View source for 2024 DOCK tutorial 3 with PDBID 1Y0X

= Introduction =
Proteins are mechano-chemical machines that governs many functions in cells and can caused serious diseases to human when malfunction. One active branch of protein research involves the design of small molecules that can bind to proteins at designated positions and inhibit/alter/modulate the function of the protein in a desirable manner. DOCK is a wonderful tool to be utilized to perform virtual screening ( 


#Setting up 
#Preparing the protein and ligand
#Finding the binding site of the protein
#Generating the grid
#Ligand energy minimization
#Perform 3 common types of molecular docking


== Learning Objectives ==
By following this tutorial, you will be able to successfully reproduce the docking result of the demonstrated case of 1Y0X and understand the fundamentals of virtual screening using DOCK6.10. Applying the same method illustrated in this tutorial for other protein system will likely yield meaningful results but there might be some slight fine tuning from case to case.

= Preparation of the ligand and protein =
#Evaluate the structure to determine if there are any missing loops
#Prepare the protein structure
#Prepare the ligand structure

=== Evaluating the Structure===

#Select an atom at near the start of the missing section (hold the ctrl button while clicking it)
#Select another atom near the binding site (hold ctrl + shift while clicking the second atom)
#Go to Tools → Structure Analysis → Distances


===Preparing the Protein file===
#Select an atom on the protein
#Press the up arrow until the entire protein is selected
#Go to Select → Invert (all models).  This will change the selection from the protein to everything else in the structure
#Go to Actions → Atoms/Bonds → Delete
#Save the structure with a new file name (i.e. 4s0v_protein_only.pdb).  Your pdb file will now look similar to this:
#Adding hydrogens
#Adding charge
#Click on one atom anywhere on the protein
#Click on Select → Zone.  This will cause the following dialogue box to appear:
===Preparing the Ligand File===
#Select an atom on the ligand
#Press the up arrow until the entire ligand is selected (you may have to press the up arrow many times)
#Go to Select → Invert (all models).  This will change the selection from the ligand to everything else in the structure
#Go to Actions → Atom/Bonds → Delete
#Save the structure with a new file name (i.e. 4s0v_ligand_only.pdb).  The image will look similar to this:

#Add hydrogens
#Add charges


===Final Steps===

=Creating the Protein Binding Site Surface=

===Creating the Required Surface (DMS) File===


===Generating Spheres for the Binding Site===

===Binding Site Spheres===

#scp selected_spheres.sph to your local computer
#Close any open sessions you have in Chimera
#In Chimera open selected_spheres.sph
#In the current session, open the original protein/ligand complex (4s0v.pdb)
#You should see the spheres located within the binding site of the protein, similar to:

#Hold down ctrl and click on a sphere
#Press the up arrow until all spheres are selected
#Actions → Atoms/Bonds → hide
#Verify the ligand is where the spheres were


=Box and Grid Generation=
The next step in the docking process is to generate energy interactions between the atoms of the protein and ligand.  If this was done for the whole complex it would take too long to run to be useful.  To get around this computationally expensive step, dock uses a box/grid method.  We will define a box around the area of interest for the protein/ligand and DOCK will generate a grid within this box which will be used in the energy calculations.
===Generating the Box===
To generate the box we will be working again on the command line using a DOCK program called showbox.  Start by logging into Seawulf and navigating to your 003.gridbox directory.  We need to make a new file called showbox.in by typing:
    vi showbox.in

This will create a new file, with a filename of showbox.in and open it in vi.  The following commands need to be typed:
      Y
      8.0 
      ../002.surface_spheres/selected_spheres.sph
      1 
      1y0x.box.pdb

Remember to change the last line to be a filename with the number of protein you are working with.  The second line of this code (8.0) is telling dock how many angstroms from the selected spheres to draw the box.  Depending on your system you may need to modify this number.

