2021 DOCK tutorial 4 with PDBID 1EFY
Contents
Introduction
DOCK
DOCK is a member of a large suite of molecular docking packages. Notably, it was the first molecular docking package of its kind, and it was created by Irwin Kuntz’s group in the 1980s. The original authors of DOCK include Brian K. Shoichet, David A. Case, and Robert C.Rizzo. Molecular docking plays a key role in computer aided drug design (CADD). Generally, molecular docking allows us to explore the interactions between small molecules, or ligands, and proteins by evaluating the shape and energetics of the ligand. We will be using the 6.9 release of DOCK, the latest as of November 2018, for this tutorial. New features of DOCK6.9 include DOCK_DN, a new ligand searching method for ligand de novo design and fragment library generation, which allows users to generate their own fragment libraries from mol2 files. For a comprehensive list of what’s new in DOCK6.9, click here.
In this tutorial, we will be using DOCK to predict the interactions between a small molecule inhibitor and a protein target. Here is the general workflow that we will be following:
- Download a PDB file from the Protein Data Bank
- Add protons and partial charges to the small molecule inhibitor and the target (independently).
- Calculate the molecular surface of the target protein.
- Generate spheres surrounding the binding site.
- Calculate the bounding box and energy grid for the target protein.
- Match the ligand atoms to the sphere centers.
- Score the putative ligand atom centers using the energy grid.
- Rank the orientations by score.
Chimera
UCSF Chimera is a standalone program that allows us to visualize and analyze macromolecules. General features of Chimera include automatic identification of atom, hydrogen addition and partial charge assignment, measurements of distances, angles, surface area, volume, and many more. For a complete description of everything Chimera can do, see https://www.rbvi.ucsf.edu/chimera/.
1EFY
In this tutorial, we will be working with the crystal structure of a member of the Poly (ADP-Ribose) polymerase (PARP) family. Specifically, we will be working with the catalytic fragment of PARP along with a benzimidazole inhibitor. This crystal structure has a resolution of 2.20 Å and an R-Value free of 0.274. PARP family proteins are involved in DNA repair, and it has been shown that they may allow cancer cells to repair their DNA after exposure to chemotherapy, which allows for resistance to develop [1]. As such, it is of particular interest to develop novel inhibitors for PARP. The goal for this tutorial is to demonstrate an end-to-end example of docking the benzimidazole inhibitor against the PARP target, as well as perform a virtual screen to find other small molecule inhibitors of PARP.
Software Requirements
In order to successfully follow this tutorial, please make sure to have the following programs installed:
- MobaXterm (for Windows users)
- Chimera 1.15 (Please choose the right specifications for your OS)
- DOCK 6.9
We will be using a shell environment (either the built-in terminal for MacOS/Linux users or MobaXTerm for Windows users) to run several of the programs in this tutorial. As such, it may be useful to familiarize yourself with running basic UNIX commands and VIM.
In addition, this tutorial assumes you have access to a High Performance Computing cluster, i.e. Seawulf.
Directory Setup
Before we begin our molecular docking and virtual screen, it will be useful to establish our directory structure.
First ssh into your account on Seawulf:
ssh <your netid>@login.seawulf.stonybrook.edu
Navigate into your student directory using the cd command. Once you are in your student directory, create a new directory for this tutorial and name it 1EFY. The shell command to make a new directory is mkdir.
mkdir 1EFY
Now move into the 1EFY directory. Next, we will be creating the following directories:
mkdir 001.structure 002.surface_spheres 003.gridbox 004.dock 005.virtual_screen 006.virtual_screen_mpi 007.cartesian_min 008.rescore
In order to move back up the tree out of the current directory, use:
cd ../
Preparing the Ligand and Receptor
PDB Structure
For this tutorial, we will be utilizing the Protein Data Bank for information about our target protein. The Protein Databank is a database containing the structural data for biological macromolecules. Visit the Protein Data Bank and search for 1EFY. You should be redirected to a page containing the X-ray crystal structure of our protein, as well as the primary literature about the protein.
Click on Download files > PDB Format. This file contains the crystal structure of the catalytic fragment of Poly (ADP-ribose) Polymerase complexed with a benzimidazole inhibitor. It contains information about the atomic coordinates, secondary structure assignments, and atomic connectivity of the protein and ligand.
We will be visualizing the structure in Chimera. Open Chimera and click File > Open. Once we select the PDB file that we have just downloaded, we should see the following.
