plays a key role in the formation of blood clots and is therefore a drug target for anticoagulants.

The drug Apixaban is a second-generation therapy developed by Bristol Myers Squibb- it has fewer drug interactions than the more traditional Heparin and Warfarin, and therefore can be used in patients with additional complications. A docking study of this system may elucide the mechanism of this increased specificity and help design the next generation of specific inhibitors.

Directory Structure

In order to understand the necessary directories it's important to understand the workflow of this project:

Docking flowchart.jpg

We will begin with the pdb file, clean up and separate out the protein and ligand, and then use the unprotonated forms to calculate spheres representing the molecular surface. We will then use our protonated form along with the spheres to calculate the scoring grid. The blue arrows represent transformations we can make using Chimera, the red ones using the SPHGEN and sphere_selector commands, and the green ones using showbox and grid commands.

We will need directories for each step of this process- the sphere generation, grid generation, structure files, as well as for the docking, virtual screening, cartesian minimization, and rescoring after the initial preparatory phase. You can use the following commands to log into the cluster and the generate the relevant directories: 

ssh username@login.seawulf.stonybrook.edu
cd /gpfs/projects/AMS536/your_docking_tutorial_directory
mkdir structures spheres gridbox dock virtual_screen virtual_screen_mpi cartesian_min rescore

Protein and Ligand Preparation

Download the 2p16.pdb file from the pdb website, save it in the structures directory, and visualize it using Chimera. You may do so using the command line: 

cd structures
wget https://files.rcsb.org/download/2P16.pdb
chimera 2P16.pdb

Make sure you read the associated paper to get an understanding protonation states, charges, and crystallization conditions. Identify the following components- protein, ligand, ions, and solvent.

2p16.png

We will be assuming biological pH and neglecting solvent and ion effects, so you can delete the ions, solvent, and protein chain that isn't involved in ligand binding using the following Chimera commands: 

Select->Structure->ion
Actions->Atoms/Bonds->delete

Select->Structure->solvent
Actions->Atoms/Bonds->delete

Select->Chain->L
Actions->Atoms/Bonds->delete

Save the file as 2p16_cleaned.pdb

2p16 cleaned.png

Protein Preparation

To prepare the protein file delete the ligand: 

Select->Structure->ligand
Actions->Atoms/Bonds->delete

Save the file as 2p16_noh_protein.mol2 using File-> Save Mol2

2p16 noh protein.png

To add hydrogens and charge, you may use the Dockprep function in Chimera through Tools->Surface/Binding Analysis->Dock Prep. Uncheck delete solvent in the first window, as we have done this manually, and press ok until you reach the "Assign Charges" window. Ensure that AMBER ff14SB is selected in this window and AM1-BCC in the following "Specify Net Charges" window. Save the file as 2p16_dockprep_protein.mol2

2p16 dockprep protein.png

Ligand Preparation

To prepare the ligand file load 2p16_cleaned.pdb and delete the protein: 

Select->Chain->A
Actions->Atoms/Bonds->delete

Save the file as 2p16_noh_ligand.mol2 using File-> Save Mol2

2p16nohligand.png

Add hydrogens and charge using Dockprep. Save the file as 2p16_dockprep_ligand.mol2

2p16 dockprep ligand.png

Surface Spheres

The remainder of this tutorial will rely on commands from the DOCK6 program. Please ensure that you have DOCK6.10 installed and accessible in your path, which you can check using the commands: 

which dock
echo $PATH

If not, you may want to check your ~/.bashrc file and add the following lines if needed: 

export DOCKHOME="/gpfs/projects/AMS536/zzz.programs/dock6.10"
 PATH=$DOCKHOME/bin\:$PATH
export PATH=$DOCKHOME/bin\:$PATH

Save the file and activate it using: 

source ~/.bashrc

DMS Preparation

To prepare the DMS file open 2p16_noh_protein.mol2 in Chimera and do the following: 

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

Save the file as 2p16_noh_protein.dms

Sphere Generation

Transfer all the files generated so far to the structures directory in your Seawulf docking directory using: 

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

Move to the spheres directory and open an input file for the sphgen command: 

cd spheres
vim INSPH

Type in the following and save the file: 

../structures/2p16_noh_protein.dms        #input file
R                                         #spheres to be generated outside the receptor surface  
X                                         #all surface points to be used 
0.0                                       #surface clashes
4.0.                                      #max sphere radius
1.4.                                      #min sphere radius
2p16_protein.sph                          #output file

Run sphgen using: 

sphgen -i INSPH -o OUTSPH

Open your output file 2p16_protein.sph along with your 2016_noh_protein.mol2 file to check if everything looks ok.

