Difference between revisions of "2014 DOCK tutorial with HIV Protease"

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(→‎III. Generating Receptor Surface and Spheres)
(→‎VI. Virtual Screening)
 
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===HIV Protease===
 
===HIV Protease===
  
−
Joe
+
HIV protease (HIVPR) is a protein involved in viral maturation during the life cycle of HIV. HIVPR is an approximately 22 kDa homodimer with 99 residues per chain. Inhibition of this protein has been shown to be an effective form of treatment of HIV. Currently-available HIVPR inhibitors generally take the form of a symmetric cyclic urea compound. For more information, see [http://www.ncbi.nlm.nih.gov/pubmed/8278812?dopt=Abstract Lam et al.]
  
 
===Organizing Directories===
 
===Organizing Directories===
Line 33: Line 33:
 
==II. Preparing the Receptor and Ligand==
 
==II. Preparing the Receptor and Ligand==
  
−
Tianao
+
 
 +
'''Downloading the PDB Structure (1HVR)'''
 +
 
 +
Go to PDB (Protein Data Bank) website (http://www.rcsb.org/pdb/home/home.do) enter the protein ID (1HVR), search for the PDB file and download it as a text form.
 +
 
 +
'''Preparing the ligand and receptor in Chimera'''
 +
 
 +
Put the 1HVR PDB file in 00.file/folder. If you are in the 00.files/directory, then tap the command:
 +
  cp ~/Downloads/1HVR.pdb ./
 +
When you are preparing your PDB files, you have to make some modifications on your original file. Make a copy of your original PDB file, and rename it as "1HVR.modified.pdb" in the 00.files/. Here we changed the atom name from "CSO" to "CYS" and deleted two lines "OD" and "HD". Here is an example to change all "CSO" to "CYS":
 +
  :%s/CSO/CYS/gc
 +
And then we will create 4 files in 01.dockprep/ directory:
 +
  1HVR.dockprep.mol2 
 +
  1HVR.ligand.mol2 
 +
  1HVR.receptor.mol2 
 +
  1HVR.receptor.noH.pdb
 +
''Create the dockprep file''
 +
 
 +
For the "1HVR.dockprep.mol2" file: open the 1HVR.modified.pdb in Chimera; delete the water molecules ; delete the original hydrogen atoms; add the charge by go to Tools->structure editing->add charge ''(Note when adding the charge to the ligand, you can choose AMBER ff99SB as the charge model and chose gasteiger as the charge method. In this 1HVR case, we set Net Charge to 0. You may have to consider the chemistry of the ligand when assigning a charge state)''. Add the hydrogen atoms manually by Tools->structure editing->Add H . Or you can do all of the above by clicking Tools -> Structure Editing -> Dock Prep.
 +
 
 +
Finally save the file as a mol2 format in Chimera.
 +
 
 +
''Create the ligand file''
 +
 
 +
To create the ligand file: Open 1HVR.dockprep.mol2 in Chimera and select the ligand chain "XK2", then Select->invert, then go to Action->Atoms/bonds->Delete
 +
 
 +
''Create the receptor file''
 +
 
 +
Select the ligand residue "XK2" and delete it and save it as a mol2 file.
 +
 
 +
''Create the receptor without hydrogen atoms''
 +
 
 +
Open the "1HVR.receptor.mol2" file and delete all of the hydrogen atoms in Chimera by go to Select->Chemistry->Element->H and then Action->Atoms/bonds->Delete.Save it as a pdb file.
  
 
==III. Generating Receptor Surface and Spheres==
 
==III. Generating Receptor Surface and Spheres==
Line 55: Line 87:
  
 
| In the new window save the surface as ''1HVR.receptor.dms''
 
| In the new window save the surface as ''1HVR.receptor.dms''
 +
 +
[[Image:2014_1HVR_receptor_surface.png|thumb|center|375px|1HVR Receptor surface]]
  
 
=== Placing Spheres ===
 
=== Placing Spheres ===
Line 75: Line 109:
  
 
Input File Details:
 
Input File Details:
−
| 1HVR.receptor.dms - surface file from the previous step
+
  1HVR.receptor.dms - surface file from the previous step
−
| R - tells SPHGEN to place spheres either outside of the surface (R) or inside the surface (L)
+
  R - tells SPHGEN to place spheres either outside of the surface (R) or inside the surface (L)
−
| X - tells SPHGEN the subset of surface points to be used (X=all points)
+
  X - tells SPHGEN the subset of surface points to be used (X=all points)
−
| 0.0 - prevents generation of large spheres with close surface contacts(defalut=0.0)
+
  0.0 - prevents generation of large spheres with close surface contacts(defalut=0.0)
−
| 4.0 - maximum sphere radius in angstroms (default=4.0)
+
  4.0 - maximum sphere radius in angstroms (default=4.0)
−
| 1.4 - minimum sphere radius in angstroms (default=radius of probe)
+
  1.4 - minimum sphere radius in angstroms (default=radius of probe)
−
| 1HVR.receptor.sph - clustered spheres file that we want to generate
+
  1HVR.receptor.sph - clustered spheres file that we want to generate
 +
 
 +
'''2.''' Run the sphgen program from the terminal:
 +
 
 +
sphgen -i INSPH -o OUTSPH
 +
 
 +
'''-i''' tells sphgen where the input file '''INSPH''' is
 +
'''INSPH''' tells sphgen what to do
 +
'''-o''' tells sphgen what to call the oputput file
 +
'''OUTSPH''' is the output file containing the sphere information
 +
 
 +
'''3.''' ''(optional)'' To look at the spheres generated, you need to put them into PDB format.
 +
 
 +
Run '''showsphere''', by typing the following into the terminal:
 +
 
 +
showsphere
 +
 
 +
You will be prompted with the following questions:
 +
 
 +
Enter name of sphere cluster file:
 +
      '''1HVR.receptor.sph'''
 +
Enter cluster number to process (<0 = all):
 +
      '''-1'''
 +
Generate surfaces as well as pdb files (<N>/Y)?
 +
      '''N'''
 +
Enter name for output file prefix:
 +
      '''output_spheres'''
 +
Process cluster 0 (contains ALL spheres) (<N>/Y)?
 +
      '''N'''
 +
 
 +
You can then open the receptor file in Chimera as well as the output_spheres.pdb file and should see many spheres placed all over the receptor surface.
 +
 
 +
[[Image:All_spheres_surface_2014.png|thumb|center|375px|1HVR Receptor surface with spheres]]
 +
 
 +
'''4.''' Run the '''sphere_selector''' program from in the terminal to select spheres of interest ''(note: not all of the spheres in the previous image are in the active site so we want to eliminate them)'':
 +
 
 +
sphere_selector 1HVR.receptor.sph ../01.dockprep/1HVR.ligand.mol2 8.0
 +
 
 +
This program goes to select the spheres within a user-defined radius (8.0 here) of a target molecule from a previously obtained file: '''1HVR.receptor.sph'''. In turn, a new file '''selected_spheres.sph''' will be generated.
 +
 
 +
'''5.''' Run '''showsphere''' to visualize the spheres:
 +
 
 +
showsphere
 +
 
 +
When prompted on the command line, answering the questions as follows:
 +
 
 +
Enter name of sphere cluster file:
 +
      '''selected_spheres.sph'''
 +
Enter cluster number to process (<0 = all):
 +
      '''-1'''
 +
Generate surfaces as well as pdb files (<N>/Y)?
 +
      '''N'''
 +
Enter name for output file prefix:
 +
      '''output_spheres_selected'''
 +
Process cluster 0 (contains ALL spheres) (<N>/Y)?
 +
      '''N'''
 +
 