We can see the receptor, the benzimidazole inhibitor, and two water molecules. In order to see the complex from different angles or positions, we may perform three dimensional manipulations. It is possible to rotate, translate, or scale the the view of the structures using the mouse. Please refer to this documentation to see which mouse controls are appropriate for your setup.
We will next describe how to separate the receptor and ligand into two separate MOL2 formatted files.
Preparing the Receptor
In order to prepare our system for DOCK, we must perform some preprocessing steps, since DOCK can only process Tripos MOL2 formatted files as input. Luckily, we can use Chimera to create two new MOL2 files out of the original PDB file: one for the receptor and one for the ligand. In addition, prior to docking, we must also use Chimera to specify the charges for every atom in the receptor and ligand.
Let's first create the MOL2 file for the receptor. Using the original PDB structure, we will strip everything but the receptor. In Chimera, choose
Select > Residue > all nonstandard
The benzimidazole inhibitor and the two water molecules should now be highlighted like this:
Again in Chimera, select
Actions > Atoms/Bonds > delete
This leaves us with a “cleaned” version of the receptor:
We can now save the receptor as a MOL2 file. Click
File > Save Mol2…
Save the file as 1EFY_rec_woH.mol2 where woH indicates the receptor without hydrogens. Next we must add hydrogens and charges, which is required for docking. Select
Tools > Structure Editing > AddH
A window should pop up like this:
Select OK. Now each residue in the receptor should be properly protonated. However, it is important that we validate the hydrogens that Chimera has added, as they may not be correct.
Similarly, we must add partial charges to each atom in the receptor. Select
Tools > Structure Editing > Add Charge.
Make sure that AM1-BCC is selected in the window that pops up and select OK. Now each atom of the receptor should have the appropriate partial charge. As with the protonation states, it is important to double check Chimera’s work.
This receptor, which has now been prepared for DOCK, can be saved as a MOL2 file named 1EFY_rec_dockprep.mol2.
Ligand Preparation
We will now prepare the ligand in a similar fashion. Open the original PDB structure in Chimera. In this case, we will be deleting all of the constituents in the structure except for the benzimidazole inhibitor. Click
Select > Residue > BZC.
This should highlight the inhibitor. Click
Select > Invert (all models).
This should highlight everything except the inhibitor. Click
Actions > Atoms/Bonds > delete
Only the ligand should now be present:
We will save this structure as MOL2 file.
File > save as mol2 > 1EFY_ligand_noH.mol2
where noH indicates no hydrogens. Just as before, we must now add the hydrogens and charge.
Tools > Structure Editing > AddH
The ouput should look like:
In its current state, the ligand protonation is incorrect and will lead to complications during docking. To fix this it is important to forcibly protonate the N4 atom.
Tool > Structure Editing > Build Structure
Set the number of bonds at position N4 to 3
Click Apply. Add charges as shown previously We will save this charged and protonated structure as a MOL2 file:
File > save as mol2 > 1EFY_ligand_H.mol2
Now that we have prepared our files for DOCK, we can scp or rsync all four of the files into the 001.structure directory.
Surface Generation and Sphere Selection
Identification of the binding site on a receptor is driven by identifying its concave surfaces. Once the molecular surface has been identified, we can generate overlapping spheres to describe the negative image of the molecular surface. The centers of the spheres composing the largest cluster represent potential positions where the ligand can bind.
Calculate the Molecular Surface
In Chimera, we can calculate the molecular surface according to the following steps: Open 1EFY_rec_woH.mol2 in Chimera. Select
Actions > Surface > Show
This shows the solvent excluded molecular surface of the receptor. We can use DMS, an open source program, to calculate the molecular surface of the receptor. Select
Tools > Structure Editing > Write DMS
Save the file name as 1EFY_rec_surphace.dms This file can be uploaded to 002.surface_spheres.
Generate Spheres
Now that the molecular surface has been calculated and written to the DMS file, we can construct spheres to fill in any concave regions of the surface of the receptor by using the sphgen program in DOCK. The centers of the spheres represent putative positions of ligand atoms. Navigate to the 002.surface_spheres directory. The template for sphere generation using sphgen looks like the following:
INPUT FILE*: rec.ms #molecular surface file R #sphere outside of surface (R) or inside surface (L) X #specifies subset of surface points to be used (X=all points) 0.0 #prevents generation of large spheres with close surface contacts (default=0.0) 4.0 #maximum sphere radius in angstroms (default=4.0) 1.4 #minimum sphere radius in angstroms (default=radius of probe) rec.sph #clustered spheres file
Our file looked like the following:
1efy_rec_surface.dms R X 0.0 4.0 1.4 1EFY_rec_woH.sph
You can run the sphgen program like this:
sphgen -i INSPH -o OUTSPH
Chimera can read the output of the sphgen program.
