2023.04.11.6OIM - tbalius/teb_docking_test_sets GitHub Wiki
DOCK 6 tutorial using KRAS in complex with Sotorasib/AMG 510 (PDB ID 6OIM)
By Y. Stanley Tan, Mayukh Chakrabarti, and Trent E. Balius
Based on 2022 Rizzo lab tutorial.
Getting started
Command line
This tutorial expects a basic understanding of Unix.
Here is a Unix tutorial: http://www.ee.surrey.ac.uk/Teaching/Unix/
Chimera
This tutorial will use UCSF Chimera to manually prepare files for docking and to visualize dock results.
To show Chimera's command line, select Tools -> General Controls -> Command Line in the menu bar.
Files can be opened in Chimera by selecting File -> Open... in the menu bar at the top. Structures can be saved by selecting File -> Save Mol2... in the menu bar to save as a Mol2 file, or by selecting File -> Save PDB... to save as a PDB file. Save a Chimera session by selecting File -> Save Session As... in the menu bar. This will create a Python script that restores open models and any modifications made at the time of saving. To close the current session, select File -> Close Session in the menu bar.
DOCK results should be visualized using Chimera's ViewDock. To open an output Mol2 file with ViewDock, select Tools -> Surface/Binding Analysis -> ViewDock in the menu bar and select the file in the Open Dock Results window that opens. In the Type Selection dialog box, select Dock 4, 5 or 6 for DOCK 6 results. The ViewDock tool allows the poses stored in the Mol2 file to be viewed one at a time instead of all at once. Select Column -> Show in the menu bar of the ViewDock window and select a value to display it in the table.
Here is a Chimera tutorial.
DOCK
Here is the UCSF DOCK website.
Organizing file directories
Make a directory to house the files for this tutorial:
>> mkdir 6OIM_tutorial
Inside the created 6GJ8_tutorial directory, make subdirectories to keep files organized in:
>> mkdir 001_structure 002_surface_spheres 003_gridbox 004_dock 005_enrichment
Dock Prep
Download the 6OIM PDB file from the RCSB Protein Data Bank by going to https://rcsb.org/structure/6OIM and selecting "PDB Format" in the Download Files drop-down menu in the top right. Save the file in the 001_structure directory. Alternatively, while in the 001_structure directory, run the following command in the terminal:
>> wget https://files.rcsb.org/download/6OIM.pdb --no-check-certificate
Prepare the receptor and cofactors
Open 6OIM.pdb in Chimera.
Isolate the receptor and cofactors
Select the ligand by typing select #0: MOV in the command line. The selected atoms should be highlighted in green. Delete the selected atoms by selecting Actions -> Atoms/Bonds -> delete in the menu bar, or by typing delete selected in the command line.
Mutate the cysteine to an alanine
Select residue 12 by typing select #0: 12 in the command line. This will select the cysteine that the ligand was covalently bonded to. Mutate the selected cysteine to an alanine by first selecting Tools -> Structure Editing -> Rotamers in the menu bar. In the "Choose Rotamer Parameters" dialog box that opens, make sure that "ALA" is selected next to "Rotamer type:" and apply. Alternatively, type swapaa ala sel in the command line after selecting the cysteine.
Add hydrogens and charges
Chimera misplaces a hydrogen on GDP when adding hydrogens, the misplaced hydrogen must be corrected before adding charges. Open Dock Prep by first selecting Surface/Binding Analysis -> Dock Prep in the menu bar. Deselect the "Add charges" and "Write Mol2 file" options so that Chimera stops after adding hydrogens and charges are not added. Make sure that the delete solvent, delete alternate side chain locations, and mutate incomplete side chains steps are selected in addition to adding hydrogens so that all the necessary Dock Prep steps are run. In the "Add Hydrogens for Dock Prep" dialog box that opens, select "Unspecified (determined by method)" and click OK. Select the misplaced hydrogen (H3) and delete it. Select the nitrogen that should be protonated (N1) and select Tools -> Structure Editing -> Build Structure in the menu bar. In the "Build Structure" dialog box that opens, change the mode from "Start Structure" to "Modify Structure" at the top. After "Modify Structure" is selected, under "Change selected atoms to...", change the element to nitrogen and change the number of bonds to 3. Under "Residue Name", select "Change the modified residue's name to" and replace UNK with GDP. Apply the changes and close the dialog box. The newly added hydrogen is named incorrectly, select H1 and reopen Build Structure. Switch to "Modify Structure", change the element to hydrogen, set the number of bonds to 1, set the atom name to HN1, and apply.