 +
* Launch '''Chimera''', choose ''File'' -> ''Open'', choose '''1HVR.receptor.noH.pdb'''
 +
* Go ''File'' -> ''Open'', choosing '''output_spheres_selected.pdb'''
 +
* Go ''Select'' -> ''Residue'' -> ''SPH''
 +
* Finally, ''Actions'' -> ''Atoms/Bonds'' -> ''sphere''
 +
 
 +
The final image should look similar to the example below:
 +
 
 +
[[Image:Spheres_surface_receptor_1HVR_2014.png|thumb|center|375px|1HVR Receptor surface with spheres within 8A]]
  
 
==IV. Generating Box and Grid==
 
==IV. Generating Box and Grid==
 +
=== Box Generation===
 +
Mosavverul Arkin
 +
 +
* Make a new directory and name it: ''03.box-grid/''
 +
    mkdir 03.box-grid
 +
  
−
Mosavverul Arkin
+
* Make a new file in this directory and name it ''showbox.in''
 +
    vim showbox.in
 +
 
 +
* This will automatically open the file ''showbox.in''. Edit the file ''showbox.in'' as follows:
 +
 
 +
    Y                                              # Yes, generate a box
 +
    8.0                                            # Size of the box in Angstroms
 +
    ../02.surface-spheres/selected_spheres.sph      # Sphere.sph file
 +
    1                                              # Cluster number
 +
    1HVR.box.pdb                                    # Name of the output file
 +
 
 +
 
 +
* Save the file using the command:
 +
    :wq
 +
in ''esc'' mode
 +
* Run the command:
 +
    showbox < showbox.in
 +
 
 +
[[Image:1HVR_receptor_surface_2014.png|thumb|center|375px|Receptor surface with generated box]]
 +
 
 +
=== Grid computing===
 +
We now create the energy scoring grid file '' *.nrg '' using the program named ''grid''.
 +
 
 +
First you need to make sure that DOCK6 is installed, you can determine the path of DOCK installation by this command
 +
    which dock6
 +
from this location, make sure you have these three files and copy them to your local ~00.files/folder
 +
    ../parameters/vdw_AMBER_parm99.defn
 +
    ../parameters/flex.defn
 +
    ../parameters/flex_drive.tbl
 +
* Make a new file in the same directory (''03.box-grid/'') and name it: ''grid.in''
 +
    touch grid.in
 +
    grid -i grid.in
 +
 
 +
This will automatically open the file grid.in. Edit the file ''grid.in'' with the parameters and values listed in the table below.
 +
The program ''grid'' also computes a bump grid ''*.bmp'' file which identifies whether a ligand atom is in severe steric overlap with a receptor atom. For more information please refer to [http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm#Grid DOCK 6.6 Users Manual]
 +
{| border="1" cellpadding="5" cellspacing="0" align="center"
 +
|+ '''grid.in'''
 +
|-
 +
! style="background: #efefef;" | Parameter
 +
! style="background: #efefef;" | Value
 +
! style="background: #efefef;" | Description
 +
|-
 +
| compute_grids
 +
| yes
 +
| compute scoring grids (yes)
 +
|-
 +
| grid_spacing
 +
| 0.4
 +
| distance between grid points along each axis (in &Aring;).
 +
|-
 +
| output_molecule
 +
| no
 +
| write up coordinates of the receptor into a new file
 +
|-
 +
| contact_score 
 +
| no
 +
| compute contact grid? default is no
 +
|-
 +
| energy_score
 +
| yes
 +
| compute energy score? yes - we are using this method to compute force fields on probes
 +
|-
 +
| energy_cutoff_distance 
 +
| 9999
 +
| the max distance between atoms for the energy contribution to be computed
 +
|-
 +
| atom_model 
 +
| a
 +
| atom_model u means united atom model where atoms are attached to hydrogens, and a stands for all-atom model, where hydrogens on carbons are treated separately
 +
|-
 +
| attractive_exponent
 +
| 6
 +
| attractive component stands for exponent of the attractive LJ term in VDW potential
 +
|-
 +
| repulsive_exponent
 +
| 9
 +
| repulsive component stands for exponent in the repulsive LJ term in VDW potential
 +
|-
 +
| distance_dielectric
 +
| yes
 +
| distance dielectric stands for the dielectric constant to be linearly dependent on distance
 +
|-
 +
| dielectric_factor
 +
| 4
 +
| distance dielectric factor is the coefficient of the dielectric
 +
|-
 +
| bump_filter
 +
| yes
 +
| bump filter flag determines if we want to screen orientation for clashes before scoring and minimization
 +
|-
 +
| bump_overlap
 +
| 0.75
 +
| bump_overlap stands for the fraction of allowed overlap where 1 corresponds to no allowed overlap and 0 corresponds to full overlap being permitted.
 +
|-
 +
| receptor_file                     
 +
| ../01.dockprep/1HVR.receptor.mol2
 +
| our receptor file
 +
|-
 +
| box_file                           
 +
| 1HVR.box.pdb
 +
| the box file we generated in the Box section
 +
|-
 +
| vdw_definition_file         
 +
| ../00.files/vdw_AMBER_parm99.defn
 +
| van der Waals parameters file
 +
|-
 +
| style="border-bottom: 3px solid grey;" |  score_grid_prefix             
 +
| style="border-bottom: 3px solid grey;" | 1HVR.grid
 +
| style="border-bottom: 3px solid grey;" | prefix for the grid file name; all the extensions will be generated automatically.
 +
|-
  
−
1.) Make a new directory and name it: 03.box-grid/
+
|}
−
      mkdir 03.box-grid
 
  
 
==V. Docking a Single Molecule for Pose Reproduction==
 
==V. Docking a Single Molecule for Pose Reproduction==
  
−
Jess Junjie Kai
+
'''Rigid Ligand DOCKing'''
 +
 
 +
Rigid Ligand Docking is usually used to check the validity of docking program and user-defined parameters because it's much faster than flexible docking. It also helps users to identify any mistakes made during the preparation of receptor and ligand. In our case, we know the experimental structure of a receptor-ligand complex. So if we dock the ligand back to the ligand-free receptor. The pose predicted by DOCK should be very close to the experimental one. Otherwise, users should examine the parameters and all the files involved.
 +
 
 +
The following should be performed in 04.dock/file
 +
 
 +
First create an empty file and execute DOCK6. The dock.in is the name of the input file(your input file may have another name).
 +
    touch dock.in
 +
    dock6 -i dock.in
 +
Here is the input for DOCK6
 +
  ligand_atom_file                                            ../01.dockprep/1HVR.ligand.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              yes
 +
  use_rmsd_reference_mol                                      yes
 +
  rmsd_reference_filename                                      ../01.dockprep/1HVR.ligand.mol2
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../02.surface-spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  use_internal_energy                                          no
 +
  flexible_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                                      ../03.box-grid/1HVR.grid
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_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                                10.0
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              ../00.files/flex.defn
 +
  flex_drive_file                                              ../00.files/flex_drive.tbl
 +
  ligand_outfile_prefix                                        1HVR.out
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        100
 +
  write_conformations                                          no
 +
  cluster_conformations                                        no
 +
  rank_ligands                                                no
 +
 