2p16 spheres.png

Sphere Selection

We now need to narrow down the generated spheres to only those near the binding pocket.

The following command will select those within 10 angstroms of the ligand and save them to selected_spheres.sph: 

sphere_selector 2p16_protein.sph ../structures/2p16_noh_ligand.mol2 10.0
2p16 selected spheres.png

Grid Box

We will now use the selected spheres to generate a grid within which we will score the sampled molecular conformations.

Box Generation

Move to the gridbox directory and open up an input file titled showbox.in. Type in the following text: 

Y                                    #Yes to making a box    
8.0                                  #Angstroms between box boundary and any sphere
../spheres/selected_spheres.sph      #Input file
1                                    #Number of clusters of spheres to use 
2p16.box.pdb                         #Output file

Generate the box using the following command: 

showbox < showbox.in
2p16 gridbox.png

Grid Generation

Open up an input file titled grid.in and type in the following: 

allow_non_integral_charges                no
compute_grids                             yes
grid_spacing                              0.4
output_molecule                           no
contact_score                             no
energy_score                              yes
energy_cutoff_distance                    9999
atom_model                                a
attractive_exponent                       6
repulsive_exponent                        9
distance_dielectric                       yes
dielectric_factor                         4
bump_filter                               yes
bump_overlap                              0.75
receptor_file                             ../structures/2p16_dockprep_protein.mol2
box_file                                  2p16.box.pdb
vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
score_grid_prefix                         grid

Generate the grid using the following command: 

grid -i grid.in -o grid.out

This should create a grid.out, grid.nrg, and grid.bmp file. Check the grid.out file to see if your residue charges and total charge match the original protein mol2 file. If not, go back and check if you made any mistakes in earlier steps.

Ligand Minimization

Before running DOCK we need to ensure our ligand is energy minimized to prevent any steric clashes.

Switch to the dock directory and open up an input file called min.in and type in the following: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../structures/2p16_dockprep_ligand.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       ../structures/2p16_dockprep_ligand.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                                       ../gridbox/grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        yes
simplex_coefficient_restraint                                10.0
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        2p16_ligand.min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Run the minimization using: 

dock6 -i min.in -o min.out

This should save a new file called 2p16_ligand.min_scored.mol2. Open it up in Chimera along with the protein and ligand mol2 files for comparison.

2p16ligandminscored.png

You can also view the RMSD values in the header of the file using: 

head 2p16.ligand.min_scored.mol2

Footprint Analysis

We can use footprinting to visualize the protein-ligand interaction energies on a per-residue basis.

In the same dock directory open up an input file called footprint.in and type in the following: 

conformer_search_type                                        rigid
use_internal_energy                                          no
ligand_atom_file                                             2p16_ligand.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                  ../structures/2p16_dockprep_ligand.mol2
fps_score_foot_compare_type                                  Euclidean
fps_score_normalize_foot                                     no
fps_score_foot_comp_all_residue                              yes
fps_score_receptor_filename                                  ../structures/2p16_dockprep_protein.mol2
fps_score_vdw_att_exp                                        6
fps_score_vdw_rep_exp                                        9
fps_score_vdw_rep_rad_scale                                  1
fps_score_use_distance_dependent_dielectric                  yes
fps_score_dielectric                                         4.0
fps_score_vdw_fp_scale                                       1
fps_score_es_fp_scale                                        1
fps_score_hb_fp_scale                                        0
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        fps.min.output
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

This should generate two output files: fps.min.output_hbond_scored.txt and fps.min.output_footprint_scored.txt.

The latter can be visualized (in this case for 50 residues) using a python script which we can copy from the class directory: 

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

2p16 footprint.jpg

DOCKING

The goal of docking in this tutorial is to reproduce the crystal structure from x-ray diffraction. This is called “pose reproduction”; this can be used to validate the docking software and parameters of use within the software. If the pose reproduction is not successful, it might be possible that: 1.the parameters to dock the ligand needs to be changed, 2.preparation of ligand or receptor went wrong, 3.the system itself is not suitable to reproduce in the software. Having a preliminary docking pose before getting into virtual screening–a procedure docking thousands of molecules–would be helpful to validate the results you will get in the future. In DOCK6 (at the point of 2020), the docking is successful if the RMSD of the docked pose is within 2.0Å from the crystal pose. In this section, we will be working on (i)rigid docking, (ii)flexible docking, (iii)fixed anchor docking.