Now, finish running Dock Prep by adding charges and saving the structure. Select Surface/Binding Analysis -> Dock Prep in the menu bar and select the "Add charges" and "Write Mol2 file" steps before rerunning Dock Prep. The magnesium ion should have a net charge of +2. Save the structure as kras_cof.mol2 and close the session.
Prepare the ligand
Reopen 6OIM.pdb.
Isolate the ligand
Select the ligand by typing select #0: MOV in the command line. Invert the selection by typing select invert in the command line. Delete the selected receptor, cofactors, and water molecules, leaving only the ligand.
Remove the reactive group
AMG 510 is a covalent ligand that binds to the mutated cysteine residue of KRAS G12C. To prepare the ligand for noncovalent docking, we will artificially convert it to a noncovalent ligand by removing the reactive group that forms a covalent bond with the protein. In the case of AMG 510, the acrylamide group forms a bond with the sulfur in the cysteine residue. Select carbon C25 of the ligand and delete it. This removes the alkene of the acrylamide group, leaving behind a methyl in its place.
Run Dock Prep
Run Dock Prep by first selecting Tools -> Surface/Binding Analysis -> Dock Prep in the menu bar and clicking OK in the "Dock Prep" dialog box that opens. Select "Unspecified (determined by method)" and click OK in the "Add Hydrogens for Dock Prep" dialog box that opens to add hydrogens. Leave the default selections and click OK in the "Assign Charges for Dock Prep" and the "Specify Net Charges" dialog boxes to assign partial charges. The ligand should have a net charge of 0.
Save as amg510.mol2 and close the session.
Surface spheres
Generating DMS
Open kras_cof.mol2.
Run surface calculation by selecting Actions -> Surface -> Show in the menu bar or by typing surf #0 in the command line.
Save the surface as a DMS file by selecting Tools -> Structure Editing -> Write DMS in the menu bar. Save as kras_cof.dms.
Creating surface spheres
Change directories to the 002_surface_spheres directory:
>> cd ../002_surface_spheres
Create a file called INSPH by running the command and format the file as follows:
../001_structure/kras_cof.dms
R
X
0.0
4.0
1.4
6OIM.sph
This file will be the input file for the sphere generation program, sphgen. The first line of the file specifies the input DMS file. In the second line, R indicates that spheres should be generated outside the surface. In the third line, X indicates that all surface points should be used. The fourth line specifies the acceptable steric clash in angstroms, which in this case is set to 0.0 or no steric clash allowed. The fifth and sixth lines specify the maximum and minimum sphere radii in angstroms, respectively. The last line specifies the output sphere file name.
Run sphgen to generate spheres based on the parameters given in the INSPH file:
>> ${dock6_path}/bin/sphgen -i INSPH -o OUTSPH
- ${dock6_path} is a variable (placeholder) for the DOCK 6 installation path (e.g. /home/baliuste/zzz.github/dock6)
The surface spheres generated by sphgen can be visualized in Chimera. In a new Chimera session open 7RPZ.pdb and show the surface. In the same session, open the output sphere file (6OIM.sph) created by sphgen to visualize the surface spheres.
Selecting relevant spheres
To select spheres based on the distance from the ligand, run the command:
>> ${dock6_path}/bin/sphere_selector 6OIM.sph ../001_structure/amg510.mol2 3.0
The first two arguments passed to sphere_selector specify the surface sphere and ligand files, respectively. The last argument, 3.0, specifies that only spheres within 3.0 angstroms of the ligand should be selected.
The selected_spheres.sph file generated by sphere_selector can be visualized in Chimera.
Generating box/grid
Change directories to the 003_gridbox directory.
Box generation
Create an input file for box generation called showbox.in and format the file as follows:
Y
8.0
../002_surface_spheres/selected_spheres.sph
1
6OIM.box.pdb
In the first line of the file, Y (Yes) indicates that showbox should create a box. The second line specifies the distance, in angstroms, between the spheres and the box borders. The third line specifies the input sphere file. The fourth line indicates the cluster number. The last line specifies the output box file name.