 +
Note that "flexible_ligand" is turned off. "minimize_ligand" has to be on.
 +
 
 +
 
 +
You will have a file named "1HVR.out_scored.mol2" file in the directory where you run dock6. This file have the coordinates of ligand as "1HVR.ligand.mol2". Moreover, it also has energy score and rmsd value. You can open the pdb file of the complex in CHIMERA and open viewdock in Tool->surface->binding analysis. In viewdock, open the "1HVR.out_scored.mol2" file and viewdock will superimpose the predicted ligand pose onto the complex. You can have a visualized examination of the predicted pose.
 +
 
 +
[[Image:Rigid docking result.png|thumb|center|375px|Rigid docking result;RMSD = 0.9848; green: experiment; purple: predicted]]
 +
 
 +
The predicted pose and the experimental pose have an RMSD less than 2.000 which means the prediction reproduced the result from experiment. The preparation of receptor and ligand is correct and we can go to the next steps(such as flexible ligand docking and virtual screening) with some minor modification of this input file.
 +
 
 +
'''Flexible Ligand DOCKing'''
 +
 
 +
For flexible docking, some parameters need to be changed, and these changes will generate several additional questions to the user. Some changes of parameters are shown below:
 +
    calculate_rmsd                                              yes
 +
    flexible_ligand                                            yes
 +
    use_internal_energy                                        yes
 +
    minimize_ligand                                            yes
 +
    num_scored_conformers                                      10
 +
After the changes are made, execute the DOCK6:
 +
    dock6 -i dock.in
 +
Several additional questions will be asked, and answer them according to the table below:
 +
 
 +
  ligand_atom_file                                            ../01.dockprep/1HVR.ligand.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              yes
 +
  use_rmsd_reference_mol                                      yes
 +
  rmsd_reference_filename                                      ../01.dockprep/1HVR.ligand.mol2
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../02.surface-spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  flexible_ligand                                              yes
 +
  user_specified_anchor                                        no
 +
  limit_max_anchors                                            no
 +
  min_anchor_size                                              5
 +
  pruning_use_clustering                                      yes
 +
  pruning_max_orients                                          100
 +
  pruning_clustering_cutoff                                    100
 +
  pruning_conformer_score_cutoff                              100
 +
  use_clash_overlap                                            no
 +
  write_growth_tree                                            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                                      ../03.box-grod/1HVR.grid
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_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
 +
  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_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                                        yes
 +
  simplex_coefficient_restraint                                10.0
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              ../00.files/flex.defn
 +
  flex_drive_file                                              ../00.files/flex_drive.tbl
 +
  ligand_outfile_prefix                                        1HVR.output
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        100
 +
  write_conformations                                          no
 +
  cluster_conformations                                        yes
 +
  cluster_rmsd_threshold                                      2.0
 +
  rank_ligands                                                no
 +
 
 +
Note that "flexible_ligand" is on.
 +
 
 +
In the case of flexible ligand DOCKing, the ligand is allowed to be flexible. This type of docking allows the ligand to structurally rearrange in response to the receptor. Initially the largest rigid substructure of the ligand is identified; then the flexible layers are identified. The orientations are ranked according to their score, and are grouped by root mean squared deviation (RMSD). To run the docking calculation, use the program dock6 that is distributed with DOCK in the /bin directory. You need to generate an input file by answering questions interactively or manually via text file. The program will generate an output file summarizing the parameters used in the run, any warning or error messages, and summary information about the best scoring pose. It will also produce a structure (.mol2) file, containing the geometric coordinates of the best pose as well as a summary of the interaction energy of that pose.
 +
 
 +
[[Image:1HVR_flexible_dock_best.png|thumb|center|375px|Best scored ligand result]]
 +
 
 +
[[Image:1HVR_flexible_dock_worst.png|thumb|center|375px|Worst scored ligand result]]
  
 
==VI. Virtual Screening==
 
==VI. Virtual Screening==
Line 98: Line 480:
 
'''Virtual Screening Introduction'''
 
'''Virtual Screening Introduction'''
  
−
A virtual screen of various ligand allows for the comparison of both qualitative (e.g. position in binding site) and quantitative (e.g. energy scores) data pertaining to the each screened ligand with an originally docked molecule. Virtual screening is often used as a method to cut the cost of experimentation by narrowing down the ligands within a database and predicting which will exhibit the most favorable binding to a specific protein (with a pre-determined .grid file).
+
Virtual screening is a method used to predict most favorable ligand binding to a target protein within a ligand database. It also allows for comparison of both qualitative (e.g. position in binding site) and quantitative (e.g. grid scores, internal energy) data pertaining to the each screened ligand with the originally docked molecule.
 +
 
 +
To perform virtual screening, HIVPR.ligands.005.mol2, a mol2 file that contains 5 small molecules will be the virtual library based on the receptor 1HVR. This is a reasonable computational cost for a quick search, so we can conduct it on our own computer. After that, virtual screening can be conducted within a larger database, HIVPR.ligands.100.mol2, which contains 100 ligands. Since the computational cost of virtual screening is much higher, it is better for us to run it on Seawulf.
 +
 
 +
'''Running Virtual Screening'''
 +
 
 +
Before running virtual screening, a new directory 05.mini-virtual-screen is created and ligand files HIVPR.ligands.005.mol2 and HIVPR.ligands.100.mol2 are copied from Wjallen. Since most parameters of dock.in and new virtual screening input are the same, we can make few modifications to dock.in script but rename it as mini-virtual-screen.in.
 +
 
 +
  mkdir 05.mini-virtual-screen
 +
  cd 05.mini-virtual-screen
 +
  cp ~wjallen/AMS536/multi-mol2/files/HIVPR.ligands.005.mol2 ./
 +
  cp ../04.dock/dock.in/ ./
 +
  mv dock.in mini-virtual-screen.in
 +
 
 +
The content of mini-virtual-screen.in is as follows:
 +
 
 +
  ligand_atom_file                                            HIVPR.ligands.005.mol2
 +
  limit_max_ligands                                            no
 +
  skip_molecule                                                no
 +
  read_mol_solvation                                          no
 +
  calculate_rmsd                                              no
 +
  use_database_filter                                          no
 +
  orient_ligand                                                yes
 +
  automated_matching                                          yes
 +
  receptor_site_file                                          ../02.surface-spheres/selected_spheres.sph
 +
  max_orientations                                            1000
 +
  critical_points                                              no
 +
  chemical_matching                                            no
 +
  use_ligand_spheres                                          no
 +
  use_internal_energy                                          yes
 +
  internal_energy_rep_exp                                      12
 +
  flexible_ligand                                              yes
 +
  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
 +
  use_clash_overlap                                            no
 +
  write_growth_tree                                            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                                      ../03.box-grid/1HVR.grid
 +
  multigrid_score_secondary                                    no
 +
  dock3.5_score_secondary                                      no
 +
  continuous_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
 +
  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                                  1000
 +
  simplex_grow_tors_premin_iterations                          0
 +
  simplex_random_seed                                          0
 +
  simplex_restraint_min                                        no
 +
  atom_model                                                  all
 +
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
 +
  flex_defn_file                                              ../00.files/flex.defn
 +
  flex_drive_file                                              ../00.files/flex_drive.tbl
 +
  ligand_outfile_prefix                                        1HVR.output
 +
  write_orientations                                          no
 +
  num_scored_conformers                                        100
 +
  rank_ligands                                                no
 +
 
 +
'''Analyzing the Results'''
 +
 
 +
After performinging virtual screening on local machine, a file named 1HVR.output_scored.mol2 is obtained, which contains successfully docked ligands, including grid score, internal energy, etc.
 +
 
