Difference between revisions of "2016 DOCK tutorial with Beta Trypsin"

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For additional Rizzo Lab tutorials see [[DOCK Tutorials]]. Use this link [http://www.mediawiki.org/wiki/Help:Formatting Wiki Formatting] as a reference for editing the wiki. This tutorial was developed collaboratively by the AMS 536 class of 2014, using DOCK v6.6.
+
For additional Rizzo Lab tutorials see [[DOCK Tutorials]]. Use this link [http://www.mediawiki.org/wiki/Help:Formatting Wiki Formatting] as a reference for editing the wiki. This tutorial was developed collaboratively by the AMS 536 class of 2016, using DOCK v6.8.
  
 
==I. Introduction==  
 
==I. Introduction==  
Line 13: Line 13:
  
 
===Beta Trypsin===
 
===Beta Trypsin===
−
Beta trypsin is a serine protease involved in degrading peptides and proteins. The development of inhibitors of trypsin-like serine proteases has been justified because of the wide processes these enzymes regulate, among them blood coagulation, complement activation, digestion, reproduction and phagocytosis. Early developments of inhibitors for these enzymes were based on the benzamidine chemical structure. Crystals structures of these benzamidine based inhibitors in complex with beta trypsin gave new insights of the interactions with the inhibitors and pointed out he importance of the "oxyanion hole" within the catalytic site. This gave new insights of the reaction mechanism of the enzyme and revealed what interactions are required to develop inhibitors with increased potency. [http://pubs.acs.org/doi/abs/10.1021/bi981636u]     
+
Beta trypsin is a serine protease involved in degrading peptides and proteins. The development of inhibitors of trypsin-like serine proteases has been justified because of the wide processes these enzymes regulate, among them blood coagulation, complement activation, digestion, reproduction and phagocytosis. Early developments of inhibitors for these enzymes were based on the benzamidine chemical structure. Crystal structures of these benzamidine based inhibitors in complex with beta trypsin gave showed the key resides interactions with the inhibitors and pointed out the importance of the "oxyanion hole" within the catalytic site. This gave new insights of the reaction mechanism of the enzyme and revealed what interactions are required to develop inhibitors with increased potency. [http://pubs.acs.org/doi/abs/10.1021/bi981636u]     
 
    
 
    
−
In this tutorial we will use PDB website 1BJU deposited crystal structure of betatrypsin in complex with inhibitor GP6.
+
In this tutorial we will use PDB website 1BJU deposited crystal structure of beta-trypsin in complex with inhibitor GP6.
−
   
+
   
 
[[Image:2016_1BJU_receptor_surface.png|thumb|center|375px| Beta trypsin in complex with GP6 inhibitor. Residues interacting with the inhibitor are shown in spheres.]]
 
[[Image:2016_1BJU_receptor_surface.png|thumb|center|375px| Beta trypsin in complex with GP6 inhibitor. Residues interacting with the inhibitor are shown in spheres.]]
  
 
===Organizing Directories===
 
===Organizing Directories===
−
<pre>While performing docking, it is convenient to adopt a standard directory  
+
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:
−
structure / naming scheme, so that files are easy to find / identify.
+
 
−
For this tutorial, we will use something similar to the following:</pre>
 
 
{|
 
{|
 
  |<pre>~username/AMS536-Spring2016/dock-tutorial/00.files/
 
  |<pre>~username/AMS536-Spring2016/dock-tutorial/00.files/
Line 34: Line 33:
 
                                         /08.print_fps</pre>
 
                                         /08.print_fps</pre>
 
   
 
   
−
|
+
|
−
|}
+
|}
−
 
+
In addition, most of the important files that are derived from the original crystal structure will be given a prefix that is thsame as the PDB code, '1BJU'.The following sections in this tutorial will adhere to  
−
<pre>In addition, most of the important files that are derived from  
+
this directory structure/naming scheme.
−
the original crystal structure will be given a prefix that is the
 
−
same as the PDB code, '1BJU'.The following sections in this tutorial  
 
−
will adhere to this directory structure/naming scheme.</pre>
 
  
 
==II. Preparing the Receptor and Ligand==   
 
==II. Preparing the Receptor and Ligand==   
Line 54: Line 50:
 
Using vi command to open 1BJU.pdb, then deleting bottom and top lines which do not have ATOM or ATPATM in its start.
 
Using vi command to open 1BJU.pdb, then deleting bottom and top lines which do not have ATOM or ATPATM in its start.
 
Then changing all ATPATM to ATOM using command:
 
Then changing all ATPATM to ATOM using command:
−
  %s /ATPATM/ATOM  /gc
+
  :%s /ATPATM/ATOM  /gc
 
In addition, changing all GP6 to LIG B using command:
 
In addition, changing all GP6 to LIG B using command:
−
  %s /GP6  /LIG B  /gc
+
  :%s /GP6  /LIG B  /gc
 
Saving this as raw_1BJU.pdb in 00.files.
 
Saving this as raw_1BJU.pdb in 00.files.
  
Line 100: Line 96:
 
To show the surface receptor:
 
To show the surface receptor:
 
   ''Action -> Surface -> Show'' (The surface should look like the picture shown below.)
 
   ''Action -> Surface -> Show'' (The surface should look like the picture shown below.)
−
[[File:noH_surface_1BJUAMS536.png]]
+
[[File:NoH surface 1BJUAM536.png|thumb|center|375px| Surface of the receptor Beta Trypsin without hydrogens as generated by Chimera.]]
−
To save a DMS file:
+
To save a DMS file as 1BJU.rec.dms in 02.surface-sphere directory:  
−
Then do ''Tools -> Structure editing -> Write DMS'', and save the file as 1BJU.rec.dms in 02.surface-sphere directory
+
  ''Tools -> Structure editing -> Write DMS''
  
  
 +
=== Creating Spheres ===
  
 +
Using the Sphgen program complete following steps:
  
 +
'''1.''' Create an input file name '''INSPH''' to be read by Sphgen:
  
