2017 Denovo design tutorial with PDB 1BJU

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2017 Denovo design tutorial with PDB 1BJU

The Denovo module of DOCK is a relatively new feature (as of Fall 2016) that constructs new ligand molecules inside a protein active site from a library of user-specified "fragments", and then scores them based off the scoring method being utilized. These fragments are common chemical functional groups -- or building blocks -- that are typically selected from a ZINC library of millions of compounds based off of their frequency of appearance. These fragments are classified as scaffolds, linkers, or side chains, according to the number of atomic positions that are permitted to seed growth: 3, 2, and 1 atoms, respectively. Thus, a scaffold could seed growth from three different atoms, having three linkers bonded to each position, and a linker could seed growth on two positions, and a side-chain on one position.

Once the molecules are built within the active site, their interactions with the protein are scored using the user-specified method of scoring. This tutorial will walk through the steps needed to run a Denovo calculation on the Beta Trypsin system from the 2016 DOCK tutorial. This method will utilize the multi-grid scoring function, called through the descriptor score. Ensure you have all the folders and files necessary from running the 2016 tutorial, if not, they can be found on LIRed through this path: /gpfs/home/guest43/scratch/denovo/wiki_denovo/001.files/

Before running the calculation, it's worth looking through the "Things to Keep in Mind" section at the bottom for some good pieces of information.

The Files Needed For Denovo

To run the Denovo code with multigrid scoring you need these files:

fraglib_scaffold.mol2
fraglib_linker.mol2
fraglib_sidechain.mol2
anchor_library.mol2
fraglib_torenv.dat
selected_spheres.sph
primary_residues_multigrid.bmp / .nrg  
multigrid_minimized_ligand.mol2
vdw_AMBER_parm99.defn
flex.defn
flex_drive.tbl
vdw_DumHyd.defn (needed for scoring functions other than multigrid)

The fragment libraries and parameter files must be obtained prior to the Denovo calculation, and can be found on LIRed through the paths

/gpfs/home/guest43/scratch/denovo/trial_denovo/000.fraglib 
/trial_denovo/zzz.parameters/

Everything else is generated through this tutorial, prior to running the Denovo code.

Preparing The Files

Before running Denovo on Beta-Trypsin, please ensure you have gone through the DOCK 2016 tutorial and have all the resulting files. The tutorial can be accessed through here.

You should have these files in your directory:

1BJU.pdb
1BJU.lig.mol2
1BJU.rec.clean.mol2
1BJU.rec.noH.mol2
selected_spheres.sph

Additionally, you will also need these parameter files:

vdw_AMBER_parm99.defn
flex.defn
flex_drive.tbl

In order to run Denovo with multigrid scoring, we must first go through several steps:

1). Create a primary residue text file and a reference text file -- selects the primary residues of interest.

2). Make a multigrid file for each specified residue -- forms a grid for each residue specified in previous step.

3). Minimizes ligand mol2 file using multigrids from previous step.

4). Rescores ligand on multigrid to yield a minimized ligand .mol2 file. This serves as the reference ligand for Denovo calculations.

Luckily, our good friend Brian generated some extremely robust scripts to make this process easier. There is one script for each step, but we will only use the simple input files for DOCK. If you are interested in using the scripts (and a lot of debugging), they can be found on lired under: /gfps/home/guest43/scratch/denovo/trial_denovo/run/ .

DOCK Specifying Primary Residues

Create a directory within your working directory titled 008.footprint_rescore. This is where all pertinent files from this step will go, and where we will run our calculation from.