To run this file, simply type:
     showbox < showbox.in

If showbox was successful the file 1y0x.box.pdb will now be in your directory.

===Generating the Grid===
Now that we have our box defined we need to instruct DOCK to generate the grid within it.  We do this using a DOCK program called grid:
   vi grid.in

This command will generate and open a file named grid.in.  The commands to be typed into this file are:
   allow_non_integral_charges                no
   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                              ../001.structure/protein_final.mol2
   box_file                                  1y0x.box.pdb
   vdw_definition_file                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
   score_grid_prefix                         grid


The only change you need to make to the above commands is the receptor_file and box_file to reflect the files you previously generated. 

Once this file is saved, run it:
    grid -i grid.in -o 1y0xGridInfo.out

Be patient, this step might take a few minutes to run.  You will know it's worked successfully if you see:
#grid.bmp
#grid.nrg
#1y0xGridInfo.out

in your directory.  With the box and grid successfully generated we are ready to move onto the energy minimization step.

=Energy Minimization=
At its core, DOCK is finding interactions between a protein and ligand by looking at energy interactions between atoms.  In order for DOCK to give the most accurate results we need to ensure that the ligand is at its lowest energy state before docking it into the binding site of the protein.

===Ligand Minimization===
For this section we will be working in the 004.energy_min directory and will be using the dock6 command.  Again we need to generate the input file that dock6 needs:
   vim min.in

Once in vim the following lines need to be typed in:
  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../001.structure/ligand_final.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/protein_final.mol2
  use_database_filter                                          no
  orient_ligand                                                no
  bump_filter                                                  no
  score_molecules                                              yes
  contact_score_primary                                        no
  grid_score_primary                                           yes
  grid_score_rep_rad_scale                                     1
  grid_score_vdw_scale                                         1
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                       ../003.gridbox/grid
  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                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        1y0x.lig.min
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

And the file is run with the following command:
  dock6 -i min.in -o min.out

After successful completion of the program two new files will be in your directory:
*min.out
*1y0x.lig.min.mol2

scp the .mol2 file back to your local computer.  To view the changes that dock made to the structure, and open the 1y0x.lig.min.mol2 file in Chimera and in the same session, open the ligand_final.pdb file. Remember the ligand_final.pdb is the file we saved after making the protonation changes to the ligand. You should see something similar to:

[[File:En min tint originial cyan.png|thumb|center]]

The 1y0x.ligand from PDB with hydrogen and charges is cyan and the newly generated and energy minimized 1y0x.lig.min.mol2 is tint

=== Footprint Analysis===
At its core, DOCK can be thought of as a program which evaluates, minimizes, and uses the electrostatic and VDW interactions between the ligand and protein to determine how they bind to each other.  A footprint analysis is a way to visualize these interactions and possibly be used to design new ligands with the same, or better, set of energetics.  The steps in this section will be done on Seawulf in the 005.footprint directory.  We will be using the dock6 command and again need to create an input file:
  vi footprint.in

The following lines needed to be typed into the newly created file:
  conformer_search_type                                        rigid
  use_internal_energy                                          no
  ligand_atom_file                                             ../004.energy_min/1y0x.lig.min_scored.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
  grid_score_primary                                           no
  multigrid_score_primary                                      no
  dock3.5_score_primary                                        no
  continuous_score_primary                                     no
  footprint_similarity_score_primary                           yes
  fps_score_use_footprint_reference_mol2                       yes
  fps_score_footprint_reference_mol2_filename                  ../001.structure/ligand_final.mol2
  fps_score_foot_compare_type                                  Euclidean
  fps_score_normalize_foot                                     no
  fps_score_foot_comp_all_residue                              yes
  fps_score_receptor_filename                                  ../001.structure/protein_final.mol2
  fps_score_vdw_att_exp                                        6
  fps_score_vdw_rep_exp                                        9
  fps_score_vdw_rep_rad_scale                                  1
  fps_score_use_distance_dependent_dielectric                  yes
  fps_score_dielectric                                         4.0
  fps_score_vdw_fp_scale                                       1
  fps_score_es_fp_scale                                        1
  fps_score_hb_fp_scale                                        0
  minimize_ligand                                              no
  atom_model                                                   all
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        1y0x_footprint.out
  write_footprints                                             yes
  write_hbonds                                                 yes
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

to generate the footprint of the ligand/protein interaction type:
  dock6 -i footprint.in