−
=== Placing Spheres ===
+
vi INSPH
  
 +
1BJU.rec.dms #specifies the input file
 +
R            #spheres generated will be outside of teh receptor surface
 +
X            #specifies that all points won the receptor will be used
 +
0.0          #distance in angstroms (avoids steric clashes)
 +
4.0          #max surface radius of the spheres in angstroms
 +
1.4          #min surface radius of the spheres in angstroms
 +
1BJU.rec.sph #the specified outfile containing all generated spheres
  
 +
'''2.''' Run the Sphgen using the input file '''INSPH''' with the command:
  
−
==IV. Generating Box and Grid==  
+
  sphgen -i INSPH -o OUTSPH
−
Monaf Awwa
 
−
=== Box Generation===
 
−
>Start Chimera and open the RECEPTOR files 1BJU.rec.noH.pdb
 
−
Next, select the ACTION subheader, then select SURFACE, then SHOW
 
  
−
Once you can see the van der Waals surface of the protein, select the
+
'''-i''' is the flag that specifies the input file '''INSPH'''
−
TOOLS subheader, then STRUCTURE EDITING, and lastly select WRITE DMS and save the file as 1BJU.dms
+
'''INSPH''' is int eh specified input file
 +
'''-o''' is the flag to specifiy the output file
 +
'''OUTSPH''' is the file containing the generated spheres
  
−
Now that we have a surface, we can more effectively calculate regions of the protein where a small molecule can bind. To further accelerate calculations, we will specify which atoms to calculate the interactions for via the program sphgen
+
'''3.''' Visualization generated spheres with '''Chimera''':
  
−
Switch over to the 02.surfacespheres directory with the following command
+
* Launch '''Chimera''' in termal,
 +
* Choose ''File'' -> ''Open'', choose '''1BJU.rec.mol2'''
 +
* choose ''File'' -> ''Open'', choose '''1BJU.rec.sph'''
  
−
cd 02.surfacespheres
+
You should have an image like this:
  
−
Next, create an input file for sphgen to read.
+
[[Image:All_Spheres_1BJU.png|thumb|center|375px|1BJU Receptor surface with the generated surface spheres]]
  
−
vim INSPH
+
=== Selecting Spheres ===
  
−
The file should have the following information
+
To select spheres of interest run the program '''sphere_selector''' in the terminal at 8.0 angstrom radius of the target molecule or known binding site:
 +
  sphere_selector 1BJU.rec.sph ../01.dockprep/1BJU.lig.mol2 8.0
  
−
1BJU.rec.dms #the file corresponding to the surface is the input file
+
The output file generated will be '''selected_spheres.sph''' 
−
R            #we want the spheres outside the protein surface
 
−
X            #we want all subsets of spheres to be generated
 
−
0.0          #default size of proteins with surface contacts
 
−
4.0          #default maximum of the sphere radius generated (Angstroms)
 
−
1.4          #default minimum of the sphere radius generated (Angstroms) Also corresponds to probe radius
 
−
1BJU.rec.sph #output file name
 
  
−
Now we can enter the command to run sphgen
+
To visualize the spheres using '''Chimera''' as previously done:
  
−
sphgen -i INSPH -o OUTSPH
+
* Launch '''Chimera''', choose ''File'' -> ''Open'', choose '''1BJU.rec.noH.pdb'''
 +
* ''File'' -> ''Open'', choose '''output_spheres_selected.pdb'''
 +
* ''Select'' -> ''Residue'' -> ''SPH''
 +
* ''Actions'' -> ''Atoms/Bonds'' -> ''sphere''
  
−
Once completed, check the OUTSPH file to ensure no errors occured
+
The selected spheres with the receptor surface should look similar to that as seen below:
  
−
If you'd like to visually inspect the spheres generated, start Chimera and load the 1BJU.rec.mol2 file, then load the 1BJU.rec.sph file.
+
[[Image:Selected_spheres.png|thumb|center|375px|1BJU receptor surface with the selected spheres within 8.0 Angstroms]]
  
−
Now we will use the showsphere program to convert our .sph file back to a .pdb format.
+
==IV. Generating Box and Grid==
−
 
+
Monaf Awwa
−
In the same directory, enter the command
+
=== Box Generation===
−
 
 
−
showsphere
 
−
 
 
−
The following questions will appear. Answer as follows
 
−
 
 
−
Enter name of sphere cluster file:
 
−
    1BJU.rec.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 should now have a file named output_spheres.pdb in your directory. You can visualize the spheres by loading chimera and the 1BJU.rec.noH.mol2, then loading the output_spheres.pdb file.
 
−
 
 
−
For the docking calculation, we are most interesting in a subset of the spheres. To select the spheres most important to ligand binding, we will use the command
 
−
 
 
−
sphere_selector 1BJU.rec.sph ../01.dockprep/1BJU.lig.mol2 8.0
 
−
 
 
−
The 8.0 Angstrom value indicates we want to select spheres within 8 angstroms of our ligand. This hypothetically means we are designing a competitive inhibitor.
 
−
 
 
−
Now lets create the proper output file for our spheres. Enter the command
 
−
 
 
−
showsphere
 
−
 
 
−
and answer the following questions
 
−
 
 
−
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
 
−
 
 
−
This will ensure the docking calculation samples the most important regions of the protein surface
 
−
 
 
−
=== Grid computing===
 
 
Since the electronic interactions between two objects in space can be decomposed into pairwise interactions, it is highly efficient to fully calculate a proteins electric contribution to binding energy before DOCKing a ligand.
 
Since the electronic interactions between two objects in space can be decomposed into pairwise interactions, it is highly efficient to fully calculate a proteins electric contribution to binding energy before DOCKing a ligand.
−
 
 
switch to the 03.box-grid/ directory
 
switch to the 03.box-grid/ directory
−
 
+
  cd 03.box-grid/
−
cd 03.box-grid/
 
−
 
 
 
create an input file named showbox.in
 
create an input file named showbox.in
−
 
+
 
−
vim showbox.in
+
  vim showbox.in
−
 
 
 
enter the following information
 
enter the following information
−
 
+
  Y                                              # Do you want to calculate a box? (YES)
−
  Y                                              # Do you want to calculate a box? (YES)
+
  8.0                                            # How long should the box length be?
−
  8.0                                            # How long should the box length be?
+
  ../02.surface-spheres/selected_spheres.sph      # which sphere file is our input?
−
  ../02.surface-spheres/selected_spheres.sph      # which sphere file is our input?
+
  1                                              # How many clusters for the file?
−
  1                                              # How many clusters for the file?
+
  1BJU.box.pdb                                    # What do we want to name the output file?
−
  1BJU.box.pdb                                    # What do we want to name the output file?
 