The input file for this step should be titled 1BJU.footprint_rescore.in, and should look like (substitute your own working directory for ${WORKDIR}) :

conformer_search_type                                        rigid
use_internal_energy                                          no
ligand_atom_file                                             ${WORKDIR}/001.files/1BJU.lig.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_use_footprint_reference_mol2                             yes
fps_footprint_reference_mol2_filename                        ${WORKDIR}/001.files/1BJU.lig.mol2
fps_foot_compare_type                                        d
fps_normalize_foot                                           no
fps_foot_comp_all_residue                                    no
fps_choose_foot_range_type                                   threshold
fps_vdw_threshold                                            1
fps_es_threshold                                             0.5
fps_hb_threshold                                             0.5
fps_use_remainder                                            yes
fps_receptor_filename                                        ${WORKDIR}/001.files/1BJU.rec.clean.mol2
fps_vdw_att_exp                                              6
fps_vdw_rep_exp                                              12
fps_vdw_rep_rad_scale                                        1
fps_use_distance_dependent_dielectric                        yes
fps_dielectric                                               4.0
fps_vdw_fp_scale                                             1
fps_es_fp_scale                                              1
fps_hb_fp_scale                                              0
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_score_secondary                              no
amber_score_secondary                                        no
minimize_ligand                                              no
atom_model                                                   all
vdw_defn_file                                                ${WORKDIR}/001.files/vdw_AMBER_parm99.defn
flex_defn_file                                               ${WORKDIR}/001.files/flex.defn
flex_drive_file                                              ${WORKDIR}/001.files/flex_drive.tbl
ligand_outfile_prefix                                        output
write_footprints                                             yes
write_hbonds                                                 no
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no


This calculation should be done very quickly (<10 seconds), and upon finishing you will have three output files:

1BJU.footprint_rescore.out 
output_footprint_scored.txt
output_scored.mol2


Now, we must declare the primary residues in the active site and generate a grid file for each. Create a new file in the text editor named 1BJU.primary_residues.sh. Write this inside of it (copied from Brian's script *.fpsrescore.qsub.sh):

#!/bin/bash
grep -A 1 "range_union" 1BJU.footprint_rescore.out |
grep -v "range_union" |
grep -v "\-" |
sed -e '{s/,/\n/g}' |
sed -e '{s/ //g}' |
sed '/^$/d' |
sort -n |
uniq > temp.dat
for i in `cat temp.dat`; do printf "%0*d\n" 3 $i; done > 1BJU.primary_residues.dat
for RES in `cat temp.dat`
do
        grep " ${RES} " output_footprint_scored.txt  |
        awk -v temp=${RES} '{if ($2 == temp) print $0;}' |
        awk '{print $1 "  " $3 "  " $4}' >> reference.txt
done
grep "remainder" output_footprint_scored.txt |
sed -e '{s/,/  /g}' |
tr -d '\n' |
awk '{print $2 "  " $3 "  " $6}' >> reference.txt
mv reference.txt 1BJU.reference.txt
rm temp.dat

Run the script and you should have two new files:

1BJU.primary_residues.dat
1BJU.reference.txt 

These are our primary residues! Now we need to generate a grid for each one.

Generating the Grids

We must now generate a grid file for each residue. To do so, we will need the aid of another one of Brian's scripts: 1BJU.make_multigrids.qsub.sh. But before we can use his script, we need to generate two input files for Dock which will be called upon by the script. Create a file named 1BJU.multigrid.in inside your 007.multigrid folder with the following inside it:

compute_grids                  yes
grid_spacing                   0.4
output_molecule                yes
contact_score                  no
chemical_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                  temp.mol2
box_file                       ../001.files/1bju.box.pdb
vdw_definition_file            ../001.files/vdw_AMBER_parm99.defn
chemical_definition_file       ../001.files/chem.defn
score_grid_prefix              temp.rec
receptor_out_file              temp.rec.grid.mol2

Additionally, create a file named 1BJU.reference_multigrid.in:

