View source for 2017 Denovo design tutorial 1 with PDB 1BJU

==2017 Denovo design tutorial 1 with PDB 1BJU==

The de novo module of DOCK6 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 de novo 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 De novo===

To run the de novo 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

The fragment libraries and parameter files must be obtained prior to the de novo growth, and can be found on LIRed through the paths 

 /PATH/trial_denovo/000.fraglib 
 /PATH/DOCK6/parameters/

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

==Preparing The Files==

Before running de novo 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 [http://ringo.ams.sunysb.edu/index.php/2016_DOCK_tutorial_with_Beta_Trypsin 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 found in the parameters directory fo DOCK6:
 vdw_AMBER_parm99.defn
 flex.defn
 flex_drive.tbl
In order to run de novo 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 de novo calculations.

There is one script for each step, but we will only use the simple input files for DOCK6. If you are interested in using the scripts (and a lot of debugging), they can be found on lired under: /PATH/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 de novo 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:

 [[File: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 De novo ==

We can now run de novo growth! 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                                        nova
 dn_fraglib_scaffold_file                                     /PATH/trial_denovo/000.fraglib/fraglib_scaffold.mol2
 dn_fraglib_linker_file                                       /PATH/trial_denovo/000.fraglib/fraglib_linker.mol2
 dn_fraglib_sidechain_file                                    /PATH/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                                              /PATH/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 De novo 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 de novo 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 de novo 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 de novo 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 de novo 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 de novo 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 de novo code.

We have ran the de novo code here '''linearly''', that is with each anchor being ran to completion before beginning the next. The de novo 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 de novo DOCK. I could not have figured it out without his groundwork. Thanks Brian!

===Length of Denovo Growth===

The de novo 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 de novo calculations take, below are some details from different runs on Beta-Trypsin (the .out file from the de novo 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