Rigid Docking

Rigid docking does not conduct pose searching with dihedral rotations or bond length perturbations. It takes 3 translational and 3 rotational degrees of freedom to do its pose searching. Therefore, rigid docking is less computationally expensive compared to flex docking. Since we already have the crystal structure of the ligand bound at the active site, it is expected to have a good reproduction using rigid docking.

The first step is to create an input file by rigid.in

Insert the following into that file: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100
ligand_atom_file                                             2p16.ligand.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                                      2p16.ligand.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           selected_spheres.sph
max_orientations                                             2000
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                                       grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                              
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        rigid.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


Make sure to (i)have all the files you need (selected_spheres.sph and 2p16.ligand.min_scored.mol2) and (ii)inputs are correct.

Run the input file with the following command: 

dock6 -i rigid.in -o rigid.out


After the job is complete, you should see a file named “rigid.out_scored.mol2”. This is the output that shows the docked poses (if you rank it, it will show from the highest to lowest docking score). If you open the file, in the first part, you will see general information about the docked pose including the grid scores. Make sure that the output file is not empty. The pose can be visualized using Chimera. After you load the protein you prepared (simply open the file), you can go to: Tools -> Surface/Binding Analysis -> ViewDock -> *choose the file* -> Dock4,5, or 6. Along with the visualized pose, you will see a small window popping out with the quantitative information as you saw in the first part of the output file. Since we took a crystal structure of the ligand, minimized, and rigidly docked into the same protein, it is expected that the produced pose is well-reproduced. If you want to check the reproducibility, you could check out the RMSD (root-mean-square-deviation) values for the pose.

The crystal reference ligand is colored in pink

RMSD: 7.4 Å relative to the minimized crystal pose

From the result pose, you can see that the pose reproduction was not successful. From the scored.mol2 file, the RMSD was indicated to be 7.4 Å, which is above 2.0 Å cutoff for a successful pose reproduction. Although this was an unexpected results from the initial assumption, but we can try using two other methods: flex and fixed anchor docking.

Fixed Anchor Docking

As it is said “fixed anchor” docking, this is a docking method that docks while maintaining the orientation of the initially placed structure. For this reason, “comformer_search_type” is flexible, while “write orientation” is turned off. This allows more flexibility in its conformational change compared to rigid docking methods while maintaining the general placement. You can also pick specific fragments within the molecules to be the anchor.

Not to mess up the outputs with other types of docking methods, make a new folder by

mkdir anchor_dock


In this directory, create an input file: 


vi fixed.in


Insert the following: 

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                                          10000
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                                             2p16.ligand.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                                      2p16.ligand.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           yes
grid_score_secondary                                         no
grid_score_rep_rad_scale                                     1
grid_score_vdw_scale                                         1
grid_score_es_scale                                          1
grid_score_grid_prefix                                       grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
minimize_anchor                                              yes
minimize_flexible_growth                                     yes
use_advanced_simplex_parameters                              no
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1
simplex_trans_step                                           1
simplex_rot_step                                             0.1
simplex_tors_step                                            10
simplex_anchor_max_iterations                                500
simplex_grow_max_iterations                                  500
simplex_grow_tors_premin_iterations                          0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        2P16_fixed
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


Start the docking by: 

dock6 -i fixed.in -o fixed.out


The resulting docked pose:

RMSD: 0.22 Å relative to the minimized crystal pose

The RMSD in this result was 0.22 Å, which is well within 2.0 Å (success cutoff). From the rigid docking result, we could see that the orientation of the entire molecule was flipped from the minimized crystal structure. By having a part of the molecule anchored, it seemed that the big rotation of this molecule did not occur. For a case of new compounds, ones that do not have a crystal structures, this method allows more chemical exploration.

Flex Docking

Finally, flex docking is a docking method that allows flexibility in the rotatable bonds in the ligand and orientation of the compound. This method has the largest exploration of the pose for the ligand, although the calculation time takes the longest. To speed up the calculation time, it is necessary to use cpus provided in seawulf. To use the cpus, we need to submit our run to the queue.

Create a directory: 

mkdir flex_dock


First, start with creating an input file, flex.in. 