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.files/1BJU.lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../001.files/1BJU.lig.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                                           no
grid_score_secondary                                         no
multigrid_score_primary                                      yes
multigrid_score_secondary                                    no
multigrid_score_rep_rad_scale                                1
multigrid_score_vdw_scale                                    1
multigrid_score_es_scale                                     1
multigrid_score_number_of_grids                              19
multigrid_score_grid_prefix0                                 ../007.multigrid/1BJU.resid_001
multigrid_score_grid_prefix1                                 ../007.multigrid/1BJU.resid_040
multigrid_score_grid_prefix2                                 ../007.multigrid/1BJU.resid_081
multigrid_score_grid_prefix3                                 ../007.multigrid/1BJU.resid_084
multigrid_score_grid_prefix4                                 ../007.multigrid/1BJU.resid_171
multigrid_score_grid_prefix5                                 ../007.multigrid/1BJU.resid_172
multigrid_score_grid_prefix6                                 ../007.multigrid/1BJU.resid_173
multigrid_score_grid_prefix7                                 ../007.multigrid/1BJU.resid_174
multigrid_score_grid_prefix8                                 ../007.multigrid/1BJU.resid_176
multigrid_score_grid_prefix9                                 ../007.multigrid/1BJU.resid_177
multigrid_score_grid_prefix10                                ../007.multigrid/1BJU.resid_191
multigrid_score_grid_prefix11                                ../007.multigrid/1BJU.resid_192
multigrid_score_grid_prefix12                                ../007.multigrid/1BJU.resid_193
multigrid_score_grid_prefix13                                ../007.multigrid/1BJU.resid_194
multigrid_score_grid_prefix14                                ../007.multigrid/1BJU.resid_196
multigrid_score_grid_prefix15                                ../007.multigrid/1BJU.resid_197
multigrid_score_grid_prefix16                                ../007.multigrid/1BJU.resid_204
multigrid_score_grid_prefix17                                ../007.multigrid/1BJU.resid_206
multigrid_score_grid_prefix18                                /gpfs/home/guest43/scratch/denovo/wiki_denovo/007.multigrid/1BJU.resid_remaining
multigrid_score_fp_ref_mol                                   no
multigrid_score_fp_ref_text                                  yes
multigrid_score_footprint_text                               /gpfs/home/guest43/scratch/denovo/wiki_denovo/008.footprint_rescore/1BJU.reference.txt
multigrid_score_use_euc                                      yes
multigrid_score_use_norm_euc                                 no
multigrid_score_use_cor                                      no
multigrid_vdw_euc_scale                                      1
multigrid_es_euc_scale                                       1
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
ph4_score_secondary                                          no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_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                                5.0
atom_model                                                   all
vdw_defn_file                                                ../zzz.parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               ../zzz.parameters/flex.defn
flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
ligand_outfile_prefix                                        output
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Now that we have our input files, we can form the script that will call upon them to generate the grid files for each specified residue. Create a blank file named 1BJU.make_multigrids.qsub.sh in your 007.multigrid folder. Then transcribe into it:

cd /gpfs/home/guest43/scratch/denovo/trial_denovo/009.make-mg/
export PRIMARY_RES=` cat ../008.footprint_rescore/1BJU.primary_residues.dat | sed -e 's/\n/ /g' `
export DOCKHOME="/gpfs/home/guest43/local/dock.6.7_2015-02-17.denovo_paper.2016.05.04/"
python /gpfs/home/guest43/local/dock.6.7_2015-02-17.denovo_paper.2016.05.04/bin/multigrid_fp_gen.py ../001.files/1BJU.rec.clean.mol2 1BJU.resid 1BJU.multigrid.in ${PRIMARY_RES}
rm temp.mol2
rm 1BJU.resid_*.rec.grid.mol2
/gpfs/home/guest43/local/dock.6.7_2015-02-17.denovo_paper.2016.05.04/bin/dock6 -i 1BJU.reference_multigridmin.in -o 1BJU.reference_multigridmin.out
mv output_scored.mol2 1BJU.lig.multigridmin.mol2
cp 1BJU.lig.multigridmin.mol2 ../001.files/