 +
 
 +
  ##########          Name: NONAME
 +
  ##########    Grid Score:          -25.904999
 +
  ##########      Grid_vdw:          -25.904999
 +
  ##########      Grid_es:            0.000000
 +
  ##########    Int_energy:            0.062399
 +
  @<TRIPOS>MOLECULE
 +
  NONAME
 +
  20 20 1 0 0
 +
  SMALL
 +
  NO_CHARGES
 +
  @<TRIPOS>ATOM
 +
      1 O          -6.5962  19.4021  23.0405 O.2    1  <1>        0.0000
 +
      2 C          -6.8355  18.4953  22.2343 C.2    1  <1>        0.0000
 +
      3 N          -7.8272  17.5802  22.4935 N.am    1  <1>        0.0000
 +
      4 H          -8.3181  17.7085  23.3731 H      1  <1>        0.0000
 +
      5 C          -8.2659  16.4500  21.6933 C.3    1  <1>        0.0000
 +
      6 H          -9.1047  15.9926  22.2333 H      1  <1>        0.0000
 +
      7 H          -7.4773  15.6904  21.6784 H      1  <1>        0.0000
 +
      8 C          -8.7214  16.8444  20.2915 C.3    1  <1>        0.0000
 +
      9 H          -9.4627  17.6481  20.3875 H      1  <1>        0.0000
 +
    10 O          -9.4130  15.7379  19.7151 O.3    1  <1>        0.0000
 +
    11 H        -10.1762  15.5384  20.2849 H      1  <1>        0.0000
 +
    12 C          -7.5968  17.3421  19.3635 C.3    1  <1>        0.0000
 +
    13 H          -7.8639  18.3502  19.0239 H      1  <1>        0.0000
 +
    14 O          -7.5032  16.5445  18.1836 O.3    1  <1>        0.0000
 +
    15 H          -8.3854  16.5076  17.7791 H      1  <1>        0.0000
 +
    16 C          -6.2182  17.4085  20.0113 C.3    1  <1>        0.0000
 +
    17 H          -5.4732  17.6806  19.2547 H      1  <1>        0.0000
 +
    18 H          -5.8956  16.4394  20.4044 H      1  <1>        0.0000
 +
    19 N          -6.1245  18.4045  21.0622 N.am    1  <1>        0.0000
 +
    20 H          -5.4272  19.1287  20.9184 H      1  <1>        0.0000
 +
  @<TRIPOS>BOND
 +
    1    1    2    2
 +
    2    2    3  am
 +
    3    2    19  am
 +
    4    3    4    1
 +
    5    3    5    1
 +
    6    5    6    1
 +
    7    5    7    1
 +
    8    5    8    1
 +
    9    8    9    1
 +
    10    8    10    1
 +
    11    8    12    1
 +
    12    10    11    1
 +
    13    12    13    1
 +
    14    12    14    1
 +
    15    12    16    1
 +
    16    14    15    1
 +
    17    16    17    1
 +
    18    16    18    1
 +
    19    16    19    1
 +
    20    19    20    1
 +
 
 +
When virtual screening with a larger ligand database runs completely in Seawulf, files 1HVR.output_scored.mol2  which contains the mol2 files of all successfully docked ligands, virtual_screen.out which contains the dock results of successfully docked ligands, and virtual_screen.out.1 to virtual_screen.out.3 which contain dock results from different nodes you use (the number is related with the number of nodes you use, here we use 4 nodes) are produced. One of the successfully docked ligands is as follows:
 +
 +
  Molecule: CHEMBL171551
 +
  Anchors:              3
 +
    Orientations:          3000
 +
    Conformations:        359
 +
      Grid Score:          -60.092117
 +
        Grid_vdw:          -62.332535
 +
          Grid_es:            2.240418
 +
      Int_energy:          13.718960
 +
 
 +
These files should be copied to local computers if we want to use Chimera to visually check the results. In Seawulf, move to 06.virtual-screen/ directory,
 +
 
 +
  scp –r username@herbie.mathlab.sunysb.edu:~/AMS536/dock_tutorial/
 +
 
 +
In herbie, open the receptor 1HVR.receptor.mol2 and reference ligand 1HVR.ligand.mol2 and then how the ligands fitting the receptor can be checked by ViewDock function in Chimera. Go to Tools -> Surface/Binding Analysis -> ViewDock and choose 1HVR.output_scored.mol2.In the pup-up window, we can go to Column ->show -> Grid Score to show the grid score.
  
 
==VII. Running DOCK in Parallel on Seawulf==
 
==VII. Running DOCK in Parallel on Seawulf==
  
−
Fengfei Lu
 
  
−
==VIII. Frequently Encountered Problems==
+
The [http://www.stonybrook.edu/seawulfcluster/ Seawulf Cluster] is a custom-built 470-processor (235 dual processor nodes and 2 processors per node) Linux Cluster capable of highly parallel processing. It severs as to provide computational resources and expertise for basic scientific research to the faculty and students of Stony Brook University.
 +
 
 +
 
 +
Typically, to dock one ligand, one processor on a single node will be used, while for docking multiple ligands, more than one processor can be used in parallel mode. However, the number of processors used should be less than the number of ligands for dock.
 +
 
 +
 
 +
Before running DOCK on Seawulf, we need to copy our whole dock-tutorial files from Herbie to Seawulf. Log into the Seawulf through Herbie, and then type,
 +
 
 +
  scp -r username@herbie.mathlab.sunysb.edu:~/AMS536/dock-tutorial/ ~/AMS536/
 +
 
 +
 
 +
First, making a file named qsub.csh with the following context,
 +
 
 +
  #!/bin/csh
 +
  #PBS -l nodes=2:ppn=2
 +
  #PBS -l walltime=10:00:00
 +
  #PBS -N 1HVR.vs
 +
  #PBS -o 1HVR.output
 +
  #PBS -j oe
 +
  #PBS –V
 +
 
 +
  cd /nfs/user03/username/AMS536/dock-tutorial/06.virtual-screen
 +
  mpirun -np 4 /nfs/user03/wjallen/local/dock6/bin/dock6.mpi -i virtual_screen.in -o virtual_screen.out
 +
 
 +
 
 +
Explanation of the commands,
 +
 
 +
'''#!/bin/csh''': Execute script with tcsh
 +
 
 +
'''#PBS -l nodes=2:ppn=2''': Use 2 nodes, and 2 processors per node, so 4 processors
 +
 
 +
'''#PBS -l walltime=10:00:00''': Allow 10 hours for your job run
 +
 
 +
'''#PBS -N 1HVR.vs''': Name of your job
 +
 
 +
'''#PBS -o 1HVR.output''': Name of your output file
 +
 
 +
'''#PBS -j oe''': Combine the output and error streams into a single output file
 +
 
 +
'''#PBS –V''': Show more information about what is happening for the users
 +
 
 +
'''cd /nfs/user03/username/AMS536/dock-tutorial/06.virtual-screen''': Change to your home directory and folder with dock files
 +
 
 +
'''mpirun -np 4 /nfs/user03/wjallen/local/dock6/bin/dock6.mpi -i virtual_screen.in -o virtual_screen.out''': This line specifies path to dock executable and provide input and output filenames. Notice that in order to run DOCK in parallel (on 4 processors here), we need to use dock6.mpi instead of dock6. Message passing interface ( [http://www.mpi-forum.org/docs/docs.html MPI]) is basically a program that allows programs like DOCK to run in parallel.
 +
 
 +
 
 +
Then, we can run the dock virtual screen on Seawulf by submitting the DOCK experiment to the Seawulf queue,
 +
 
 +
  qsub virtual_screen.in
 +
 
 +
 
 +
You can use the command '''qstat''' to show the status of pbs batch jobs,
 +
 
 +
  qstat #the whole list of submitted jobs in queue
 +
 
 +
  qstat -u username #the whole list of jobs in queue you submitted
  
−
=== Tianao ===
 
  
−
=== Mike ===
+
For more information, see [http://ringo.ams.sunysb.edu/index.php/PBS_Queue PBS Queue].
  