−
 
 
 
save the file, then enter the following command to generate the box
 
save the file, then enter the following command to generate the box
 +
    showbox < showbox.in
 +
The box can be visualized in Chimera by loading the output_spheres_selected.pdb file, then loading the 1BJU.box.pdb file
  
−
showbox > showbox.in
+
=== Grid computing===
−
 
 
−
The box can be visualized in Chimera by loading the output_spheres_selected.pdb file, then loading the 1BJU.box.pdb file
 
  
 
Next, we will calculate the grid energies
 
Next, we will calculate the grid energies
Line 224: Line 179:
 
Create the input file for grid. Enter the command
 
Create the input file for grid. Enter the command
  
−
vim grid.in
+
  vim grid.in
  
 
Run grid with the command
 
Run grid with the command
  
−
grid - grid.in
+
  grid -i grid.in
  
 
This allows you to go back and fix the input file in case there is a mistake when answer the questions prompted by grid.
 
This allows you to go back and fix the input file in case there is a mistake when answer the questions prompted by grid.
  
 +
    grid.in
  
−
grid.in
+
  Parameter         Value Description
−
Parameter         Value Description
 
  
−
compute_grids            yes we want to compute grids
+
  compute_grids            yes we want to compute grids
  
−
grid_spacing         0.4 resolution of grid energetic contributions (Angstroms). The smaller the value, the more accurate the calculation
+
  grid_spacing         0.4 resolution of grid energetic contributions (Angstroms). The smaller the value, the more accurate the calculation
  
−
output_molecule         no we don't want to rewrite ligand output file
+
  output_molecule         no we don't want to rewrite ligand output file
  
−
contact_score         no we don't want to deal with contact score
+
  contact_score         no we don't want to deal with contact score
  
−
energy_score         yes we want to compute energetic interaction based on force field
+
  energy_score         yes we want to compute energetic interaction based on force field
  
−
energy_cutoff_distance 9999 what distance should we stop computing atomic interactions in angstroms
+
  energy_cutoff_distance 9999 what distance should we stop computing atomic interactions in angstroms
  
−
atom_model         a corresponds to all atom model rather than united atom
+
  atom_model         a corresponds to all atom model rather than united atom
  
−
attractive_exponent 6 see Lennard-Jones attraction term
+
  attractive_exponent 6 see Lennard-Jones attraction term
  
−
repulsive_exponent 12 see Lennard-Jones repulsion term
+
  repulsive_exponent 12 see Lennard-Jones repulsion term
  
−
distance_dielectric yes linear decrease in electric force
+
  distance_dielectric yes linear decrease in electric force
  
−
dielectric_factor 4 how much will electric force decrease based on atomic distance in angstroms
+
  dielectric_factor 4 how much will electric force decrease based on atomic distance in angstroms
  
−
bump_filter         yes
+
  bump_filter         yes
  
−
bump_overlap         0.75
+
  bump_overlap         0.75
  
−
receptor_file         ../01.dockprep/1BJU.rec.mol2
+
  receptor_file         ../01.dockprep/1BJU.rec.mol2
  
−
box_file         4TKG.box.pdb
+
  box_file         4TKG.box.pdb
  
−
vdw_definition_file ../zzz.parameters/vdw_AMBER_parm99.defn characteristic van der Waal radii parameter file
+
  vdw_definition_file ../zzz.parameters/vdw_AMBER_parm99.defn characteristic van der Waal radii parameter file
  
−
score_grid_prefix grid ONLY THE PREFIX OF THE GRID FILE NAME
+
  score_grid_prefix grid ONLY THE PREFIX OF THE GRID FILE NAME
  
 
==V. Docking a Single Molecule for Pose Reproduction==   
 
==V. Docking a Single Molecule for Pose Reproduction==   
−
Agatha Lyczek & Haoyue Guo
 
 
===Minimization===
 
===Minimization===
 +
One can use the DOCK software to perform a energy minimization of a ligand in the receptor's binding site.
 +
 +
Create an empty file (min.in) and run dock6:
 +
touch min.in
 +
dock6 -i min.in
 +
 +
The dock program will start and it will begin to ask you a series of questions. The answers will be saved to the min.in file, so next time you don't have to go through all the questions again. You can just edit the min.in file directly.
 +
 
Dock Minimization Input file: min.in
 
Dock Minimization Input file: min.in
 
  conformer_search_type                                        rigid
 
  conformer_search_type                                        rigid
Line 326: Line 288:
 
  num_scored_conformers                                        1
 
  num_scored_conformers                                        1
 
  rank_ligands                                                no
 
  rank_ligands                                                no
−
Result:
+
 
 +
Note that "conformer_search_type" is set to "rigid".
 +
 
 +
Note that "simplex_restraint_min" is set to "yes". This is a flag to perform RMSD tethering, which restrains the conformer sampling to not move too far away from the initial docking site.
 +
 
 +
The output file prefix is "1BJU.min". The output file "1BJU.min_scored.mol2" is saved in the directory where you ran dock6 (04.dock). This file contains the predicted pose coordinates, internal energy, grid score, and RMSD values.
 +
 
 +
To view your results, you can open the 1BJU.min_scored.mol2 file in CHIMERA and compare it to your original ligand file in the 01.dockprep directory.
 +
 
 +
Minimization result:
 
[[Image:Min.png|thumb|center|400px|Minimization result;RMSD = 0.3177; blue: experiment; green: predicted]]
 
[[Image:Min.png|thumb|center|400px|Minimization result;RMSD = 0.3177; blue: experiment; green: predicted]]
 +
 +
The energy minimized pose has an RMSD of 0.3177, meaning that it was minimally moved from it's original experimental ligand pose (1BJU.lig.mol2).
  