Change the path to Dock and your primary residue file if necessary, and ensure you are using a version of Dock with the Denovo code. If you get an error that says something like "cannot stat *.nrg / *.bmp" etc, check to make sure your directories are all pointing to the right places in your two input files. After running this script, you should be given a plethora of different files. If you are running on the 1BJU system, you should have 19 different residues: 18 individual residues, and a 19th file containing the grid for the rest of the residues. You will have four files for each residue: a .bmp file, a .mol2 file, a .nrg file, and a .out file (for each residue!). Additionally you should have two other files: 1BJU.lig.multigridmin.mol2, 1BJU.reference_multigridmin.out. Check your output file for any errors and to make sure everything ran to completion. Visualize your ligand in Chimera to make sure it contains atoms and looks like a real chemical structure. You should have something that looks like this:

1BJU.multigridmin.png

Minimizing Ligand on Grids

We're taking the input here straight from Brian's script for this part (run.003e.mg_rescore.sh). Create an input file named 1BJU.parents_multigridmin.in with this inside it:

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
ligand_atom_file                                             ../001.files/1BJU.lig.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                                      yes
multigrid_score_secondary                                    no
multigrid_score_rep_rad_scale                                1
multigrid_score_vdw_scale                                    1
multigrid_score_es_scale                                     1
multigrid_score_number_of_grids                              19
multigrid_score_grid_prefix0                                 ../007.multigrid/1BJU.resid_001
multigrid_score_grid_prefix1                                 ../007.multigrid/1BJU.resid_040
multigrid_score_grid_prefix2                                 ../007.multigrid/1BJU.resid_081
multigrid_score_grid_prefix3                                 ../007.multigrid/1BJU.resid_084
multigrid_score_grid_prefix4                                 ../007.multigrid/1BJU.resid_171
multigrid_score_grid_prefix5                                 ../007.multigrid/1BJU.resid_172
multigrid_score_grid_prefix6                                 ../007.multigrid/1BJU.resid_173
multigrid_score_grid_prefix7                                 ../007.multigrid/1BJU.resid_174
multigrid_score_grid_prefix8                                 ../007.multigrid/1BJU.resid_176
multigrid_score_grid_prefix9                                 ../007.multigrid/1BJU.resid_177
multigrid_score_grid_prefix10                                ../007.multigrid/1BJU.resid_191
multigrid_score_grid_prefix11                                ../007.multigrid/1BJU.resid_192
multigrid_score_grid_prefix12                                ../007.multigrid/1BJU.resid_193
multigrid_score_grid_prefix13                                ../007.multigrid/1BJU.resid_194
multigrid_score_grid_prefix14                                ../007.multigrid/1BJU.resid_196
multigrid_score_grid_prefix15                                ../007.multigrid/1BJU.resid_197
multigrid_score_grid_prefix16                                ../007.multigrid/1BJU.resid_204
multigrid_score_grid_prefix17                                ../007.multigrid/1BJU.resid_206
multigrid_score_grid_prefix18                                ../007.multigrid/1BJU.resid_remaining
multigrid_score_fp_ref_mol                                   no
multigrid_score_fp_ref_text                                  yes
multigrid_score_footprint_text                               ../008.footprint_rescore/1BJU.reference.txt
multigrid_score_use_euc                                      yes
multigrid_score_use_norm_euc                                 no
multigrid_score_use_cor                                      no
multigrid_vdw_euc_scale                                      1
multigrid_es_euc_scale                                       1
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
ph4_score_secondary                                          no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_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                                5.0
atom_model                                                   all
vdw_defn_file                                                /gpfs/home/guest43/scratch/denovo/wiki_denovo/001.files/vdw_AMBER_parm99.defn
flex_defn_file                                               /gpfs/home/guest43/scratch/denovo/wiki_denovo/001.files/flex.defn
flex_drive_file                                              /gpfs/home/guest43/scratch/denovo/wiki_denovo/001.files/flex_drive.tbl
ligand_outfile_prefix                                        output
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

After running this with dock6 you should have an output file (which should be checked for errors, as always) and a .mol2 file named output_scored.mol2. Rename this to 1BJU.parents_multigridmin.mol2, and visualize it in Chimera, to ensure you still have a realistic molecule. This is the mol2 file of the ligand minimized using the multigrid scoring. This will serve as our reference molecule for guided growth!