Generate a box based on the parameters given in showbox.in by running showbox as follows:
>> ${dock6_path}/bin/showbox < showbox.in
The 6OIM.box.pdb file generated by showbox can be visualized in Chimera.
Grid generation
Create the input file for grid generation called grid.in:
compute_grids yes
grid_spacing 0.4
output_molecule no
contact_score no
energy_score yes
energy_cutoff_distance 9999
atom_model a
attractive_exponent 6
repulsive_exponent 9
distance_dielectric yes
dielectric_factor 4
allow_non_integral_charges no
bump_filter yes
bump_overlap 0.75
receptor_file ../001_structure/kras_cof.mol2
box_file 6OIM.box.pdb
vdw_definition_file ${dock6_path}/parameters/vdw_AMBER_parm99.defn
score_grid_prefix grid
Generate grids by running:
>> ${dock6_path}/bin/grid -i grid.in -o grid.out
Docking and auxiliary calculations
Change directories to the 004_dock directory.
Single point calculation
This calculation performs an energy evaluation on the crystallographic ligand conformation without any movement.
Create the input file for the single point calculation called single_point.in:
conformer_search_type rigid
use_internal_energy yes
internal_energy_rep_exp 12
internal_energy_cutoff 100.0
ligand_atom_file ../001_structure/amg510.mol2
limit_max_ligands no
skip_molecule no
read_mol_solvation no
calculate_rmsd yes
use_rmsd_reference_mol yes
rmsd_reference_filename ../001_structure/amg510.mol2
use_database_filter no
orient_ligand no
bump_filter no
score_molecules yes
contact_score_primary no
contact_score_secondary no
grid_score_primary yes
grid_score_secondary no
grid_score_rep_rad_scale 1
grid_score_vdw_scale 1
grid_score_es_scale 1
grid_score_grid_prefix ../003_gridbox/grid
gist_score_secondary no
multigrid_score_secondary no
dock3.5_score_secondary no
continuous_score_secondary no
footprint_similarity_score_secondary no
pharmacophore_score_secondary no
hbond_score_secondary no
descriptor_score_secondary no
gbsa_zou_score_secondary no
gbsa_hawkins_score_secondary no
SASA_score_secondary no
amber_score_secondary no
minimize_ligand no
atom_model all
vdw_defn_file ${dock6_path}/parameters/vdw_AMBER_parm99.defn
flex_defn_file ${dock6_path}/parameters/flex.defn
flex_drive_file ${dock6_path}/parameters/flex_drive.tbl
ligand_outfile_prefix single_point.out
write_orientations no
num_scored_conformers 1
rank_ligands no
To run the single point calculation:
>> ${dock6_path}/bin/dock6 -i single_point.in -o single_point.out
Energy minimization
This calculation performs a minimization run on the crystallographic ligand conformation, allowing the ligand to subtly adjust its conformation (internal degrees of freedom) and orientation (3 translational and 3 orientational degrees of freedom).
Create the input file for energy minimization called min.in:
conformer_search_type rigid
use_internal_energy yes
internal_energy_rep_exp 12
internal_energy_cutoff 100.0
ligand_atom_file ../001_structure/amg510.mol2
limit_max_ligands no
skip_molecule no
read_mol_solvation no
calculate_rmsd yes
use_rmsd_reference_mol yes
rmsd_reference_filename ../001_structure/amg510.mol2
use_database_filter no
orient_ligand no
bump_filter no
score_molecules yes
contact_score_primary no
contact_score_secondary no
grid_score_primary yes
grid_score_secondary no
grid_score_rep_rad_scale 1
grid_score_vdw_scale 1
grid_score_es_scale 1
grid_score_grid_prefix ../003_gridbox/grid
gist_score_secondary no
multigrid_score_secondary no
dock3.5_score_secondary no
continuous_score_secondary no
footprint_similarity_score_secondary no
pharmacophore_score_secondary no
hbond_score_secondary no
descriptor_score_secondary no
gbsa_zou_score_secondary no
gbsa_hawkins_score_secondary no
SASA_score_secondary no
amber_score_secondary no
minimize_ligand yes
simplex_max_iterations 1000
simplex_tors_premin_iterations 0
simplex_max_cycles 1
simplex_score_converge 0.1
simplex_cycle_converge 1.0
simplex_trans_step 1.0
simplex_rot_step 0.1
simplex_tors_step 10.0
simplex_random_seed 0
simplex_restraint_min yes
simplex_coefficient_restraint 10.0
atom_model all
vdw_defn_file ${dock6_path}/parameters/vdw_AMBER_parm99.defn
flex_defn_file ${dock6_path}/parameters/flex.defn
flex_drive_file ${dock6_path}/parameters/flex_drive.tbl
ligand_outfile_prefix 6OIM.ligand.min
write_orientations no
num_scored_conformers 1
rank_ligands no
Run energy minimization:
>> ${dock6_path}/bin/dock6 -i min.in -o min.out
To view the RMSD of the minimized ligand conformation, head the generated MOL2 file:
>> head 6OIM.ligand.min_scored.mol2
The minimized ligand conformation can be visualized in Chimera by opening the output MOL2 file with ViewDock.