 
===Rigid docking===
 
===Rigid docking===
−
To run the input file:
+
Rigid Ligand Docking is typically used to check the validity of docking program, user-defined parameters, and help identify any mistakes in the receptor and ligand prep files. Here, we dock the ligand back to the ligand-free receptor. The pose predicted by DOCK should be very close to the crystal structure of the ligand-receptor complex.
−
  dock6 -i rigid_nr.in
+
 
−
Rigid docking input file: rigid_nr.in
+
The following should be performed in the 04.dock directory.
 +
 
 +
Create an empty file (rigid_r.in) and run DOCK6:
 +
 
 +
touch rigid_r.in
 +
  dock6 -i rigid_r.in
 +
Rigid docking input file: rigid_r.in
 
  conformer_search_type                                        rigid
 
  conformer_search_type                                        rigid
 
  use_internal_energy                                          yes
 
  use_internal_energy                                          yes
Line 382: Line 361:
 
  simplex_random_seed                                          0
 
  simplex_random_seed                                          0
 
  simplex_restraint_min                                        yes
 
  simplex_restraint_min                                        yes
 +
simplex_coefficient_restraint                                10.0
 
  atom_model                                                  all
 
  atom_model                                                  all
 
  vdw_defn_file                                                ../zzz.parameters/vdw.defn
 
  vdw_defn_file                                                ../zzz.parameters/vdw.defn
 
  flex_defn_file                                              ../zzz.parameters/flex.defn
 
  flex_defn_file                                              ../zzz.parameters/flex.defn
 
  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
 
  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
−
  ligand_outfile_prefix                                        1BJU.rigid_nr
+
  ligand_outfile_prefix                                        1BJU.rigid_restraint
 
  write_orientations                                          no
 
  write_orientations                                          no
 +
num_scored_conformers                                        40
 +
write_conformations                                          no
 +
cluster_conformations                                        yes
 +
cluster_rmsd_threshold                                      2.0
 +
rank_ligands                                                no
 +
 +
Note that "conformer_search_type" is set to "rigid".
 +
 +
Note that simplex_restraint_min is set to "yes". This is a flag to perform RMSD tethering, which restrains the conformer sampling to not move too far away from the initial docking site.
 +
 +
The output  file prefix is "1BJU.rigid_restraint". The output file "1BJU.rigid_restraint_scored.mol2" is saved in the directory where you ran dock6 (04.dock). This file contains the ligand coordinates, energy score, and RMSD values.
 +
 +
To view your results, you can open the pdb files of the receptor and ligand in CHIMERA from your 01.dockprep directory. The open your rigid docking results using viewdock:
 +
Tools > surface/binding analysis > viewdock > 1BJU.rigid_restraint_scored.mol2.
 +
 +
You can rank the conformers based on internal energy, grid score, or RMSD.
 +
 
Best scored rigid docking result:
 
Best scored rigid docking result:
 
[[Image:Rigid.png|thumb|center|400px|Rigid docking result;RMSD = 0.7946; blue: experiment; pink: predicted]]
 
[[Image:Rigid.png|thumb|center|400px|Rigid docking result;RMSD = 0.7946; blue: experiment; pink: predicted]]
 +
 +
The predicted pose and the experimental pose have an RMSD less than 2.000,  meaning that we were able to reproduce the experimental ligand pose (crystal structure).
  
 
===Flexible Docking===
 
===Flexible Docking===
−
To run the input file:
+
Flexible docking samples internal degrees of freedom.
 +
 
 +
The following should be performed in the 04.dock directory.
 +
 
 +
Create an empty file (flex.in) and run DOCK6:
 +
touch flex.in
 
  dock6 -i flex.in
 
  dock6 -i flex.in
 
Flexible docking input file: flex.in
 
Flexible docking input file: flex.in
Line 450: Line 454:
 
  simplex_cycle_converge                                      1.0
 
  simplex_cycle_converge                                      1.0
 
  simplex_trans_step                                          1.0
 
  simplex_trans_step                                          1.0
 +
simplex_rot_step                                            0.1
 +
simplex_tors_step                                            10.0
 +
simplex_anchor_max_iterations                                500
 +
simplex_grow_max_iterations                                  500
 +
simplex_grow_tors_premin_iterations                          0
 +
simplex_random_seed                                          0
 +
simplex_restraint_min                                        no
 +
atom_model                                                  all
 +
vdw_defn_file                                                ../zzz.parameters/vdw.defn
 +
flex_defn_file                                              ../zzz.parameters/flex.defn
 +
flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
 +
ligand_outfile_prefix                                        1BJU.flex
 +
write_orientations                                          no
 +
num_scored_conformers                                        100
 +
write_conformations                                          no
 +
cluster_conformations                                        yes
 +
cluster_rmsd_threshold                                      2.0
 +
rank_ligands                                                no
 +
 +
Note that "conformer_search_type" is set to "flex".
 +
 +
The output file prefix is "1BJU.flex" and the output file is saved as "1BJU.flex_scored.mol2" in the 04.dock directory. The file "1BJU.flex_scored.mol2" contains the ligand coordinates, energy score, and RMSD values.
 +
To view your results, you can open the pdb files of the receptor and ligand in CHIMERA from your 01.dockprep directory. Then open your flexible docking results using viewdock:
 +
Tools > surface/binding analysis > viewdock > 1BJU.flex_scored.mol2.
 +
You can rank the conformers based on internal energy, grid score, or RMSD.
 +
 
Best scored flexible docking result:
 
Best scored flexible docking result:
 
[[Image:Flex.png|thumb|center|400px|Flexible docking result;RMSD = 0.45; blue: experiment; pink: predicted]]
 
[[Image:Flex.png|thumb|center|400px|Flexible docking result;RMSD = 0.45; blue: experiment; pink: predicted]]
 +
 +
The predicted pose and the experimental pose have an RMSD less than 2.000, meaning that we were able to reproduce the experimental ligand pose (crystal structure). If your top scored conformer has a high RMSD, this is considered a DOCKing failure. In the event of a DOCKing failure, consider setting "simplex_random_seed" to a different value. This is a random number generator that determines the starting point for conformer sampling. Alternatively, you could increase sampling.
  