Running Denovo

We can now run a Denovo calculation! Rejoice! Compared to the previous steps, this part is fairly straight forward. Simply create the input file, and create a script to submit it to the cluster.

Creating The Input File

Create a folder named 010.denovo. Then, inside this directory, create an input file with the following inside it:

conformer_search_type                                        denovo
dn_fraglib_scaffold_file                                     /gpfs/home/guest43/scratch/denovo/trial_denovo/000.fraglib/fraglib_scaffold.mol2
dn_fraglib_linker_file                                       /gpfs/home/guest43/scratch/denovo/trial_denovo/000.fraglib/fraglib_linker.mol2
dn_fraglib_sidechain_file                                    /gpfs/home/guest43/scratch/denovo/trial_denovo/000.fraglib/fraglib_sidechain.mol2
dn_user_specified_anchor                                     yes
dn_fraglib_anchor_file                                       03_anchors_byfreq.mol2
dn_use_torenv_table                                          yes
dn_torenv_table                                              /gpfs/home/guest43/scratch/denovo/trial_denovo/000.fraglib/fraglib_torenv.dat
dn_sampling_method                                           graph
dn_graph_max_picks                                           30
dn_graph_breadth                                             3
dn_graph_depth                                               2
dn_graph_temperature                                         100
dn_pruning_conformer_score_cutoff                            100.0
dn_pruning_conformer_score_scaling_factor                    2.0
dn_pruning_clustering_cutoff                                 100.0
dn_constraint_mol_wt                                         750
dn_constraint_rot_bon                                        15
dn_constraint_formal_charge                                  2.0
dn_heur_unmatched_num                                        1
dn_heur_matched_rmsd                                         2.0
dn_unique_anchors                                            3
dn_max_grow_layers                                           9
dn_max_root_size                                             25
dn_max_layer_size                                            25
dn_max_current_aps                                           5
dn_max_scaffolds_per_layer                                   1
dn_write_checkpoints                                         yes
dn_write_prune_dump                                          yes
dn_write_orients                                             no
dn_write_growth_trees                                        no
dn_output_prefix                                             1BJU.final
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
internal_energy_cutoff                                       100.0
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../001.files/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                                           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
ph4_score_primary                                            no
ph4_score_secondary                                          no
descriptor_score_primary                                     yes
descriptor_score_secondary                                   no
descriptor_use_grid_score                                    no
descriptor_use_multigrid_score                               yes
descriptor_use_pharmacophore_score                           no
descriptor_use_tanimoto                                      no
descriptor_use_hungarian                                     no
descriptor_multigrid_score_rep_rad_scale                     1.0
descriptor_multigrid_score_vdw_scale                         1.0
descriptor_multigrid_score_es_scale                          1.0
descriptor_multigrid_score_number_of_grids                   22
descriptor_multigrid_score_grid_prefix0                      ../007.multigrid/1BJU.resid_001
descriptor_multigrid_score_grid_prefix1                      ../007.multigrid/1BJU.resid_040
descriptor_multigrid_score_grid_prefix2                      ../007.multigrid/1BJU.resid_081
descriptor_multigrid_score_grid_prefix3                      ../007.multigrid/1BJU.resid_084
descriptor_multigrid_score_grid_prefix4                      ../007.multigrid/1BJU.resid_171
descriptor_multigrid_score_grid_prefix5                      ../007.multigrid/1BJU.resid_172
descriptor_multigrid_score_grid_prefix6                      ../007.multigrid/1BJU.resid_173
descriptor_multigrid_score_grid_prefix7                      ../007.multigrid/1BJU.resid_174
descriptor_multigrid_score_grid_prefix8                      ../007.multigrid/1BJU.resid_176
descriptor_multigrid_score_grid_prefix9                      ../007.multigrid/1BJU.resid_177
descriptor_multigrid_score_grid_prefix10                     ../007.multigrid/1BJU.resid_189
descriptor_multigrid_score_grid_prefix11                     ../007.multigrid/1BJU.resid_190
descriptor_multigrid_score_grid_prefix12                     ../007.multigrid/1BJU.resid_191
descriptor_multigrid_score_grid_prefix13                     ../007.multigrid/1BJU.resid_192
descriptor_multigrid_score_grid_prefix14                     ../007.multigrid/1BJU.resid_193
descriptor_multigrid_score_grid_prefix15                     ../007.multigrid/1BJU.resid_194
descriptor_multigrid_score_grid_prefix16                     ../007.multigrid/1BJU.resid_195
descriptor_multigrid_score_grid_prefix17                     ../007.multigrid/1BJU.resid_196
descriptor_multigrid_score_grid_prefix18                     ../007.multigrid/1BJU.resid_197
descriptor_multigrid_score_grid_prefix19                     ../007.multigrid/1BJU.resid_204
descriptor_multigrid_score_grid_prefix20                     ../007.multigrid/1BJU.resid_206
descriptor_multigrid_score_grid_prefix21                     ../007.multigrid/1BJU.resid_remaining
descriptor_multigrid_score_fp_ref_mol                        yes
descriptor_multigrid_score_footprint_ref                     ../07.multigrid/1BJU.parents.multigridmin.mol2
descriptor_multigrid_score_use_euc                           yes
descriptor_multigrid_score_use_norm_euc                      no
descriptor_multigrid_score_use_cor                           no
descriptor_multigrid_vdw_euc_scale                           1.0
descriptor_multigrid_es_euc_scale                            1.0
descriptor_weight_multigrid_score                            1
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_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                                                ../001.files/vdw_AMBER_parm99.defn
flex_defn_file                                               ../001.files/flex.defn
flex_drive_file                                              ../001.files/flex_drive.tbl