Again, you will know the program successfully ran if the following three files are now in your directory:
*1y0x_footprint.out_footprint_scored.txt
*1y0x_footprint.out_hbond_scored.txt
*1y0x_footprint.out_scored.mol2

In order to view/analyze these results we need to use a python script that is located in the class directory.  Copy it over to your 005.footprint directory with the following command:
   cp /gpfs/projects/AMS536/zzz.programs/plot_footprint_single_magnitude.py .

To run this script, first we load the python using:
  module load py/2.7.15
Then, type:
  python plot_footprint_single_magnitude.py 1y0x_footprint.out_footprint_scored.txt  50


Once the script is completed you will see a .pdf file in your directory.  scp this file back to your local computer.  The file contains two plots that shows the energetic signature for the 50 most significant residues and will look similar to:
[[File:1y0x footprint.png|thumb|center]]

=DOCK=
We will be exploring three different options of the DOCK program:
#Rigid Docking
#Fixed Anchor Docking
#Flexible Docking

Each option has it's own pros and cons and your individual system should dictate which option you choose.  Note:  In the input file for each of these options has a line:
  num_scored_conformers                                        1

Changing this number will change the number of conformations saved from the docking session.
And for each options, to generate input file, the recommended way is to create a file first by typing:
  touch example.in
then to make the file executable, type:
  chmod+x example.in
then fill the input file using:
  dock6 -i example.in
This will ask a few questions regarding the type of docking and other parameters and populate the input file accordingly. The input files for three different docking can be generated by answering those questions. In the next sections, the put files are pasted which was created from answering those questions.

 
===Rigid Docking===
When using rigid docking, the DOCK program does not sample different conformations of the ligand to try and find the most energetically stable.  It simply treats the ligand as a rigid structure and tries to fit it into the binding site of the protein.  For this step we will be using the energy minimized ligand file we generated above. This section will again be completed on Seawulf, please move to the 006.rigid_docking directory.

Again we need an input file that can be created using the command:
    vi rigid.in

The following lines need to be typed into the file:
  conformer_search_type                                        rigid
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../004.energy_min/1y0x.lig.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                                      ../004.energy_min/1y0x.lig.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
  grid_score_primary                                           yes
  grid_score_rep_rad_scale                                     1
  grid_score_vdw_scale                                         1
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                       ../003.gridbox/grid
  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/dock6.10/parameters/vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        rigid.out
  write_orientations                                           no
  num_scored_conformers                                        10
  write_conformations                                          yes
  cluster_conformations                                        yes
  cluster_rmsd_threshold                                       2.0
  rank_ligands                                                 no


Remember to again change the necessary lines to point to your specific files.  Once this is completed run the program by typing:
  dock6 -i rigid.in -o rigid.out

Again, be patient as this may take a few minutes to complete. Once it's done running you will see two new files in your directory:
*rigid.out_scored.mol2 
*rigid.out

scp the .mol2 file over to your local computer.  Start a new session in Chimera and open the energy minimized ligand file from the previous step and the rigid docked ligand you just created (1y0x.lig.min_scored.mol2 and rigid.out_scored.mol2) to view the differences between the energy minimized ligand and DOCK's attempt to rigid dock that structure into the protein.
[[File:1y0x rigid.png|thumb|center]]
The 1y0x.lig.min_scored.mol2 is tint and the newly generated fixed.out_scored.mol2 is cyan.

Looking at this file we see that the two are very similar to each other.