 
==VI. Virtual Screening==   
 
==VI. Virtual Screening==   
−
Katie Maffucci
+
'''Katie Maffucci'''
 +
 
 +
Virtual screening uses libraries of small molecules to identify novel chemical structures that are most likely to bind a drug target. The search is narrowed to a few "hit" compounds that can be synthesized or purchased and tested against the target. Structure-based virtual screening is computationally demanding, and requires a parallel computing infrastructure to handle the task. LIRED is a Linux-based computing cluster well suited for this purpose.
  
 
===DOCK Preparation===
 
===DOCK Preparation===
  
 +
To set up the virtual screen, make a directory using the command: "mkdir 05.largevirtualscreen" Print your working directory using the "pwd" command and copy this dock directory into your LIRED account: "scp -r /gfps/home/username/AMS536/docktutorial/05.largevirtualscreen ."
 +
 +
== QSUB File ==
 +
To submit your job to the cluster, a qsub c chell script must be made so that the job can be executed. Its contents should be as follows:
 +
 +
'''
 +
    #! /bin/tcsh
 +
    #PBS -l nodes=2:ppn=2
 +
    #PBS -l walltime=24:00:00
 +
    #PBS -o zzz.qsub.out
 +
    #PBS -e zzz.qsub.err
 +
    #PBS -N large_vs
 +
    #PBS -M your.name@stonybrook.edu
 +
    #PBS -V
 +
cd /gfps/home/username/AMS536/docktutorial/ /gpfs/software/intel_2016_update1/impi/5.1.2.150/bin64/mpirun np 4 \ /home/tmcgee/local/AMS_536/dock6/bin/dock6.mpi \ '''
 +
 +
Where -l = resources required for the job, -o = defines output,-e = defines error, ppn = processors per node, -N = name of job, walltime = how long the job can run, and -M = to set up an email alert when the job is complete
 +
 +
== Input File ==
 +
 +
An input file must also be made. "vi large_vs.in":
 +
 +
'''
 +
 +
    ligand_atom_file                                            yourligandmol2file.mol2 <br>
 +
    limit_max_ligands                                            no <br>
 +
    skip_molecule                                                no <br>
 +
    read_mol_solvation                                          no <br>
 +
    calculate_rmsd                                              no<br>
 +
    use_database_filter                                          no<br>
 +
    orient_ligand                                                yes<br>
 +
    automated_matching                                          yes<br>
 +
    receptor_site_file                                          ../02.surface-spheres/selected_spheres.sph<br>
 +
    max_orientations                                            1000<br>
 +
    critical_points                                              no<br>
 +
    chemical_matching                                            no<br>
 +
    use_ligand_spheres                                          no<br>
 +
    use_internal_energy                                          yes<br>
 +
    internal_energy_rep_exp                                      12<br>
 +
    flexible_ligand                                              yes<br>
 +
    user_specified_anchor                                        no<br>
 +
    limit_max_anchors                                            no<br>
 +
    min_anchor_size                                              5<br>
 +
    pruning_use_clustering                                      yes<br>
 +
    pruning_max_orients                                          1000<br>
 +
    pruning_clustering_cutoff                                    100<br>
 +
    pruning_conformer_score_cutoff                              100<br>
 +
    use_clash_overlap                                            no<br>
 +
    write_growth_tree                                            no<br>
 +
    bump_filter                                                  no<br>
 +
    score_molecules                                              yes<br>
 +
    contact_score_primary                                        no<br>
 +
    contact_score_secondary                                      no<br>
 +
    grid_score_primary                                          yes<br>
 +
    grid_score_secondary                                        no<br>
 +
    grid_score_rep_rad_scale                                    1<br>
 +
    grid_score_vdw_scale                                        1<br>
 +
    grid_score_es_scale                                          1<br>
 +
    grid_score_grid_prefix                                      ../03.box-grid/grid<br>
 +
    multigrid_score_secondary                                    no<br>
 +
    dock3.5_score_secondary                                      no<br>
 +
    continuous_score_secondary                                  no<br>
 +
    descriptor_score_secondary                                  no<br>
 +
    gbsa_zou_score_secondary                                    no<br>
 +
    gbsa_hawkins_score_secondary                                no<br>
 +
    SASA_descriptor_score_secondary                              no<br>
 +
    amber_score_secondary                                        no<br>
 +
    minimize_ligand                                              yes<br>
 +
    minimize_anchor                                              yes<br>
 +
    minimize_flexible_growth                                    yes<br>
 +
    use_advanced_simplex_parameters                              no<br>
 +
    simplex_max_cycles                                          1<br>
 +
    simplex_score_converge                                      0.1<br>
 +
    simplex_cycle_converge                                      1.0<br>
 +
    simplex_trans_step                                          1.0<br>
 +
    simplex_rot_step                                            0.1<br>
 +
    simplex_tors_step                                            10.0<br>
 +
    simplex_anchor_max_iterations                                500<br>
 +
    simplex_grow_max_iterations                                  500<br>
 +
    simplex_grow_tors_premin_iterations                          0<br>
 +
    simplex_random_seed                                          0<br>
 +
    simplex_restraint_min                                        no<br>
 +
    atom_model                                                  all<br>
 +
    vdw_defn_file                                                ../docktutorial/zzz.parameters/vdw_AMBER_parm99.defn<br>
 +
    flex_defn_file                                              ../docktutorial/zzz.parameters/flex.defn<br>
 +
    flex_drive_file                                              ../docktutorial/zzz.parameters/flex_drive.tbl<br>
 +
    ligand_outfile_prefix                                        large_vs<br>
 +
    write_orientations                                          no<br>
 +
    num_scored_conformers                                        1<br>
 +
    rank_ligands                                                no<br>
 +
'''
 +
 +
==Post Processing==
 +
 +
After the virtual screen, the top hits and poses will be rank ordered according to the scoring function(s) chosen in previous input files. Depending on the size of the database, the top 1% of molecules can still number in the hundreds. To further refine the screen, cartesian minimization should be performed. To do this, create an input file 1BJU.cart_min.ligs.in:
 +
 +
'''
 +
 +
  conformer_search_type                                        rigid <br>
 +
  use_internal_energy                                          yes <br>
 +
  internal_energy_rep_exp                                      12 <br>
 +
  ligand_atom_file                                            ../06.large-virtual-screen/1BJU.output_scored.mol2 <br>
 +
  limit_max_ligands                                            no <br>
 +
  skip_molecule                                                no <br>
 +
  read_mol_solvation                                          no<br>
 +
  calculate_rmsd                                              no<br>
 +
  use_database_filter                                          no<br>
 +
  orient_ligand                                                no<br>
 +
  automated_matching                                          no<br>
 +
  critical_points                                              no<br>
 +
  chemical_matching                                            no<br>
 +
  use_ligand_spheres                                          no<br>
 +
  bump_filter                                                  no<br>
 +
  score_molecules                                              yes<br>
 +
  contact_score_primary                                        no<br>
 +
  contact_score_secondary                                      no<br>
 +
  grid_score_primary                                          no<br>
 +
  grid_score_secondary                                        no<br>
 +
  multigrid_score_primary                                      no<br>
 +
  multigrid_score_secondary                                    no<br>
 +
  dock3.5_score_primary                                        no<br>
 +
  dock3.5_score_secondary                                      no<br>
 +
  continuous_score_primary                                    yes<br>
 +
  continuous_score_secondary                                  no<br>
 +
  cont_score_rec_filename                                      ../01.dockprep/1BJU.rec.mol2<br>
 +
  cont_score_att_exp                                          6<br>
 +
  cont_score_rep_exp                                          12<br>
 +
  cont_score_rep_rad_scale                                    1<br>
 +
  cont_score_use_dist_dep_dielectric                          yes<br>
 +
  cont_score_dielectric                                        4.0<br>
 +
  cont_score_vdw_scale                                        1<br>
 +
  cont_score_es_scale                                          1<br>
 +
  footprint_similarity_score_secondary                        no<br>
 +
  ph4_score_secondary                                          no<br>
 +
  descriptor_score_secondary                                  no<br>
 +
  gbsa_zou_score_secondary                                    no<br>
 +
  gbsa_hawkins_score_secondary                                no<br>
 +
  SASA_descriptor_score_secondary                              no<br>
 +
  amber_score_secondary                                        no<br>
 +
  minimize_ligand                                              yes<br>
 +
  simplex_max_iterations                                      1000<br>
 +
  simplex_tors_premin_iterations                              0<br>
 +
  simplex_max_cycles                                          1<br>
 +
  simplex_score_converge                                      0.1<br>
 +
  simplex_cycle_converge                                      1.0<br>
 +
  simplex_trans_step                                          1.0<br>
 +
  simplex_rot_step                                            0.1<br>
 +
  simplex_tors_step                                            10.0<br>
 +
  simplex_random_seed                                          0<br>
 +
  simplex_restraint_min                                        yes<br>
 +
  simplex_coefficient_restraint                                10.0<br>
 +
  atom_model                                                  all<br>
 +
  vdw_defn_file                                                ../zzz.parameters/vdw.defn<br>
 +
  flex_defn_file                                              ../zzz.parameters/flex.defn<br>
 +
  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl<br>
 +
  ligand_outfile_prefix                                        1BJU.cartmin<br>
 +
  write_orientations                                          no<br>
 +
  num_scored_conformers                                        1<br>
 +
  rank_ligands                                                no<br>
 +
 +
'''
  