There are a few things to note here: you must specify a .mol2 file for the scaffold, linker, and sidechain libraries; you must specify your anchor library, which must be tailored prior to the calculation to include the specific anchors you would like to seed from; finally, even though we call upon the descriptor score, we only do so to call our multigrid scoring function -- we are not using descriptor grid score. This would be the same as running with descriptor_score = no and multigrid_score = yes, but it is standard protocol to call any and all scoring functions through descriptor score, regardless of if you're using it or not.

Creating a Script to Submit Denovo to LIRED

Now we want to generate a script that will call Dock to run the input file we just generated. Why do we need to make a script instead of submitting it directly to Dock? Denovo calculations take a good amount of time and can get very computationally expensive, thus we will want to submit it to a cluster using qsub. Generate a script with the following inside it:

#!/bin/bash
#PBS -l walltime=48:00:00
#PBS -l nodes=1:ppn=24
#PBS -q long
#PBS -N 1BJU.denovo
#PBS -V
/gpfs/home/guest43/local/dock.6.7_2015-02-17.denovo_paper.2016.05.04/bin/dock6 -i 1BJU.denovo_mg.in -o 1BJU.denovo_mg.out

Submit this to the queue by typing:

qsub <script_name>

Monitor the output file to see which anchor/layer the calculation is at. Run this calculation from your 010.denovo directory.