−
=== Ivan ===
+
==VIII. Frequently Encountered Problems==
  
−
=== Junjie ===
+
* You should always be clear about what the input file and output file of one program are and what format they are.
  
−
=== Kai ===
+
* Sometimes, it's easy to make mistakes if you are confused about your current location, or "path", so make sure you know that all the time.
  
−
=== Jess ===
+
* The most common problem experienced in UNIX is having the wrong file path. '''ALWAYS CHECK TO MAKE SURE THAT YOUR FILE PATH IS CORRECT''' This can be facilitated with the Tab button which will auto complete what you have started to type.
  
−
=== Arkin ===
+
* Make sure you are opening the correct files for each visualization step (e.g. opening the receptor file with the ligand still there may make analyzing your docking results a bit more challenging).
  
−
=== Yan ===
+
* Use the auto-fill (tab) function but double check to make sure the correct file was auto-filled (e.g. if you type ''1H'' then hit tab you might get ''1HVR_selected_spheres'' but the file you want actually is ''1HVR_selected_spheres.sph'').
  
−
=== Yao ===
+
* For users with no UNIX experience and limited programming experience - it's a good idea to run through a couple tutorials before you start using UNIX. Even if you type in the commands without fully understanding their implications, become familiar with the basic commands and shortcuts. It will help a lot when you actually start using UNIX!
  
−
=== Lu ===
+
* If you are not so familiar with UNIX environment, it is better for you to practice the tutorial with basic commands: http://www.ee.surrey.ac.uk/Teaching/Unix/
  
−
=== Fengfei ===
+
* When you are trying a command, but have a typo (so this command will not work), we can always go back to the last command by press UP arrow button go back to the command that just typed and modify that command.
  
−
=== Mosavverul ===
+
* Insert button can allows us to retype the command instead of rewrite it.
  
−
=== Joe ===
+
* Example for writing text, writing a command, and importing an image:
  
 
Write some text here..
 
Write some text here..
Line 136: Line 727:
 
   command or input file
 
   command or input file
  
−
[[Image:1HVR_receptor_surface_2014.png|thumb|center|375px|Receptor surface]]
+
[[Image:1HVR_surface_2014.png|thumb|center|375px|Receptor surface]]

Latest revision as of 17:05, 17 December 2014

For additional Rizzo Lab tutorials see DOCK Tutorials. Use this link Wiki Formatting as a reference for editing the wiki. This tutorial was developed collaboratively by the AMS 536 class of 2013, using DOCK v6.6.

I. Introduction

DOCK

DOCK is a molecular docking program used in drug discovery. It was developed by Irwin D. Kuntz, Jr. and colleagues at UCSF (see UCSF DOCK). This program, given a protein binding site and a small molecule, tries to predict the correct binding mode of the small molecule in the binding site, and the associated binding energy. Small molecules with highly favorable binding energies could be new drug leads. This makes DOCK a valuable drug discovery tool. DOCK is typically used to screen massive libraries of millions of compounds against a protein to isolate potential drug leads. These leads are then further studied, and could eventually result in a new, marketable drug. DOCK works well as a screening procedure for generating leads, but is not currently as useful for optimization of those leads.

DOCK 6 uses an incremental construction algorithm called anchor and grow. It is described by a three-step process:

  1. Rigid portion of ligand (anchor) is docked by geometric methods.
  2. Non-rigid segments added in layers; energy minimized.
  3. The resulting configurations are 'pruned' and energy re-minimized, yielding the docked configurations.

HIV Protease

HIV protease (HIVPR) is a protein involved in viral maturation during the life cycle of HIV. HIVPR is an approximately 22 kDa homodimer with 99 residues per chain. Inhibition of this protein has been shown to be an effective form of treatment of HIV. Currently-available HIVPR inhibitors generally take the form of a symmetric cyclic urea compound. For more information, see Lam et al.

Organizing Directories

While performing docking, it is convenient to adopt a standard directory structure / naming scheme, so that files are easy to find / identify. For this tutorial, we will use something similar to the following: 

~username/AMS536/dock-tutorial/00.files/
                              /01.dockprep/
                              /02.surface-spheres/
                              /03.box-grid/
                              /04.dock/
                              /05.mini-virtual-screen/
                              /06.virtual-screen/

In addition, most of the important files that are derived from the original crystal structure will be given a prefix that is the same as the PDB code, '1HVR'. The following sections in this tutorial will adhere to this directory structure / naming scheme.

II. Preparing the Receptor and Ligand

Downloading the PDB Structure (1HVR)

Go to PDB (Protein Data Bank) website (http://www.rcsb.org/pdb/home/home.do) enter the protein ID (1HVR), search for the PDB file and download it as a text form.

Preparing the ligand and receptor in Chimera

Put the 1HVR PDB file in 00.file/folder. If you are in the 00.files/directory, then tap the command: 

 cp ~/Downloads/1HVR.pdb ./

When you are preparing your PDB files, you have to make some modifications on your original file. Make a copy of your original PDB file, and rename it as "1HVR.modified.pdb" in the 00.files/. Here we changed the atom name from "CSO" to "CYS" and deleted two lines "OD" and "HD". Here is an example to change all "CSO" to "CYS": 

 :%s/CSO/CYS/gc

And then we will create 4 files in 01.dockprep/ directory: 

 1HVR.dockprep.mol2  
 1HVR.ligand.mol2  
 1HVR.receptor.mol2  
 1HVR.receptor.noH.pdb

Create the dockprep file

For the "1HVR.dockprep.mol2" file: open the 1HVR.modified.pdb in Chimera; delete the water molecules ; delete the original hydrogen atoms; add the charge by go to Tools->structure editing->add charge (Note when adding the charge to the ligand, you can choose AMBER ff99SB as the charge model and chose gasteiger as the charge method. In this 1HVR case, we set Net Charge to 0. You may have to consider the chemistry of the ligand when assigning a charge state). Add the hydrogen atoms manually by Tools->structure editing->Add H . Or you can do all of the above by clicking Tools -> Structure Editing -> Dock Prep.

Finally save the file as a mol2 format in Chimera.

Create the ligand file

To create the ligand file: Open 1HVR.dockprep.mol2 in Chimera and select the ligand chain "XK2", then Select->invert, then go to Action->Atoms/bonds->Delete

Create the receptor file

Select the ligand residue "XK2" and delete it and save it as a mol2 file.

Create the receptor without hydrogen atoms

Open the "1HVR.receptor.mol2" file and delete all of the hydrogen atoms in Chimera by go to Select->Chemistry->Element->H and then Action->Atoms/bonds->Delete.Save it as a pdb file.

