Ligand Enrichment Tutorial

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The purpose of this tutorial is to develop a uniform method to test ligand enrichment across the Rizzo lab with the DOCK software.

I.Introduction

Ligand Enrichment is a common experiment used to evaluate how well a docking program is capable of accurately modeling in vitro experiments. This experiment uses active ligands and decoy ligands to access a docking programs ability to successfully dock to a target site. These active and decoy ligands are roughly the same size and differ due to chemical similarities. These active ligands should bind more favorably(Have a lower energy score) then the decoy ligands if the docking program can accurately model these binding site and ligand interactions.


The 3 major outcomes for this experiment are early enrichment, random enrichment, and late enrichment. Early enrichment indicates the active ligands dock more successful in the experiment(The goal for all docking programs). The second is random enrichment indicating that the docking program can differentiate between active and decoy. Late enrichment indicating that docking software gives the lowest energy scores to the decoys which is the worst outcome. The other factor to consider is the degree of early and late enrichment

II.Prepping systems

-The first step is to create a directory for the system you are preparing

    mkdir 1Q4X

-The first step is to obtain the active and decoy ligand test set systems which can be found on the Schoichet DUD-E test set website http://dude.docking.org/targets

-Once these targets are obtained unzip these files using the gzip command to get the active and decoy forms

gzip -d actives_final.mol2.gz 
gzip -d decoys_final.mol2.gz 
   

-Prepare the target receptor by either using the official test set ligands or manually prepare a receptor target from scratch

Following all these steps your directory should look like the following using the 1QRX system

    actives_final.mol2
    decoys_final.mol2
    1Q4X.rec.clean.mol2

III.Docking molecules

-After completing this step a virtual screen will be conducted using mpi for both the active and decoy ligands seperately

-The input parameters are as follows for the active ligands

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                                             actives_final.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                                           /gpfs/projects/rizzo/ccorbo/2020_DUDE_0.3_gridspacing/DUDE_Good_to_go/1Q4X/1Q4X.rec.clust.close.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                                       /gpfs/projects/rizzo/ccorbo/2020_DUDE_0.3_gridspacing/DUDE_Good_to_go/1Q4X/1Q4X.rec
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/rizzo/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/rizzo/zzz.programs/dock6.9_release/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/rizzo/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
ligand_outfile_prefix                                        1Q4X.active.output.mpi
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

-The input parameters for the decoy ligands.

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                                             decoys_final.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                                           /gpfs/projects/rizzo/ccorbo/2020_DUDE_0.3_gridspacing/DUDE_Good_to_go/1Q4X/1Q4X.rec.clust.close.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                                       /gpfs/projects/rizzo/ccorbo/2020_DUDE_0.3_gridspacing/DUDE_Good_to_go/1Q4X/1Q4X.rec
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/rizzo/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/projects/rizzo/zzz.programs/dock6.9_release/parameters/flex.defn
flex_drive_file                                              /gpfs/projects/rizzo/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
ligand_outfile_prefix                                        1Q4X.decoy.output.mpi
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


-Then submit the script to the qsub to dock the molecule in parallel. Some of the ligand active and decoy testsets are quite large so mpi submission is recommended.

#!/bin/bash
#SBATCH --partition=rn-long-40core
#SBATCH --time=48:00:00
#SBATCH --nodes=4
#SBATCH --ntasks=160
#SBATCH --job-name=1B9V_mpi_runs
#SBATCH --output=1B9V_mpi_runs
cd $SLURM_SUBMIT_DIR
module load intel/mpi/64/2018/18.0.3
mpirun -np 160 dock6.mpi -i 1Q4X_active_mpi.in -o 1Q4X_decoy_mpi.out
mpirun -np 160 dock6.mpi -i 1Q4X_decoy_mpi.in -o 1Q4X_decoy_mpi.out

IV.Ligand Enrichment Analysis

-Lastly, 2 scripts were developed to analyze the results. One script to generate a CSV file and a secondary script that uses the CSV data to create a graph.

-The script that generates the CSV file takes three parameters, the list of systems, name of decoy ligands mol2 file, name of active ligands mol2 file. (NOTE: This script can generate multiple CSV files for different ligand experiments, but the naming of the active and decoy mol2 files must be the same,) The 1Q4X.txt file has the following text

1Q4X

If your creating multiple csvs for multiple systems the format fill be

1Q4X
1LRU
1SYN
etc

This script is run one directory before the 1Q4X directory(Not in the 1Q4X directory)

1Q4X/

Example:

python roc_curve_lig_enrichment_v2.py 1Q4X.txt decoys_final.mol2 actives_final.mol2

This produces the csv file in the 1Q4X

1Q4X_lig_enrichment.csv

Following this a python script is used to create a graph to analyze the results First change directory into the 1Q4X directory

cd 1Q4X

Then run the script make_roc_curve.py CSV_file Name (Note: Name can be anything) Example:

python ../make_roc_curve.py 1Q4X_lig_enrichment.csv DOCK6.9
1Q4X ligand enrichment DOCK6.9.png

This ROC curve is generated using 2 formulas for the graph For Decoy, the x-axis

# of Docked Decoys/Total Decoys

For Active, the y-axis

# of Docked Actives/Total Actives

These ROC curves start with no active and decoy ligands docked.

# of Docked Decoys=0
# of Docked Actives=0

Then these are sorted from lowest to highest energy score, if active is lower then 1 is added to active

# of Docked Decoys=# of Docked Decoys
# of Docked Actives=# of Docked Actives + 1

If decoys are lower than 1 is added to the decoy list

# of Docked Decoys=# of Docked Decoys + 1
# of Active Decoys=# of Actives Decoys

These are all continued until all of these active and decoys ligands are added til graph is [1,1] (Note: If all molecules aren't docked 1,1 is appended to the end of the csv as the final location)