[ image ]
Rigid docking
To test orienting without considering ligand flexibility, we perform rigid docking. We use the crystallographic ligand conformation for this test. This is primarily used for testing purposes only, not for screening.
Create the input file for rigid docking called rigid.in:
conformer_search_type rigid
use_internal_energy yes
internal_energy_rep_exp 12
internal_energy_cutoff 100
ligand_atom_file 6OIM.ligand.min_scored.mol2
limit_max_ligands no
skip_molecule no
read_mol_solvation no
calculate_rmsd yes
use_rmsd_reference_mol yes
rmsd_reference_filename 6OIM.ligand.min_scored.mol2
use_database_filter no
orient_ligand yes
automated_matching yes
receptor_site_file ../002_surface_spheres/selected_spheres.sph
max_orientations 2000
critical_points no
chemical_matching no
use_ligand_spheres no
bump_filter no
score_molecules yes
contact_score_primary no
contact_score_secondary no
grid_score_primary yes
grid_score_secondary no
grid_score_rep_rad_scale 1
grid_score_vdw_scale 1
grid_score_es_scale 1
grid_score_grid_prefix ../003_gridbox/grid
gist_score_secondary no
multigrid_score_secondary no
dock3.5_score_secondary no
continuous_score_secondary no
footprint_similarity_score_secondary no
pharmacophore_score_secondary no
hbond_score_secondary no
descriptor_score_secondary no
gbsa_zou_score_secondary no
gbsa_hawkins_score_secondary no
SASA_score_secondary no
amber_score_secondary no
minimize_ligand yes
simplex_max_iterations 1000
simplex_tors_premin_iterations 0
simplex_max_cycles 1
simplex_score_converge 0.1
simplex_cycle_converge 1.0
simplex_trans_step 1.0
simplex_rot_step 0.1
simplex_tors_step 10.0
simplex_random_seed 0
simplex_restraint_min no
atom_model all
vdw_defn_file ${dock6_path}/parameters/vdw_AMBER_parm99.defn
flex_defn_file ${dock6_path}/parameters/flex.defn
flex_drive_file ${dock6_path}/parameters/flex_drive.tbl
ligand_outfile_prefix rigid.out
write_orientations no
num_scored_conformers 1
rank_ligands no
Run rigid docking:
>> ${dock6_path}/bin/dock6 -i rigid.in -o rigid.out
To view the RMSD of the docked ligand conformation, head the generated MOL2 file:
>> head rigid.out_scored.mol2
Fixed anchor docking
To test the ligand flexibility algorithm without considering orienting, we perform fixed anchor docking. We use the crystallographic ligand placement for this test. This is primarily used for testing purposes only, not for screening, as a ligand in a database will be centered elsewhere, not in the site.
