Revision as of 15:32, 7 March 2022

Introduction

DOCK

System Preparation

Fetching 1X70

Open Chimera and do the following to grab the protein:

 File > Fetch By ID > 1X70

Biological assembly 1 of 1X70.

The first thing to notice is that this is a dimer and the ligand, 715, is not bound at the dimer interface. Thus, one of the monomers is entirely redundant and should be deleted.

 Select > Chain > A

 Actions > Atoms > Delete

Next it is important to remove cofactors, ions, and water molecules not involved in the binding interactions. This can be checked by reading through the paper associated with the PDB.

 Select > Residue > NAG > Actions > Atoms > Delete

 Select > Residue > NDG > Actions > Atoms > Delete

 Select > Residue > HOH > Actions > Atoms > Delete

This will leave us with just the ligand and receptor.

1 monomer of 1X70 with all waters and non standard residues deleted except for the ligand 715, which is colored light blue for clarity.

Receptor Prep

Receptor 1X70 without Hydrogens

Now that we the receptor by itself, we have to clean up the rest of the receptor by adding any missing side chains, dealing with multiple occupancies and mutated residues, and protonating and calculating partial charges.

For 1X70, there are several residues with multiple occupancies.

One way to check for these residues is to grep the number of alpha carbons from the pdb in the command line.

 In the terminal, type: grep -e CA 1X70_noH.pdb

Two potential occupancies shown for CYS649

To clean this up and protonate/charge the receptor:

 Tools > Structure Editing > Dock Prep

Using dock prep to fill in missing atoms/sidechains, add Hydrogens and charges.

Make sure that the protonation makes sense for residues in the active site or coordinated with metals (none here), especially histidines, by checking the paper and chimera for any nitrogens coordinating with metals are not protonated or residues in the active site with differing protonation states. Also make sure that all residues have integral charge (i.e. the charge is an integer). Chimera should give a warning after the dockprep if any residue charges are non-integral.

Ligand Prep

First you have to save the ligand as a separate file. You can do this in Chimera by deleting all of the protein and saving that as a separate file: "715_noH.pdb".

 Select > Residues > 715 > Invert Selection

 Actions > Atoms > delete

 File > save PDB 

Now you should just have the ligand.

715 without Hydrogens added

Next Hydrogens and partial atomic charges need to be added and saved as "715_H.mol2". It will ask for the ligands overall charge, which you should verify using chemical knowledge.

 tools > structure editing > addH 

 tools > structure editing > addCharge > Select the correct charge for your ligand, Use AM1-BCC. 

 File > save Mol2

715 with Hydrogens

Surface Generation & Spheres

This section details the generation of sphere files which will be used to describe where you are trying to DOCK to on your protein.

Surface Generation

 Load 1X70 w/o Hydrogens in Chimera

 actions > show > surface

The VDW surface for 1X70 that will be used to generate the sphere files.

Then you will write a DMS (Molecular Surface) File With the surface generated in Chimera:

 Tools > Structure Editing > Write DMS

Save this as 1X70_dms.dms, and now you should have a DMS file for the next step.

Sphere Generation To generate spheres make the following input file: "INSPH" -

 1X70_dms.dms      #Molecular Surface File 
 R                 #Whether to generate spheres outside of surface (R) or inside (L) 
 X                 #Surface points from the DMS file to use in sphere generation 
 0                 #Minimum radius between spheres
 4.0               #Maximum radius of sphere
 1.4               #Minimum radius of sphere
 1X70_wo_H.sph     #Output sphere file

For more information on sphere generation see: https://dock.compbio.ucsf.edu/DOCK_6/tutorials/sphere_generation/generating_spheres.htm

The .sph file should give you something similar to the following image if you load it up over your protein in Chimera:

The spheres representing the empty space within the protein.

Sphere Selection

Making The Infamous Grid

Making the box

In order to make the grid we first have to determine how big our grid will be. To do this:

 cd 03.grid
 showbox 
   #automatically construct box to enclose spheres [Y/N]
   Y
   #extra margin to also be enclosed (angstroms)?
    #(this will be added in all 6 directions)
   8.0
   #sphere file-
   ./../02.spheres/selected_spheres.sph
   #cluster number-
   1
   #output filename?
   1X70.box.pdb

You can also copy these inputs into "showbox.in" (none of the commented lines) and then type "showbox < showbox.in"

Making the grid

Making the following input file "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.
 bump_filter                               yes
 bump_overlap                              0.75
 receptor_file                             ../01.structures/1X70_rec.mol2
 box_file                                  ./1X70.box.pdb
 vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 score_grid_prefix                         grid

Now generate the grid. It should take several minutes.

 grid -i grid.in -o gridinfo.out

Once it is done, vi into gridinfo.out and make sure the charges are all integer and that the calculation finished. If they are not, that likely means there is something wrong with your 1X70_H.mol2, and you may want to go back to the dockprep step and remake this file.

You should get two files:

 grid.nrg (energy grid)
 grid.bmp (bump grid)

Try loading them into chimera over your protein.

The energy grid for 1X70.

The bump grid for 1X70.

Energy Minimization for the ligand

Docking & Virtual Screening

Now that the ligand and receptor files are set up, we can begin docking the ligand back into the receptor. This makes sure that files are set up correctly, since we should expect the docking results to closely match the crystal structure from the PDB.

Rigid Docking

The first docking method we will be using is rigid docking. With this method, the ligand is kept in a fixed position while the algorithm tries to move/rotate the entire molecule to reduce energy. This is the least computationally intensive method for docking. We can start by creating the input file with the command

 vim rigid.in

And using the following parameters.

 conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100
 ligand_atom_file                                             1x70.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                                      1x70.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
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 simplex_max_iterations                                       1000
 simplex_tors_premin_iterations                               0
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.9_release/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        rigid.out
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Fixed Anchor Docking

Flexible Docking

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