III. Generating Receptor Surface and Spheres

Generating the Receptor Surface

Check to make sure 02.surface-spheres directory exists under dock-tutorial. If not then make the following directory: 

mkdir 02.surface-sphgen
cd 02.surface-sphgen

The following steps will be carried out to generate the receptor surface using Chimera:

Open Chimera by simply typing chimera into the terminal window

| Go File -> Open and choose the PDB file of the protein containing no hydrogens (1HVR.receptor.noH.pdb) from 01.dockprep

| Further, Actions -> Surface -> Show

| Go Tools -> Structure Editing -> Write DMS in order to obtain a dms file, which we will need to place spheres

| In the new window save the surface as 1HVR.receptor.dms

1HVR Receptor surface

Placing Spheres

We will be using SPHGEN to generate spheres: see the DOCK online owners manual for additional information:

<http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm>

The following steps will be used to place the spheres on the receptor surface:

1. Create a file called INSPH and fill it out as follows, then save it. This input file tells SPHGEN what to do, details of each line are below: 

1HVR.receptor.dms
R
X
0.0
4.0
1.4
1HVR.receptor.sph

Input File Details: 

 1HVR.receptor.dms - surface file from the previous step
 R - tells SPHGEN to place spheres either outside of the surface (R) or inside the surface (L)
 X - tells SPHGEN the subset of surface points to be used (X=all points)
 0.0 - prevents generation of large spheres with close surface contacts(defalut=0.0)
 4.0 - maximum sphere radius in angstroms (default=4.0)
 1.4 - minimum sphere radius in angstroms (default=radius of probe)
 1HVR.receptor.sph - clustered spheres file that we want to generate

2. Run the sphgen program from the terminal: 

sphgen -i INSPH -o OUTSPH

-i tells sphgen where the input file INSPH is
INSPH tells sphgen what to do
-o tells sphgen what to call the oputput file
OUTSPH is the output file containing the sphere information

3. (optional) To look at the spheres generated, you need to put them into PDB format.

Run showsphere, by typing the following into the terminal: 

showsphere

You will be prompted with the following questions: 

Enter name of sphere cluster file:
     1HVR.receptor.sph
Enter cluster number to process (<0 = all):
     -1
Generate surfaces as well as pdb files (<N>/Y)?
     N
Enter name for output file prefix:
     output_spheres
Process cluster 0 (contains ALL spheres) (<N>/Y)? 
     N

You can then open the receptor file in Chimera as well as the output_spheres.pdb file and should see many spheres placed all over the receptor surface.

1HVR Receptor surface with spheres

4. Run the sphere_selector program from in the terminal to select spheres of interest (note: not all of the spheres in the previous image are in the active site so we want to eliminate them): 

sphere_selector 1HVR.receptor.sph ../01.dockprep/1HVR.ligand.mol2 8.0

This program goes to select the spheres within a user-defined radius (8.0 here) of a target molecule from a previously obtained file: 1HVR.receptor.sph. In turn, a new file selected_spheres.sph will be generated.

5. Run showsphere to visualize the spheres: 

showsphere

When prompted on the command line, answering the questions as follows: 

Enter name of sphere cluster file:
     selected_spheres.sph
Enter cluster number to process (<0 = all):
     -1
Generate surfaces as well as pdb files (<N>/Y)?
     N
Enter name for output file prefix:
     output_spheres_selected
Process cluster 0 (contains ALL spheres) (<N>/Y)? 
     N
  • Launch Chimera, choose File -> Open, choose 1HVR.receptor.noH.pdb
  • Go File -> Open, choosing output_spheres_selected.pdb
  • Go Select -> Residue -> SPH
  • Finally, Actions -> Atoms/Bonds -> sphere

The final image should look similar to the example below:

1HVR Receptor surface with spheres within 8A

IV. Generating Box and Grid

Box Generation

Mosavverul Arkin

  • Make a new directory and name it: 03.box-grid/
   mkdir 03.box-grid


  • Make a new file in this directory and name it showbox.in
   vim showbox.in
  • This will automatically open the file showbox.in. Edit the file showbox.in as follows:
   Y                                               # Yes, generate a box
   8.0                                             # Size of the box in Angstroms
   ../02.surface-spheres/selected_spheres.sph      # Sphere.sph file
   1                                               # Cluster number
   1HVR.box.pdb                                    # Name of the output file


  • Save the file using the command:
    :wq

in esc mode

  • Run the command:
    showbox < showbox.in
Receptor surface with generated box

Grid computing

We now create the energy scoring grid file *.nrg using the program named grid.

First you need to make sure that DOCK6 is installed, you can determine the path of DOCK installation by this command 

   which dock6

from this location, make sure you have these three files and copy them to your local ~00.files/folder 

   ../parameters/vdw_AMBER_parm99.defn
   ../parameters/flex.defn
   ../parameters/flex_drive.tbl
  • Make a new file in the same directory (03.box-grid/) and name it: grid.in
   touch grid.in
   grid -i grid.in

This will automatically open the file grid.in. Edit the file grid.in with the parameters and values listed in the table below. The program grid also computes a bump grid *.bmp file which identifies whether a ligand atom is in severe steric overlap with a receptor atom. For more information please refer to DOCK 6.6 Users Manual

grid.in
Parameter Value Description
compute_grids yes compute scoring grids (yes)
grid_spacing 0.4 distance between grid points along each axis (in Å).
output_molecule no write up coordinates of the receptor into a new file
contact_score no compute contact grid? default is no
energy_score yes compute energy score? yes - we are using this method to compute force fields on probes
energy_cutoff_distance 9999 the max distance between atoms for the energy contribution to be computed
atom_model a atom_model u means united atom model where atoms are attached to hydrogens, and a stands for all-atom model, where hydrogens on carbons are treated separately
attractive_exponent 6 attractive component stands for exponent of the attractive LJ term in VDW potential
repulsive_exponent 9 repulsive component stands for exponent in the repulsive LJ term in VDW potential
distance_dielectric yes distance dielectric stands for the dielectric constant to be linearly dependent on distance
dielectric_factor 4 distance dielectric factor is the coefficient of the dielectric
bump_filter yes bump filter flag determines if we want to screen orientation for clashes before scoring and minimization
bump_overlap 0.75 bump_overlap stands for the fraction of allowed overlap where 1 corresponds to no allowed overlap and 0 corresponds to full overlap being permitted.
receptor_file ../01.dockprep/1HVR.receptor.mol2 our receptor file
box_file 1HVR.box.pdb the box file we generated in the Box section
vdw_definition_file ../00.files/vdw_AMBER_parm99.defn van der Waals parameters file
score_grid_prefix 1HVR.grid prefix for the grid file name; all the extensions will be generated automatically.

V. Docking a Single Molecule for Pose Reproduction

Rigid Ligand DOCKing

Rigid Ligand Docking is usually used to check the validity of docking program and user-defined parameters because it's much faster than flexible docking. It also helps users to identify any mistakes made during the preparation of receptor and ligand. In our case, we know the experimental structure of a receptor-ligand complex. So if we dock the ligand back to the ligand-free receptor. The pose predicted by DOCK should be very close to the experimental one. Otherwise, users should examine the parameters and all the files involved.

The following should be performed in 04.dock/file

First create an empty file and execute DOCK6. The dock.in is the name of the input file(your input file may have another name). 

   touch dock.in
   dock6 -i dock.in

Here is the input for DOCK6 

  ligand_atom_file                                             ../01.dockprep/1HVR.ligand.mol2
  limit_max_ligands                                            no
  skip_molecule                                                no
  read_mol_solvation                                           no
  calculate_rmsd                                               yes
  use_rmsd_reference_mol                                       yes
  rmsd_reference_filename                                      ../01.dockprep/1HVR.ligand.mol2
  use_database_filter                                          no
  orient_ligand                                                yes
  automated_matching                                           yes
  receptor_site_file                                           ../02.surface-spheres/selected_spheres.sph
  max_orientations                                             1000
  critical_points                                              no
  chemical_matching                                            no
  use_ligand_spheres                                           no
  use_internal_energy                                          no
  flexible_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                                       ../03.box-grid/1HVR.grid
  multigrid_score_secondary                                    no
  dock3.5_score_secondary                                      no
  continuous_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                                10.0
  atom_model                                                   all
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
  flex_defn_file                                               ../00.files/flex.defn
  flex_drive_file                                              ../00.files/flex_drive.tbl
  ligand_outfile_prefix                                        1HVR.out
  write_orientations                                           no
  num_scored_conformers                                        100
  write_conformations                                          no
  cluster_conformations                                        no
  rank_ligands                                                 no

Note that "flexible_ligand" is turned off. "minimize_ligand" has to be on.