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                                          5000
pruning_clustering_cutoff                                    2500
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                                             
../Rigid_dock/2p16.ligand.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                                     
../Rigid_dock/2p16.ligand.min_scored.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../Rigid_dock/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                                       ../Rigid_dock/grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
minimize_anchor                                              yes
minimize_flexible_growth                                     yes
use_advanced_simplex_parameters                              no
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_anchor_max_iterations                                500
simplex_grow_max_iterations                                  500
simplex_grow_tors_premin_iterations                          0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              
/gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        flex.out
write_orientations                                           yes
num_scored_conformers                                        10
write_conformations                                          yes
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Now that we have our .in file, we need to make a slurm script to run it. To make a script, first run the following: 


 vi flex.sh


Inside of this file, copy-paste the following: 


 #!/bin/bash
 #SBATCH --time=10:00:00
 #SBATCH --nodes=1
 #SBATCH --ntasks=28
 #SBATCH --job-name=2p16_flex_dock
 #SBATCH --output=2p16_flex_dock
 #SBATCH -p long-28core

 dock6 -i flex.in -o 2p16_flex.out


You can submit the queue by 

sbatch flex.sh

Once complete, you will see three .mol2 files: (1)flex.out_scored.mol2: best poses scored from the highest to lowest (2)flex.out_conformers.mol2: poses with all conformations of the ligand (3)flex.out_orients.mol2: poses with all orientations of the ligand

To check the best pose, check the flex.out_scored.mol2. You can open it on Chimera using viewdock. Compare with the crystal structure - is the pose with freedom of orientation and rotatable bonds matching with the binding pose in real life?

RMSD: 0.9235 Å relative to the minimized crystal pose

By looking at the comparison (green is the flex output), you can see that the pose reproduction went successfully. The output file showed the RMSD of 0.92 Å, which also indicates a success. This structure was found in the first in scored.mol2 showing that this pose was in fact the best pose after rotational bond and orientation searching.

Virtual Screening

Virtual screening is a computational method of docking and ranking potential small molecule drug candidates. Leveraging the knowledge of the three-dimensional structure of the target receptor, virtual screening involves the systematic evaluation of a large library of small molecules by simulating their interactions with the target. By employing van der Waals, electrostatic, and interal energy scoring functions, virtual screening provides a way to rank and evaluate potential small molecule ligands.

For this tutorial, you will be using the VS_library_5K.mol2, located in /gpfs/projects/AMS536/zzz.programs/ and copied to our virtual screening directory. You can create a virtual screening directory (and then go into it) if you haven’t already: 

mkdir 005.virtual_screen
cd 005.virtual_screen

After that, create a new input file, vs.in: 

search_type                                                  flex
write_fragment_libraries                                     no
user_specified_anchor                                        no
limit_max_anchors                                            no
min_anchor_size                                              5
pruning_use_clustering                                       yes
pruning_max_orients                                          1000
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               100.0
pruning_conformer_score_scaling_factor                       1.0
use_clash_overlap                                            no
write_growth_tree                                            no
use_internal_energy                                          yes
internal_energy_rep_exp                                      9
internal_energy_cutoff                                       100.0
ligand_atom_file                                             VS_library_5K.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               no
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../002.surface_spheres/selected_spheres.sph
max_orientations                                             1000
critical_points                                              no
chemical_matching                                            no
use_ligand_spheres                                           no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           yes
grid_score_secondary                                         no
grid_score_rep_rad_scale                                     1
grid_score_vdw_scale                                         1
grid_score_es_scale                                          1
grid_score_grid_prefix                                       ../003.gridbox/grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
pharmacophore_score_secondary                                no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              yes
minimize_anchor                                              yes
minimize_flexible_growth                                     yes
use_advanced_simplex_parameters                              no
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_anchor_max_iterations                                500
simplex_grow_max_iterations                                  500
simplex_grow_tors_premin_iterations                          0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        2p16_virtual_5k.out
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

To make sure the virtual screen finishes in a timely manner, you will be utilizing MPI parallelization, which will allow you to use multiple nodes simultaneously for more processing power. Create a new file, 2p16_VS_5K.slurm, to give SLURM instructions on carrying out the virtual screen. It should look something like this: 

#!/bin/sh 
#SBATCH --partition=long-28core
#SBATCH --time=2-00:00:00
#SBATCH --nodes=4
#SBATCH --ntasks=112
#SBATCH --job-name=2p16_5k_vs
#SBATCH --output=log_2p16_5k_vs.txt
 
echo "starting DOCK6.10 Simulation"
module load intel_stack
 
mpirun -v -np 112 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i vs.in -o vs.out
echo "done"

This might be a bit overkill, so you may want to reduce the number of nodes or use a queue with fewer cores. The "echo" lines are there to provide an idea of whether the job started/finished properly.