The generated spheres should look like the following:
Now, the juxtaposition of the surface spheres with the receptor should look like the following: Notice how the spheres are clustered near the active site.
Generating Box and Grid
Many individual floating point calculations are required for the computation of Lennard-Jones and Coulombic Interactions requisite for docking. In order to make these calculations feasible, we can calculate the overlapping volume of two sets of atoms using a grid based method. The intuition behind this method is the following:
- Generate a bounding box that contains all of the atoms.
- Generate grid points with a predefined separation.
Generating the Box
We will be using the DOCK's accessory programs showbox and grid in order to generate the grids required for grid based scoring (which is based on the non-bonded terms of the molecular mechanic force field). The first step is to generate a box enclosing the active site of the receptor. Create an input file for showbox in the 003.gridbox directory.
touch showbox.in
10.0 ../002.surface_spheres/selected_spheres.sph 1 receptor.box.pdb
We can pipe in the input file into the showbox with the command showbox < box.in
The output of the program will be a PDB file containing the structure of the bounding box, which can be visualized in Chimera.
Generating the Grid
Next we will be generating the grid. In the same directory, create an input file called grid.in
touch grid.in
Now, edit the input file such that it looks like the following:
compute_grids yes grid_spacing 0.4 output_molecule no contact_score no energy_score yes energy_cutoff_distance 9999 atom_model a attractive_exponent 6 repulsive_exponent 9 distance_dielectric yes dielectric_factor 4 bump_filter yes bump_overlap 0.75 receptor_file ../001.structure/1EFY_rec_dockprep.mol2 box_file 1EFY.box.pdb vdw_definition_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn score_grid_prefix grid
In order to run the grid program, use the following command:
grid -i grid.in -o gridinfo.out
Note that the grid calculation can take up to 45 minutes.
The output of the showbox and grid calculation can be visualized in Chimera as the following:
Single Molecule Docking and Pose Reproduction
Energy Minimization
In order to meaningfully dock new ligands into the receptor, the current model must be verified for its accuracy. To accomplish this, the co-crystallized ligand will be energy minimized and docked into the 1efy receptor. The resulting pose should closely resemble the crystal structure. If significant deviations are noted, the ligand and receptor should be scrutinized for correct charge, protonation, and resonance.
Move to the 004.dock directory and create the input file:
touch min.in
dock6 -i min.in
Input the following
conformer_search_type rigid use_internal_energy yes internal_energy_rep_exp 9 internal_energy_cutoff 100.0 ligand_atom_file ../001.structure/1EFY_ligand_H.mol2 limit_max_ligands no skip_molecule no read_mol_solvation no calculate_rmsd yes use_rmsd_reference_mol yes rmsd_reference_filename ../001.structure/1EFY_ligand_H.mol2 use_database_filter no orient_ligand no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary yes grid_score_secondary no grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix ../003.gridbox/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes simplex_max_iterations 1000 simplex_tors_premin_iterations 0 simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_random_seed 0 simplex_restraint_min yes simplex_coefficient_restraint 10.0 atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn flex_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn flex_drive_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl ligand_outfile_prefix 1EFY_ligand_min write_orientations no num_scored_conformers 1 rank_ligands no
Rigid Docking
Rigid docking is the simplest and lowest cost of the three main docking methods, as it essentially attempts to dock the desired ligand in its native pose, only allowing 3d transformation and rotations, as well as very minor torsional corrections.