−
===Post Processing===
+
You'll need to make another minimization input file with similar contents:
 +
 
 +
'''
 +
  conformer_search_type                                        rigid <br>
 +
  use_internal_energy                                          yes<br>
 +
  internal_energy_rep_exp                                      12<br>
 +
  ligand_atom_file                                            ../01.dockprep/1BJU.lig.mol2<br>
 +
  limit_max_ligands                                            no<br>
 +
  skip_molecule                                                no<br>
 +
  read_mol_solvation                                          no<br>
 +
  calculate_rmsd                                              no<br>
 +
  use_database_filter                                          no<br>
 +
  orient_ligand                                                no<br>
 +
  bump_filter                                                  no<br>
 +
  score_molecules                                              yes<br>
 +
  contact_score_primary                                        no<br>
 +
  contact_score_secondary                                      no<br>
 +
  grid_score_primary                                          no<br>
 +
  grid_score_secondary                                        no<br>
 +
  multigrid_score_primary                                      no<br>
 +
  multigrid_score_secondary                                    no<br>
 +
  dock3.5_score_primary                                        no<br>
 +
  dock3.5_score_secondary                                      no<br>
 +
  continuous_score_primary                                    yes<br>
 +
  continuous_score_secondary                                  no<br>
 +
  cont_score_rec_filename                                      ../01.dockprep/1BJU.rec.mol2<br>
 +
  cont_score_att_exp                                          6<br>
 +
  cont_score_rep_exp                                          12<br>
 +
  cont_score_rep_rad_scale                                    1<br>
 +
  cont_score_use_dist_dep_dielectric                          yes<br>
 +
  cont_score_dielectric                                        4.0<br>
 +
  cont_score_vdw_scale                                        1<br>
 +
  cont_score_es_scale                                          1<br>
 +
  footprint_similarity_score_secondary                        no<br>
 +
  ph4_score_secondary                                          no<br>
 +
  descriptor_score_secondary                                  no<br>
 +
  gbsa_zou_score_secondary                                    no<br>
 +
  gbsa_hawkins_score_secondary                                no<br>
 +
  SASA_descriptor_score_secondary                              no<br>
 +
  amber_score_secondary                                        no<br>
 +
  minimize_ligand                                              yes<br>
 +
  simplex_max_iterations                                      1000<br>
 +
  simplex_tors_premin_iterations                              0<br>
 +
  simplex_max_cycles                                          1<br>
 +
  simplex_score_converge                                      0.1<br>
 +
  simplex_cycle_converge                                      1.0<br>
 +
  simplex_trans_step                                          1.0<br>
 +
  simplex_rot_step                                            0.1<br>
 +
  simplex_tors_step                                            10.0<br>
 +
  simplex_random_seed                                          0<br>
 +
  simplex_restraint_min                                        yes<br>
 +
  simplex_coefficient_restraint                                10.0<br>
 +
  atom_model                                                  all<br>
 +
  vdw_defn_file                                                ../zzz.parameters/vdw.defn<br>
 +
  flex_defn_file                                              ../zzz.parameters/flex.defn<br>
 +
  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl<br>
 +
  ligand_outfile_prefix                                        1BJU.cartmin_ref<br>
 +
  write_orientations                                          no<br>
 +
  num_scored_conformers                                        1<br>
 +
  rank_ligands                                                no<br>
 +
 