Now we can look at the other file that dock generated, rigid.out.  If we scroll to the bottom you will see grid scoring for this run:
[[File:1y0x rigid grid score.png|thumb|center]]

===Fixed Anchor Docking===
The next DOCK option we will be looking at is called 'Fixed Anchor Docking'.  For this option, DOCK cuts the ligand into fragments along its rotatable bonds.  It then chooses the largest fragment as the 'anchor' and orients this fragment into the binding site of the protein.  Once this is completed, the next fragments are added to the ligand, orientating them such that their energy is minimized but keeping them as rigid bodies.  Comparing this to Rigid Docking, we see that more conformational sampling is being done but still within limits since each fragment is considered rigid.

We'll be working on the command line again, please move to the 007.fixed_anchor_docking directory and create an input file called fixed.in:
  vim fixed.in

and type the following lines:
  conformer_search_type                                        flex
  write_fragment_libraries                                     no
  user_specified_anchor                                        no
  limit_max_anchors                                            no
  min_anchor_size                                              5
  pruning_use_clustering                                       yes
  pruning_max_orients                                          1000
  pruning_clustering_cutoff                                    100
  pruning_conformer_score_cutoff                               100.0
  pruning_conformer_score_scaling_factor                       1.0
  use_clash_overlap                                            no
  write_growth_tree                                            no
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  ligand_atom_file                                             ../004.energy_min/1y0x.lig.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                                      ../004.energy_min/1y0x.lig.min_scored.mol2
  use_database_filter                                          no
  orient_ligand                                                no
  bump_filter                                                  no
  score_molecules                                              yes
  contact_score_primary                                        no
  grid_score_primary                                           yes
  grid_score_rep_rad_scale                                     1
  grid_score_vdw_scale                                         1
  grid_score_es_scale                                          1
  grid_score_grid_prefix                                       ../003.gridbox/grid
  minimize_ligand                                              yes
  minimize_anchor                                              yes
  minimize_flexible_growth                                     yes
  use_advanced_simplex_parameters                              no
  minimize_flexible_growth_ramp                                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                                            10.0
  simplex_anchor_max_iterations                                500
  simplex_grow_max_iterations                                  500
  simplex_grow_tors_premin_iterations                          0
  simplex_random_seed                                          0
  simplex_restraint_min                                        no
  atom_model                                                   all
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/  vdw_AMBER_parm99.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
  ligand_outfile_prefix                                        1y0x_fixed_output
  write_orientations                                           no
  num_scored_conformers                                        1
  rank_ligands                                                 no

Again, change the necessary lines to point to your specific files.  To run this file type:
   dock6 -i fixed.in -o fixed.out

Once it's done running you will see two new files in your directory:
*fixed.out
*1y0x_fixed_output_scored.mol2
scp the .mol2 file over to your local computer. Start a new session in Chimera and open the energy minimized ligand file from the previous step and the fixed anchor docked ligand you just created (1y0x.lig.min_scored.mol2 and 1y0x_fixed_output_scored.mol2) to view the differences between the energy minimized ligand and DOCK's attempt to place that structure using its fixed anchor algorithm.  Your image should be similar to:
[[File:1y0x fixed anchor.png|thumb|center]]

The 1y0x.lig.min_scored.mol2 is cyan and the newly generated 1y0x_fixed_output_scored.mol2 is tint.

Now we can look at the other file that dock generated, fixed.out.  If we scroll to the bottom you will see grid scoring for this run:
 Anchors:               2
 Orientations:          2
 Conformations:         111

                          Grid_Score:          -79.230011
                     Grid_vdw_energy:          -73.074615
                      Grid_es_energy:           -6.155398
           Internal_energy_repulsive:            6.240775



===Flexible Docking===

= Virtual Screening of a Ligand Library =
=Cartesian Minimization of Virtually Screened Small Molecules=
=Rescoring and Ranking Virtually Screened Molecules=