To submit this job to the queue, run: 

sbatch 2p16_VS_5K.slurm

Each core will produce its own .out output file, but the only one of importance will be vs.out. This file should be transferred over to your local computer for viewing within Chimera, along with 2p16_virtual_5k.out_scored.mol2, which will contain the positions of the highest-scored ligands. Shown below are the aforementioned results in Chimera, as well as the comparison of the currently best-scored ligand and the original ligand:

The original ligand (blue) compared to the best-scored ligand result from virtual screening (orange).
Structural comparison between the original ligand (blue) and the best-scored ligand from virtual screening (orange) Note the various differences in ring structures between both structures.

Cartesian Minimization

Following the completion of the virtual screen, Cartesian minimization plays a pivotal role in refining the ligand-receptor complexes to obtain more accurate predictions of binding affinities. This process involves rigid docking of the ligands within the binding site to minimize the potential energy of the system. To start, we must create the proper directory and enter into it: 

mkdir 006.cartesian_min
cd 006.cartesian_min

We will need to make a new input file for Cartesian minimization, min_scored.in: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100
ligand_atom_file                                             ../005.virtual_screen/2p16_virtual_5k.out_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               no
use_database_filter                                          no
orient_ligand                                                no
bump_filter                                                  no
score_molecules                                              yes
vcontact_score_primary                                       no
contact_score_secondary                                      no
grid_score_primary                                           no
grid_score_secondary                                         no
multigrid_score_primary                                      no
multigrid_score_secondary                                    no
dock3.5_score_primary                                        no
dock3.5_score_secondary                                      no
continuous_score_primary                                     yes
continuous_score_secondary                                   no
cont_score_rec_filename                                      ../001.files/2p16_rec_withH.mol2
cont_score_att_exp                                           6
cont_score_rep_exp                                           12
cont_score_rep_rad_scale                                     1
cont_score_use_dist_dep_dielectric                           yes
cont_score_dielectric                                        4.0
cont_score_vdw_scale                                         1.0
cont_score_es_scale                                          1.0
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                                           1.0
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
ligand_outfile_prefix                                        2p16_vsc_cart_min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Similar to virtual screening, you will need to run this on a Seawulf queue and utilize MPI. Create a slurm input file, cart_min.slurm, and add the following: 

#!/bin/bash
#SBATCH --time=48:00:00
#SBATCH --nodes=2
#SBATCH --ntasks=28
#SBATCH --job-name=2p16_VS_CM
#SBATCH --output=log_2p16_VS_CM.txt
#SBATCH -p long-28core

cd $SLURM_SUBMIT_DIR
echo “Starting Cartesian minimization!”
mpirun -np 56 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i min_scored.in -o min_scored.out
echo “Done!”

When you are done with the previous instructions, you can submit the job: 

sbatch cart_min.slurm

We used fewer nodes for this job compared to the virtual screen, but remember to change queue, number of cores, and number of nodes based on what works best: too many nodes or submitting to a popular queue may incur a significant wait time before the job starts. Too few cores or nodes and you may be waiting quite a while for the job to finish.

Once finished, you can export 2p16_vsc_cart_min_scored.mol2 and min_scored.out to view the results. As you can see, the best grid-scored VS ligand may now be docked in a different position compared to the virtual screen results:

The previously shown ligand shown before Cartesian minimization (orange) and after (pink).

Rescoring

After Cartesian minimization, our virtually screened ligands and their conformations should now be rescored. The various conformations and ligands can be scored (and eventually sorted) by other scoring functions, such as pharmacophore score, footprint similarity score, Hungarian score, etc. To start the final step of virtual screening, you will need to create a directory for rescoring: 

mkdir 007.rescore
cd 007.rescore

As you can guess, we then need to create an input file: 