 +
'''
 +
 
 +
To execute the job, you'll need to make a cartminligs.qsub.tcsh script similar to the previous one:
 +
 
 +
'''
 +
    #!/bin/tcsh
 +
    #PBS -l walltime=09:00:00
 +
    #PBS -l nodes=1:ppn=24
 +
    #PBS -q medium
 +
    #PBS -N 1BJU
 +
    #PBS -V
 +
    #PBS -M youremail@stonybrook.edu
 +
    #PBS -j oe
 +
    cd /gpfs/home/username/AMS536/docktutorial/07.cartesian_min
 +
    /gpfs/software/intel_2016_update1/impi/5.1.2.150/bin64/mpirun -np 24 \
 +
  /home/tmcgee/local/AMS_536/dock6/bin/dock6.mpi \
 +
  -i 1BJU.cart_min_ligs.in \
 +
  -o 1BJU.cart_min_ligs.out
 +
 
 +
'''
  
 
==VIII. Frequently Encountered Problems==
 
==VIII. Frequently Encountered Problems==
 
EVERYONE
 
EVERYONE
 +
 +
You may see that your vdw scores are zero - this is highly unlikely to actually be the case. If you are using an older version of Chimera and used the "dockprep" button to add charges and hydrogens to your receptor, the charges were likely not saved. You must add the charges and hydrogens manually.
 +
 +
When submitting the qsub script, make sure that there are no spaces after the " \ " on the mpirun line - the job will not run in parallel if there are spaces after the backslash.

Latest revision as of 16:53, 30 January 2017

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 2016, using DOCK v6.8.

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.

Beta Trypsin

Beta trypsin is a serine protease involved in degrading peptides and proteins. The development of inhibitors of trypsin-like serine proteases has been justified because of the wide processes these enzymes regulate, among them blood coagulation, complement activation, digestion, reproduction and phagocytosis. Early developments of inhibitors for these enzymes were based on the benzamidine chemical structure. Crystal structures of these benzamidine based inhibitors in complex with beta trypsin gave showed the key resides interactions with the inhibitors and pointed out the importance of the "oxyanion hole" within the catalytic site. This gave new insights of the reaction mechanism of the enzyme and revealed what interactions are required to develop inhibitors with increased potency. [1]

In this tutorial we will use PDB website 1BJU deposited crystal structure of beta-trypsin in complex with inhibitor GP6.

Beta trypsin in complex with GP6 inhibitor. Residues interacting with the inhibitor are shown in spheres.

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-Spring2016/dock-tutorial/00.files/
                                         /01.dockprep/
                                         /02.surface-spheres/
                                         /03.box-grid/
                                         /04.dock/
                                         /05.large-virtual-screen/
                                         /06.virtual-screen/
                                         /07.footprint/
                                         /08.print_fps

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

II. Preparing the Receptor and Ligand

Download the PDB File (1BJU)

Go to the PDB website to download 1BJU.pdb file(PDB code: 1BJU). Using command or WinSCP to transfer 1BJU.pdb to 00.files. If you are in 00.files now, the command is: 

cp ~/Downloads/1BJU.pdb .

Edit the PDB File (1BJU)

Using vi command to open 1BJU.pdb, then deleting bottom and top lines which do not have ATOM or ATPATM in its start. Then changing all ATPATM to ATOM using command: 

:%s /ATPATM/ATOM  /gc

In addition, changing all GP6 to LIG B using command: 

:%s /GP6  /LIG B  /gc

Saving this as raw_1BJU.pdb in 00.files.

Prepare ligand and receptor files for dock

Create dockprep file

Open raw_1BJU.pdb file in chimera, in Tools, find structure editing, then click AddH to add hydrogen in this system.

Also in structure editing, click Add charge, then, changing AMBER ff14SB to AMBER ff99SB and changing net charge to +1.

Saving it as 1BJU.dockprep.mol2 in 01.dockprep.

Create receptor file

Open 1BJU.dockprep.mol2 in chimera, then click select to choose ligand, click action in Atoms/Bonds click delete.

Saving this molecule as 1BJU.rec.mol2 in 01.dockprep.

Create ligand file

Open 1BJU.dockprep.mol2 in chimera, using the same method delete the protein molecule.

Saving it as 1BJU.lig.mol2 in 01.dockprep.

Create no hydrogen receptor file

Open 1BJU.rec.mol2 in chimera, click select, then choosing chemistry H to select hydrogen in protein, Using action button to delete H.

Saving it as 1BJU.rec.noH.pdb in 01.dockprep.

III. Generating Receptor Surface and Spheres

Generating the Receptor Surface

Create the directory where the following steps will be done: 

  mkdir 02.surface-sphere
  cd 02.surface-sphere

Open Chimera in the terminal to view the receptor surface: 

 Chimera

In Chimera complete the following steps to open the receptor file in the 01.dockprep directory: 

  File -> Open -> 1BJU.rec.noH.pdb

To show the surface receptor: 

  Action -> Surface -> Show (The surface should look like the picture shown below.)
Surface of the receptor Beta Trypsin without hydrogens as generated by Chimera.