Create the input file
touch rigid.in
dock6 -i rigid.in
conformer_search_type rigid use_internal_energy yes internal_energy_rep_exp 12 internal_energy_cutoff 100.0 ligand_atom_file 1EFY_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 1EFY_ligand_min_scored.mol2 use_database_filter no orient_ligand yes automated_matching yes receptor_site_file ../002.surface_spheres/selected_spheres.sph max_orientations 1000 critical_points no chemical_matching no use_ligand_spheres no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary yes grid_score_secondary no grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix ../003.gridbox/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes simplex_max_iterations 1000 simplex_tors_premin_iterations 0 simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_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
At this point, it is important to visualize the resulting pose in comparison to the crystallized ligand. Open both in Chimera along with the receptor file to ensure no major changes have occurred
Fixed Anchor Docking
In fixed anchor docking, the ligand of interest is "anchored" into the binding site of the receptor, and rotatable bonds and torsions are adjusted to minimize the energy of the structure. As this method allows for greater variance of the ligand, but still maintains a particular location in the receptor, this method blends accuracy and efficiency when compared to rigid and flexible methods.
Create an input file
touch fixed.in
dock6 -i fixed.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 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 1EFY_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 1EFY_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 ../003.gridbox/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes minimize_anchor yes minimize_flexible_growth yes use_advanced_simplex_parameters no simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1 simplex_trans_step 1 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_anchor_max_iterations 500 simplex_grow_max_iterations 500 simplex_grow_tors_premin_iterations 0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_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 1 rank_ligands no
Flexible Docking
In flexible docking, the rotatable bonds and torsions, as well as the ligand location is searched within the defined box and grid. For this reason, flexible methods are typically the most meaningful of the three methods when docking new ligands to the receptor of interest.
Create and input file:
touch flex.in
dock6 -i 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 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 1EFY_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 1EFY_ligand_min_scored.mol2 use_database_filter no orient_ligand yes automated_matching yes receptor_site_file ../002.surface_spheres/selected_spheres.sph max_orientations 1000 critical_points no chemical_matching no use_ligand_spheres no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary yes grid_score_secondary no grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix ../003.gridbox/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes minimize_anchor yes minimize_flexible_growth yes use_advanced_simplex_parameters no simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_anchor_max_iterations 500 simplex_grow_max_iterations 500 simplex_grow_tors_premin_iterations 0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn flex_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn flex_drive_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl ligand_outfile_prefix flex.out write_orientations no num_scored_conformers 1 rank_ligands no
Molecular Footprint
A typical molecular footprint plot shows the van der waals and electrostatic interaction between the ligand and nearby amino acid residues. This is useful for determining which interactions are important for suitable affinity of the ligand. This also serves as an additional method to ensure the ligand wasn't significantly distorted in the minimization process.
First, generate an input file
touch footprint.in
dock6 -i footprint.in
conformer_search_type rigid use_internal_energy no ligand_atom_file 1EFY_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 ../001.structure/1EFY_ligand_H.mol2 fps_score_foot_compare_type Euclidean fps_score_normalize_foot no fps_score_foot_comp_all_residue yes fps_score_receptor_filename ../001.structure/1efy_rec_dockprep.mol2 fps_score_vdw_att_exp 6 fps_score_vdw_rep_exp 9 fps_score_vdw_rep_rad_scale 1 fps_score_use_distance_dependent_dielectric yes fps_score_dielectric 4.0 fps_score_vdw_fp_scale 1 fps_score_es_fp_scale 1 fps_score_hb_fp_scale 0 pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn flex_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn flex_drive_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl ligand_outfile_prefix footprint.out write_footprints yes write_hbonds yes write_orientations no num_scored_conformers 1 rank_ligands no
Upon completion, several .txt files should be produced. In order to convert these to .pdf files for easy viewing, use the plot_footprint_single_magnitude.py python script to show the results for the 50 highest scoring residues. The result should look similar to this:
Virtual Screen
Virtual screening is a widely used method for screening many drug-like molecules from a curated library for binding affinity via Dock in order to identify potential lead molecules. In this example we will be utilizing the VS_library_5k.mol2 library which contains over 5,000 drug-like molecules from the ZINC database. This library can be found in the /gpfs/projects/AMS536/2021/ directory. Copy this file into the 005.virtual_screen directory.