vi rescore.in

Into this file, paste the following: 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100
ligand_atom_file                                             ../007.cartesianmin/2p16_vsc_min_scored.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               no
use_database_filter                                          no
orient_ligand                                                no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           no
grid_score_secondary                                         no
multigrid_score_primary                                      no
multigrid_score_secondary                                    no
dock3.5_score_primary                                        no
dock3.5_score_secondary                                      no
continuous_score_primary                                     no
continuous_score_secondary                                   no
footprint_similarity_score_primary                           no
footprint_similarity_score_secondary                         no
pharmacophore_score_primary                                  no
pharmacophore_score_secondary                                no
descriptor_score_primary                                     yes
descriptor_score_secondary                                   no
descriptor_use_grid_score                                    no
descriptor_use_multigrid_score                               no
descriptor_use_continuous_score                              no
descriptor_use_footprint_similarity                          yes
descriptor_use_pharmacophore_score                           yes
descriptor_use_hungarian                                     yes
descriptor_use_volume_overlap                                yes
descriptor_fps_score_use_footprint_reference_mol2            yes
descriptor_fps_score_footprint_reference_mol2_filename       ../004.dock/2p16.lig.min_scored.mol2
descriptor_fps_score_foot_compare_type                       Euclidean
descriptor_fps_score_normalize_foot                          no
descriptor_fps_score_foot_comp_all_residue                   yes
descriptor_fps_score_receptor_filename                       ../001.files/2p16_rec_withH.mol2
descriptor_fps_score_vdw_att_exp                             6
descriptor_fps_score_vdw_rep_exp                             12
descriptor_fps_score_vdw_rep_rad_scale                       1
descriptor_fps_score_use_distance_dependent_dielectric       yes
descriptor_fps_score_dielectric                              4.0
descriptor_fps_score_vdw_fp_scale                            1
descriptor_fps_score_es_fp_scale                             1
descriptor_fps_score_hb_fp_scale                             0
descriptor_fms_score_use_ref_mol2                            yes
descriptor_fms_score_ref_mol2_filename                       ../004.dock/2p16.lig.min_scored.mol2
descriptor_fms_score_write_reference_pharmacophore_mol2      no
descriptor_fms_score_write_reference_pharmacophore_txt       no
descriptor_fms_score_write_candidate_pharmacophore           no
descriptor_fms_score_write_matched_pharmacophore             no
descriptor_fms_score_compare_type                            overlap
descriptor_fms_score_full_match                              yes
descriptor_fms_score_match_rate_weight                       5.0
descriptor_fms_score_match_dist_cutoff                       1.0
descriptor_fms_score_match_proj_cutoff                       .7071
descriptor_fms_score_max_score                               20
descriptor_fingerprint_ref_filename                          ../004.dock/2p16.lig.min_scored.mol2
descriptor_hms_score_ref_filename                            ../004.dock/2p16.lig.min_scored.mol2
descriptor_hms_score_matching_coeff                          -5
descriptor_hms_score_rmsd_coeff                              1
descriptor_volume_score_reference_mol2_filename              ../004.dock/2p16.lig.min_scored.mol2
descriptor_volume_score_overlap_compute_method               analytical
descriptor_weight_fps_score                                  1
descriptor_weight_pharmacophore_score                        1
descriptor_weight_fingerprint_tanimoto                       -1
descriptor_weight_hms_score                                  1
descriptor_weight_volume_overlap_score                       -1
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_score_secondary                                         no
amber_score_secondary                                        no
minimize_ligand                                              no
atom_model                                                   all
vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
chem_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
ligand_outfile_prefix                                        2p16_rescored.out
write_footprints                                             yes
write_hbonds                                                 yes
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

This is going to be a larger job, so you should run this parallelized on Seawulf: 

vi rescore.sh

Here’s another example slurm batch file that should cover the running of your job: 

#!/bin/bash
#SBATCH --time=4:00:00
#SBATCH --nodes=2
#SBATCH --ntasks=28
#SBATCH --job-name=2p16_VS_rescore
#SBATCH --output=log_2p16_rescore.txt
#SBATCH -p short-28core

cd $SLURM_SUBMIT_DIR
mpirun -np 56 /gpfs/projects/AMS536/zzz.programs/dock6.10/bin/dock6.mpi -i rescore.in -o rescore.out

Once saved, submit it to Slurm via: 

sbatch rescore.sh

Each core will produce its own .out output file, but the only rescore.out file of importance will be the unnumbered rescore.out. However, the “xxxx_rescored.out_scored.mol2” will contain all of the scored ligands, along with all of their descriptor scores. There will also be “xxx_rescored.out_descriptor_scored.txt” files that provide more details about the ligands, including potential hydrogen bonding interactions and footprints. These files should be transferred over to your local computer for viewing within Chimera via ViewDock, along with “xxxx_rescored.out_scored.mol2”.

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