You will have a file named "1HVR.out_scored.mol2" file in the directory where you run dock6. This file have the coordinates of ligand as "1HVR.ligand.mol2". Moreover, it also has energy score and rmsd value. You can open the pdb file of the complex in CHIMERA and open viewdock in Tool->surface->binding analysis. In viewdock, open the "1HVR.out_scored.mol2" file and viewdock will superimpose the predicted ligand pose onto the complex. You can have a visualized examination of the predicted pose.

Rigid docking result;RMSD = 0.9848; green: experiment; purple: predicted

The predicted pose and the experimental pose have an RMSD less than 2.000 which means the prediction reproduced the result from experiment. The preparation of receptor and ligand is correct and we can go to the next steps(such as flexible ligand docking and virtual screening) with some minor modification of this input file.

Flexible Ligand DOCKing

For flexible docking, some parameters need to be changed, and these changes will generate several additional questions to the user. Some changes of parameters are shown below: 

   calculate_rmsd                                              yes
   flexible_ligand                                             yes
   use_internal_energy                                         yes
   minimize_ligand                                             yes
   num_scored_conformers                                       10

After the changes are made, execute the DOCK6: 

   dock6 -i dock.in

Several additional questions will be asked, and answer them according to the table below: 

  ligand_atom_file                                             ../01.dockprep/1HVR.ligand.mol2
  limit_max_ligands                                            no
  skip_molecule                                                no
  read_mol_solvation                                           no
  calculate_rmsd                                               yes
  use_rmsd_reference_mol                                       yes
  rmsd_reference_filename                                      ../01.dockprep/1HVR.ligand.mol2
  use_database_filter                                          no
  orient_ligand                                                yes
  automated_matching                                           yes
  receptor_site_file                                           ../02.surface-spheres/selected_spheres.sph
  max_orientations                                             1000
  critical_points                                              no
  chemical_matching                                            no
  use_ligand_spheres                                           no
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  flexible_ligand                                              yes
  user_specified_anchor                                        no
  limit_max_anchors                                            no
  min_anchor_size                                              5
  pruning_use_clustering                                       yes
  pruning_max_orients                                          100
  pruning_clustering_cutoff                                    100
  pruning_conformer_score_cutoff                               100
  use_clash_overlap                                            no
  write_growth_tree                                            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                                       ../03.box-grod/1HVR.grid
  multigrid_score_secondary                                    no
  dock3.5_score_secondary                                      no
  continuous_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
  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_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                                        yes
  simplex_coefficient_restraint                                10.0
  atom_model                                                   all
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
  flex_defn_file                                               ../00.files/flex.defn
  flex_drive_file                                              ../00.files/flex_drive.tbl
  ligand_outfile_prefix                                        1HVR.output
  write_orientations                                           no
  num_scored_conformers                                        100
  write_conformations                                          no
  cluster_conformations                                        yes
  cluster_rmsd_threshold                                       2.0
  rank_ligands                                                 no

Note that "flexible_ligand" is on.

In the case of flexible ligand DOCKing, the ligand is allowed to be flexible. This type of docking allows the ligand to structurally rearrange in response to the receptor. Initially the largest rigid substructure of the ligand is identified; then the flexible layers are identified. The orientations are ranked according to their score, and are grouped by root mean squared deviation (RMSD). To run the docking calculation, use the program dock6 that is distributed with DOCK in the /bin directory. You need to generate an input file by answering questions interactively or manually via text file. The program will generate an output file summarizing the parameters used in the run, any warning or error messages, and summary information about the best scoring pose. It will also produce a structure (.mol2) file, containing the geometric coordinates of the best pose as well as a summary of the interaction energy of that pose.

Best scored ligand result
Worst scored ligand result

VI. Virtual Screening

Virtual Screening Introduction

Virtual screening is a method used to predict most favorable ligand binding to a target protein within a ligand database. It also allows for comparison of both qualitative (e.g. position in binding site) and quantitative (e.g. grid scores, internal energy) data pertaining to the each screened ligand with the originally docked molecule.

To perform virtual screening, HIVPR.ligands.005.mol2, a mol2 file that contains 5 small molecules will be the virtual library based on the receptor 1HVR. This is a reasonable computational cost for a quick search, so we can conduct it on our own computer. After that, virtual screening can be conducted within a larger database, HIVPR.ligands.100.mol2, which contains 100 ligands. Since the computational cost of virtual screening is much higher, it is better for us to run it on Seawulf.

Running Virtual Screening

Before running virtual screening, a new directory 05.mini-virtual-screen is created and ligand files HIVPR.ligands.005.mol2 and HIVPR.ligands.100.mol2 are copied from Wjallen. Since most parameters of dock.in and new virtual screening input are the same, we can make few modifications to dock.in script but rename it as mini-virtual-screen.in. 

  mkdir 05.mini-virtual-screen
  cd 05.mini-virtual-screen
  cp ~wjallen/AMS536/multi-mol2/files/HIVPR.ligands.005.mol2 ./
  cp ../04.dock/dock.in/ ./
  mv dock.in mini-virtual-screen.in

The content of mini-virtual-screen.in is as follows: 

  ligand_atom_file                                             HIVPR.ligands.005.mol2
  limit_max_ligands                                            no
  skip_molecule                                                no
  read_mol_solvation                                           no
  calculate_rmsd                                               no
  use_database_filter                                          no
  orient_ligand                                                yes
  automated_matching                                           yes
  receptor_site_file                                           ../02.surface-spheres/selected_spheres.sph
  max_orientations                                             1000
  critical_points                                              no
  chemical_matching                                            no
  use_ligand_spheres                                           no
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  flexible_ligand                                              yes
  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
  use_clash_overlap                                            no
  write_growth_tree                                            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                                       ../03.box-grid/1HVR.grid
  multigrid_score_secondary                                    no
  dock3.5_score_secondary                                      no
  continuous_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
  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                                  1000
  simplex_grow_tors_premin_iterations                          0
  simplex_random_seed                                          0
  simplex_restraint_min                                        no
  atom_model                                                   all
  vdw_defn_file                                                ../00.files/vdw_AMBER_parm99.defn
  flex_defn_file                                               ../00.files/flex.defn
  flex_drive_file                                              ../00.files/flex_drive.tbl
  ligand_outfile_prefix                                        1HVR.output
  write_orientations                                           no
  num_scored_conformers                                        100
  rank_ligands                                                 no

Analyzing the Results

After performinging virtual screening on local machine, a file named 1HVR.output_scored.mol2 is obtained, which contains successfully docked ligands, including grid score, internal energy, etc. 