Viewing Your Results

After the calculation has (successfully) finished, you should have in your directory a large amount of new files. These files take the form 1BJU.final_anchor_*.prune_dump_layer_*.mol2 and 1BJU.final_anchor_*.root_layer_*.mol2. For each anchor, you will have a number of both of these files equal to the number of molecules per layer you specified in the input file. Additionally, you will have an output file (which, of course, should be checked for errors), and a file named 1BJU.final.denovo_build.mol2 -- this is your final output file containing all of the constructed and scored molecules. We are going to open this in Chimera using ViewDock.

First, in Chimera, open your 1BJU.parents.multigridmin.mol2 file, then on top of that open the cleaned receptor file. Then click Tools > Surface/Binding Analysis > ViewDock and open the 1BJU.final.denovo_build.mol2 file. This file can have upwards of a thousand different molecules in it, depending on how many anchors and layers you used, and can take a little while to open. Once you select the file in ViewDock and click open, most likely Chimera will freeze, and you won't be able to do anything. It must load all the molecules at once, so give it a good five or ten minutes to load before you decide to quit the program. It will open, you just have to be patient.

Once it has loaded you can arrange your molecules based on their descriptor (multigrid) score and view them imposed over the reference ligand in the active site.

Things to Keep in Mind

When running Denovo for the first time, it is strongly encouraged that you run it through interactive mode first: that is, generate an empty input file, and run the code inputting the parameters manually for each question. This will give you a good idea of what it wants, what it's doing, and where any potential errors you may come across are originating from.

The Denovo code takes anywhere from 4-8 hours per anchor for 15 molecules per layer depending on a myriad of factors: the anchor being used, the specific system, the number of grids, the scoring function, etc.

If you submit an anchor library containing more anchors than you will use (ex: library has 100 anchors, you're only using five) the Denovo code will automatically pick the largest anchors! Thus, if you do not specify your anchors, upon finishing your calculation and reviewing your structures, you will notice a disturbing amount of large ring structures. To get around this, be sure to use an anchor library which you have personally compiled and be aware which order it will run the calculation in (it chooses the largest molecular weight anchor first).

It has been determined that the Denovo code is sequence independent. Meaning that the results do not depend on the order of their calculation. For example, if you have in your anchor library file anchors A, B, and C for a Denovo calculation, you will receive the same results (molecules, conformations, and scores) as if you had run the calculation for A, B, and C individually, with each structure in their own anchor file.

For multigrid scoring, you do not need to specify a dummy atom, or use the corresponding dummy_H parameter file. For other types of scoring functions you will have to specify in your anchor files which atoms are the dummy atoms.

Dock can be finicky about paths. Sometimes it doesn't want full paths (i.e. originating from the top directory, /gpfs), but other times it wants the explicit path in its entirety. If you keep receiving an error about a file location, and you are positive you have entered the correct path, try either reducing the path as much as possible (starting from your home directory, ~/ ) or try including the full path if you have not.

If Dock does not accept "denovo" as a conformer_search_type then you are not running a version that contains the Denovo code.

We have ran the Denovo code here linearly, that is with each anchor being ran to completion before beginning the next. The Denovo calculations finish all the layers of an anchor before moving on to the next one. For larger calculations (with more than 5-10 anchors) you may want to consider running the calculation serially, essentially making each anchor its own job, and then collating the data. This will run much quicker, but will require additional post-processing of the data. Somewhere around fifteen anchors per run is ideal.

An enormous amount of credit goes to Brian for generating the scripts and general protocol for running Denovo calculations. I could not have figured it out without his groundwork. Thanks Brian!

Length of Denovo Calculations

The Denovo code can take a large amount of time, especially as the number of anchors and layers is increased. To give an idea of how long the Denovo calculations take, below are some details from different runs on Beta-Trypsin (the .out file from the Denovo code has the total calculation time in seconds at the bottom):

1). 3 anchors with 9 molecules per layer: ~ 16.7 hours (~ 5.6 hours per anchor)
2). 15 anchors with 25 molecules per layer: ~ 107.7 hours (~7 hours per anchor)
3). 1 anchor with 25 molecules per layer: 8.1 hours