Create the input file for fixed anchor docking called fixed.in:
conformer_search_type flex
write_fragment_libraries no
user_specified_anchor no
limit_max_anchors no
min_anchor_size 5
pruning_use_clustering yes
pruning_max_orients 10000
pruning_clustering_cutoff 100
pruning_conformer_score_cutoff 100.0
pruning_conformer_score_scaling_factor 1.0
use_clash_overlap no
write_growth_tree no
use_internal_energy yes
internal_energy_rep_exp 12
internal_energy_cutoff 100000.0
ligand_atom_file ../001_structure/amg510.mol2
limit_max_ligands no
skip_molecule no
read_mol_solvation no
calculate_rmsd yes
use_rmsd_reference_mol yes
rmsd_reference_filename ../001_structure/amg510.mol2
use_database_filter no
orient_ligand no
bump_filter no
score_molecules yes
contact_score_primary no
contact_score_secondary no
grid_score_primary yes
grid_score_secondary no
grid_score_rep_rad_scale 1
grid_score_vdw_scale 1
grid_score_es_scale 1
grid_score_grid_prefix ../003_gridbox/grid
gist_score_secondary no
multigrid_score_secondary no
dock3.5_score_secondary no
continuous_score_secondary no
footprint_similarity_score_secondary no
pharmacophore_score_secondary no
hbond_score_secondary no
descriptor_score_secondary no
gbsa_zou_score_secondary no
gbsa_hawkins_score_secondary no
SASA_score_secondary no
amber_score_secondary no
minimize_ligand yes
minimize_anchor yes
minimize_flexible_growth yes
use_advanced_simplex_parameters no
simplex_max_cycles 1
simplex_score_converge 0.1
simplex_cycle_converge 1
simplex_trans_step 1
simplex_rot_step 0.1
simplex_tors_step 10
simplex_anchor_max_iterations 500
simplex_grow_max_iterations 500
simplex_grow_tors_premin_iterations 0
simplex_random_seed 0
simplex_restraint_min no
atom_model all
vdw_defn_file ${dock6_path}/parameters/vdw_AMBER_parm99.defn
flex_defn_file ${dock6_path}/parameters/flex.defn
flex_drive_file ${dock6_path}/parameters/flex_drive.tbl
ligand_outfile_prefix 6OIM_fad
write_orientations no
num_scored_conformers 100
write_conformations no
cluster_conformations yes
cluster_rmsd_threshold 2.0
rank_ligands no
Run fixed anchor docking:
>> ${dock6_path}/bin/dock6 -i fixed.in -o fixed.out
To view the RMSD of the top scoring docked ligand conformation, head the generated MOL2 file:
>> head 6OIM_fad_scored.mol2
Flexible docking
This test combines orienting and ligand flexibility, and is the most applicable to production runs of dock.
Create the input file for flexible docking called flex.in:
conformer_search_type flex
write_fragment_libraries no
user_specified_anchor no
limit_max_anchors no
min_anchor_size 5
pruning_use_clustering yes
pruning_max_orients 5000
pruning_clustering_cutoff 2500
pruning_conformer_score_cutoff 100.0
pruning_conformer_score_scaling_factor 1.0
use_clash_overlap no
write_growth_tree no
use_internal_energy yes
internal_energy_rep_exp 12
internal_energy_cutoff 100000.0
ligand_atom_file 6OIM.ligand.min_scored.mol2
limit_max_ligands no
skip_molecule no
read_mol_solvation no
calculate_rmsd yes
use_rmsd_reference_mol yes
rmsd_reference_filename 6OIM.ligand.min_scored.mol2
use_database_filter no
orient_ligand yes
automated_matching yes
receptor_site_file ../002_surface_spheres/selected_spheres.sph
max_orientations 1000
critical_points no
chemical_matching no
use_ligand_spheres no
bump_filter no
score_molecules yes
contact_score_primary no
contact_score_secondary no
grid_score_primary yes
grid_score_secondary no
grid_score_rep_rad_scale 1
grid_score_vdw_scale 1
grid_score_es_scale 1
grid_score_grid_prefix ../003_gridbox/grid
gist_score_secondary no
multigrid_score_secondary no
dock3.5_score_secondary no
continuous_score_secondary no
footprint_similarity_score_secondary no
pharmacophore_score_secondary no
hbond_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 ${dock6_path}/parameters/vdw_AMBER_parm99.defn
flex_defn_file ${dock6_path}/parameters/flex.defn
flex_drive_file ${dock6_path}/parameters/flex_drive.tbl
ligand_outfile_prefix flex.out
write_orientations no
num_scored_conformers 25
write_conformations no
cluster_conformations yes
cluster_rmsd_threshold 2
rank_ligands no
Run flexible docking:
>> ${dock6_path}/bin/dock6 -i flex.in -o flex.out
To view the RMSD of the top scoring docked ligand conformation, head the generated MOL2 file:
>> head flex.out_scored.mol2