  ##########           Name:		NONAME
  ##########    Grid Score:          -25.904999
  ##########      Grid_vdw:          -25.904999
  ##########       Grid_es:            0.000000
  ##########    Int_energy:            0.062399
  @<TRIPOS>MOLECULE
  NONAME
  20 20 1 0 0
  SMALL
  NO_CHARGES
  @<TRIPOS>ATOM
     1 O          -6.5962   19.4021   23.0405 O.2     1  <1>         0.0000
     2 C          -6.8355   18.4953   22.2343 C.2     1  <1>         0.0000
     3 N          -7.8272   17.5802   22.4935 N.am    1  <1>         0.0000
     4 H          -8.3181   17.7085   23.3731 H       1  <1>         0.0000
     5 C          -8.2659   16.4500   21.6933 C.3     1  <1>         0.0000
     6 H          -9.1047   15.9926   22.2333 H       1  <1>         0.0000
     7 H          -7.4773   15.6904   21.6784 H       1  <1>         0.0000
     8 C          -8.7214   16.8444   20.2915 C.3     1  <1>         0.0000
     9 H          -9.4627   17.6481   20.3875 H       1  <1>         0.0000
    10 O          -9.4130   15.7379   19.7151 O.3     1  <1>         0.0000
    11 H         -10.1762   15.5384   20.2849 H       1  <1>         0.0000
    12 C          -7.5968   17.3421   19.3635 C.3     1  <1>         0.0000
    13 H          -7.8639   18.3502   19.0239 H       1  <1>         0.0000
    14 O          -7.5032   16.5445   18.1836 O.3     1  <1>         0.0000
    15 H          -8.3854   16.5076   17.7791 H       1  <1>         0.0000
    16 C          -6.2182   17.4085   20.0113 C.3     1  <1>         0.0000
    17 H          -5.4732   17.6806   19.2547 H       1  <1>         0.0000
    18 H          -5.8956   16.4394   20.4044 H       1  <1>         0.0000
    19 N          -6.1245   18.4045   21.0622 N.am    1  <1>         0.0000
    20 H          -5.4272   19.1287   20.9184 H       1  <1>         0.0000
  @<TRIPOS>BOND
    1     1     2    2
    2     2     3   am
    3     2    19   am
    4     3     4    1
    5     3     5    1
    6     5     6    1
    7     5     7    1
    8     5     8    1
    9     8     9    1
   10     8    10    1
   11     8    12    1
   12    10    11    1
   13    12    13    1
   14    12    14    1
   15    12    16    1
   16    14    15    1
   17    16    17    1
   18    16    18    1
   19    16    19    1
   20    19    20    1

When virtual screening with a larger ligand database runs completely in Seawulf, files 1HVR.output_scored.mol2 which contains the mol2 files of all successfully docked ligands, virtual_screen.out which contains the dock results of successfully docked ligands, and virtual_screen.out.1 to virtual_screen.out.3 which contain dock results from different nodes you use (the number is related with the number of nodes you use, here we use 4 nodes) are produced. One of the successfully docked ligands is as follows: 

  Molecule: CHEMBL171551
  Anchors:               3
   Orientations:          3000
   Conformations:         359
      Grid Score:          -60.092117
        Grid_vdw:          -62.332535
         Grid_es:            2.240418
      Int_energy:           13.718960

These files should be copied to local computers if we want to use Chimera to visually check the results. In Seawulf, move to 06.virtual-screen/ directory, 

  scp –r username@herbie.mathlab.sunysb.edu:~/AMS536/dock_tutorial/

In herbie, open the receptor 1HVR.receptor.mol2 and reference ligand 1HVR.ligand.mol2 and then how the ligands fitting the receptor can be checked by ViewDock function in Chimera. Go to Tools -> Surface/Binding Analysis -> ViewDock and choose 1HVR.output_scored.mol2.In the pup-up window, we can go to Column ->show -> Grid Score to show the grid score.

VII. Running DOCK in Parallel on Seawulf

The Seawulf Cluster is a custom-built 470-processor (235 dual processor nodes and 2 processors per node) Linux Cluster capable of highly parallel processing. It severs as to provide computational resources and expertise for basic scientific research to the faculty and students of Stony Brook University.


Typically, to dock one ligand, one processor on a single node will be used, while for docking multiple ligands, more than one processor can be used in parallel mode. However, the number of processors used should be less than the number of ligands for dock.


Before running DOCK on Seawulf, we need to copy our whole dock-tutorial files from Herbie to Seawulf. Log into the Seawulf through Herbie, and then type, 

 scp -r username@herbie.mathlab.sunysb.edu:~/AMS536/dock-tutorial/ ~/AMS536/


First, making a file named qsub.csh with the following context, 

 #!/bin/csh
 #PBS -l nodes=2:ppn=2
 #PBS -l walltime=10:00:00
 #PBS -N 1HVR.vs
 #PBS -o 1HVR.output
 #PBS -j oe
 #PBS –V
 
 cd /nfs/user03/username/AMS536/dock-tutorial/06.virtual-screen
 mpirun -np 4 /nfs/user03/wjallen/local/dock6/bin/dock6.mpi -i virtual_screen.in -o virtual_screen.out


Explanation of the commands,

#!/bin/csh: Execute script with tcsh

#PBS -l nodes=2:ppn=2: Use 2 nodes, and 2 processors per node, so 4 processors

#PBS -l walltime=10:00:00: Allow 10 hours for your job run

#PBS -N 1HVR.vs: Name of your job

#PBS -o 1HVR.output: Name of your output file

#PBS -j oe: Combine the output and error streams into a single output file

#PBS –V: Show more information about what is happening for the users

cd /nfs/user03/username/AMS536/dock-tutorial/06.virtual-screen: Change to your home directory and folder with dock files

mpirun -np 4 /nfs/user03/wjallen/local/dock6/bin/dock6.mpi -i virtual_screen.in -o virtual_screen.out: This line specifies path to dock executable and provide input and output filenames. Notice that in order to run DOCK in parallel (on 4 processors here), we need to use dock6.mpi instead of dock6. Message passing interface ( MPI) is basically a program that allows programs like DOCK to run in parallel.


Then, we can run the dock virtual screen on Seawulf by submitting the DOCK experiment to the Seawulf queue, 

 qsub virtual_screen.in


You can use the command qstat to show the status of pbs batch jobs, 

 qstat #the whole list of submitted jobs in queue
 
 qstat -u username #the whole list of jobs in queue you submitted


For more information, see PBS Queue.

VIII. Frequently Encountered Problems

  • You should always be clear about what the input file and output file of one program are and what format they are.
  • Sometimes, it's easy to make mistakes if you are confused about your current location, or "path", so make sure you know that all the time.
  • The most common problem experienced in UNIX is having the wrong file path. ALWAYS CHECK TO MAKE SURE THAT YOUR FILE PATH IS CORRECT This can be facilitated with the Tab button which will auto complete what you have started to type.
  • Make sure you are opening the correct files for each visualization step (e.g. opening the receptor file with the ligand still there may make analyzing your docking results a bit more challenging).
  • Use the auto-fill (tab) function but double check to make sure the correct file was auto-filled (e.g. if you type 1H then hit tab you might get 1HVR_selected_spheres but the file you want actually is 1HVR_selected_spheres.sph).
  • For users with no UNIX experience and limited programming experience - it's a good idea to run through a couple tutorials before you start using UNIX. Even if you type in the commands without fully understanding their implications, become familiar with the basic commands and shortcuts. It will help a lot when you actually start using UNIX!
  • When you are trying a command, but have a typo (so this command will not work), we can always go back to the last command by press UP arrow button go back to the command that just typed and modify that command.
  • Insert button can allows us to retype the command instead of rewrite it.
  • Example for writing text, writing a command, and importing an image:

Write some text here.. 

 command or input file
File:1HVR surface 2014.png
Receptor surface
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