Create an input file:
touch virtual.in
dock6 -i virtual.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 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_25K.mol2 limit_max_ligands no skip_molecule no read_mol_solvation no calculate_rmsd no use_database_filter no orient_ligand yes automated_matching yes receptor_site_file ../002.surface_spheres/selected_spheres.sph max_orientations 1000 critical_points no chemical_matching no use_ligand_spheres no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary yes grid_score_secondary no grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix ../003.gridbox/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes minimize_anchor yes minimize_flexible_growth yes use_advanced_simplex_parameters no simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_anchor_max_iterations 500 simplex_grow_max_iterations 500 simplex_grow_tors_premin_iterations 0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_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
Allow the job to run for approximately a minute to ensure that it is working properly. Important: After ensuring the job is working, press ctrl+c to stop the job As docking several thousand molecules to the 1efy receptor requires a significant amount of computing power, we do not want to run the job on the head node. We will use slurm scripts to submit the job to the seawulf cluster. Before determining which queue to use, it may be beneficial to determine their availability. Use the command sinfo and look for queues with idle nodes. In addition, to prevent your job from being automatically rejected use [2] to determine the max time, min/max nodes, min/max cores, etc. for each particular queue. SCP the VS_library_5k.mol2 and virtual.in file into the 006.virtual_screen_mpi directory.
To submit the job to the queue create the following input file:
vi virtual.sh
#!/bin/bash #SBATCH --time=1-8:00:00 #SBATCH --nodes=1 #SBATCH --ntasks=40 #SBATCH --job-name=1EFY_vs #SBATCH --output=1EFY_vs.out #SBATCH -p extended-40core #SBATCH --mail-type=BEGIN,END #SBATCH --mail-user=EXAMPLE@stonybrook.edu
cd $SLURM_SUBMIT_DIR mpirun -np 40 dock6.mpi -i virtual.in -o virtual.out
This script will submit the job to the extended-40core queue, using 1 node, 40 cores, for a maximum of 8 hours. In addition, you can input your email address in the line #SBATCH --mail-user=EXAMPLE@stonybrook.edu , and you will be emailed at the start and end of the job.
Submit the job using :
qsub virtual.sh
You can check the status of the job using:
squeue -u NETID
And inputting your netid where the command says NETID.
Cartesian Minimization
Once we have docked the library to the receptor, it is then necessary to perform a cartesian minimization of them. Move to the 007.cartesian_min directory.
Create the following input file:
touch min.in
dock6 -i min.in
conformer_search_type rigid use_internal_energy yes internal_energy_rep_exp 9 internal_energy_cutoff 100.0 ligand_atom_file ../006.virtual_screen_mpi/virtual.out_scored.mol2 limit_max_ligands no skip_molecule no read_mol_solvation no calculate_rmsd no use_database_filter no orient_ligand no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary no grid_score_secondary no multigrid_score_primary no multigrid_score_secondary no dock3.5_score_primary no dock3.5_score_secondary no continuous_score_primary yes continuous_score_secondary no cont_score_rec_filename ../001.structures/1EFY_rec_dockprep.mol2 cont_score_att_exp 6 cont_score_rep_exp 9 cont_score_rep_rad_scale 1 cont_score_use_dist_dep_dielectric yes cont_score_dielectric 4.0 cont_score_vdw_scale 1 cont_score_es_scale 1 footprint_similarity_score_secondary no pharmacophore_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_score_secondary no amber_score_secondary no minimize_ligand yes simplex_max_iterations 1000 simplex_tors_premin_iterations 0 simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn flex_defn_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn flex_drive_file /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl ligand_outfile_prefix 1efy.virtual_screen.min write_orientations no num_scored_conformers 1 rank_ligands no
Run the cartesian min by submitting to the seawulf queue as shown in the previous section. Be sure to properly edit the names of the input and output files.
Rescore
Finally, the energy minimized structures will be ranked according to several different parameters. This will allow us to choose potential leads based on a variety of factors such as footprint overlap, hungarian, pharmacophore, tanimoto, etc. Navigate to the 008.rescore directory.
First create the input file:
touch rescore.in
dock6 -i rescore.in
conformer_search_type rigid use_internal_energy yes internal_energy_rep_exp 9 internal_energy_cutoff 100.0 ligand_atom_file ../007.cartesian_min/1efy.virtual_screen.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_tanimoto 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/1EFY_ligand_min_scored.mol2 descriptor_fps_score_foot_compare_type Euclidean descriptor_fps_score_normalize_foot no descriptor_fps_score_foot_comp_all_residue yes descriptor_fps_score_receptor_filename ../001.structure/1EFY_rec_dockprep.mol2 descriptor_fps_score_vdw_att_exp 6 descriptor_fps_score_vdw_rep_exp 9 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
Submit to the seawulf queue as shown previously.
For example, the following image depicts a comparison of the crystallized ligand with the molecule with the highest Footprint similarity score - ZINC02877436