To save a DMS file as 1BJU.rec.dms in 02.surface-sphere directory: 

 Tools -> Structure editing -> Write DMS


Creating Spheres

Using the Sphgen program complete following steps:

1. Create an input file name INSPH to be read by Sphgen: 

vi INSPH

1BJU.rec.dms #specifies the input file
R            #spheres generated will be outside of teh receptor surface 
X            #specifies that all points won the receptor will be used
0.0          #distance in angstroms (avoids steric clashes)
4.0          #max surface radius of the spheres in angstroms
1.4          #min surface radius of the spheres in angstroms
1BJU.rec.sph #the specified outfile containing all generated spheres

2. Run the Sphgen using the input file INSPH with the command: 

sphgen -i INSPH -o OUTSPH

-i is the flag that specifies the input file INSPH
INSPH is int eh specified input file
-o is the flag to specifiy the output file
OUTSPH is the file containing the generated spheres

3. Visualization generated spheres with Chimera:

  • Launch Chimera in termal,
  • Choose File -> Open, choose 1BJU.rec.mol2
  • choose File -> Open, choose 1BJU.rec.sph

You should have an image like this:

1BJU Receptor surface with the generated surface spheres

Selecting Spheres

To select spheres of interest run the program sphere_selector in the terminal at 8.0 angstrom radius of the target molecule or known binding site: 

  sphere_selector 1BJU.rec.sph ../01.dockprep/1BJU.lig.mol2 8.0

The output file generated will be selected_spheres.sph

To visualize the spheres using Chimera as previously done:

  • Launch Chimera, choose File -> Open, choose 1BJU.rec.noH.pdb
  • File -> Open, choose output_spheres_selected.pdb
  • Select -> Residue -> SPH
  • Actions -> Atoms/Bonds -> sphere

The selected spheres with the receptor surface should look similar to that as seen below:

1BJU receptor surface with the selected spheres within 8.0 Angstroms

IV. Generating Box and Grid

Monaf Awwa

Box Generation

Since the electronic interactions between two objects in space can be decomposed into pairwise interactions, it is highly efficient to fully calculate a proteins electric contribution to binding energy before DOCKing a ligand. switch to the 03.box-grid/ directory 

  cd 03.box-grid/

create an input file named showbox.in 

  vim showbox.in

enter the following information 

 Y                                               # Do you want to calculate a box? (YES)
 8.0                                             # How long should the box length be?
 ../02.surface-spheres/selected_spheres.sph      # which sphere file is our input?
 1                                               # How many clusters for the file?
 1BJU.box.pdb                                    # What do we want to name the output file?

save the file, then enter the following command to generate the box 

   showbox < showbox.in

The box can be visualized in Chimera by loading the output_spheres_selected.pdb file, then loading the 1BJU.box.pdb file

Grid computing

Next, we will calculate the grid energies

Create the input file for grid. Enter the command 

  vim grid.in

Run grid with the command 

  grid -i grid.in

This allows you to go back and fix the input file in case there is a mistake when answer the questions prompted by grid. 

   grid.in

 Parameter	        Value	Description

 compute_grids            yes	we want to compute grids

  grid_spacing	         0.4	resolution of grid energetic contributions (Angstroms). The smaller the value, the more accurate the calculation

  output_molecule	         no	we don't want to rewrite ligand output file

  contact_score	         no	we don't want to deal with contact score

  energy_score	         yes	we want to compute energetic interaction based on force field

  energy_cutoff_distance	 9999	what distance should we stop computing atomic interactions in angstroms

  atom_model	         a	corresponds to all atom model rather than united atom

  attractive_exponent	 6	see Lennard-Jones attraction term

  repulsive_exponent	 12	see Lennard-Jones repulsion term

  distance_dielectric	 yes	linear decrease in electric force

  dielectric_factor	 4	how much will electric force decrease based on atomic distance in angstroms

  bump_filter	         yes	

  bump_overlap	         0.75	

  receptor_file	        ../01.dockprep/1BJU.rec.mol2

  box_file	         4TKG.box.pdb

  vdw_definition_file	../zzz.parameters/vdw_AMBER_parm99.defn	characteristic van der Waal radii parameter file

  score_grid_prefix	grid	ONLY THE PREFIX OF THE GRID FILE NAME

V. Docking a Single Molecule for Pose Reproduction

Minimization

One can use the DOCK software to perform a energy minimization of a ligand in the receptor's binding site.

Create an empty file (min.in) and run dock6: 

touch min.in
dock6 -i min.in

The dock program will start and it will begin to ask you a series of questions. The answers will be saved to the min.in file, so next time you don't have to go through all the questions again. You can just edit the min.in file directly.

Dock Minimization Input file: min.in 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
ligand_atom_file                                             ../01.dockprep/1BJU.lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01.dockprep/1BJU.lig.mol2
use_database_filter                                          no
orient_ligand                                                no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           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/1BJU.grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
ph4_score_secondary                                          no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_score_secondary                              no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        yes
simplex_coefficient_restraint                                10.0
atom_model                                                   all
vdw_defn_file                                                ../zzz.parameters/vdw.defn
flex_defn_file                                               ../zzz.parameters/flex.defn
flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
ligand_outfile_prefix                                        1BJU.min
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Note that "conformer_search_type" is set to "rigid".

Note that "simplex_restraint_min" is set to "yes". This is a flag to perform RMSD tethering, which restrains the conformer sampling to not move too far away from the initial docking site.

The output file prefix is "1BJU.min". The output file "1BJU.min_scored.mol2" is saved in the directory where you ran dock6 (04.dock). This file contains the predicted pose coordinates, internal energy, grid score, and RMSD values.

To view your results, you can open the 1BJU.min_scored.mol2 file in CHIMERA and compare it to your original ligand file in the 01.dockprep directory.

Minimization result:

Minimization result;RMSD = 0.3177; blue: experiment; green: predicted

The energy minimized pose has an RMSD of 0.3177, meaning that it was minimally moved from it's original experimental ligand pose (1BJU.lig.mol2).

Rigid docking

Rigid Ligand Docking is typically used to check the validity of docking program, user-defined parameters, and help identify any mistakes in the receptor and ligand prep files. Here, we dock the ligand back to the ligand-free receptor. The pose predicted by DOCK should be very close to the crystal structure of the ligand-receptor complex.

The following should be performed in the 04.dock directory.

Create an empty file (rigid_r.in) and run DOCK6: 

touch rigid_r.in
dock6 -i rigid_r.in

Rigid docking input file: rigid_r.in 

conformer_search_type                                        rigid
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
ligand_atom_file                                             ../01.dockprep/1BJU.lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01.dockprep/1BJU.lig.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../02.surface-sphere/selected_spheres.sph
max_orientations                                             1000
critical_points                                              no
chemical_matching                                            no
use_ligand_spheres                                           no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           yes
grid_score_secondary                                         no
grid_score_rep_rad_scale                                     1
grid_score_vdw_scale                                         1
grid_score_es_scale                                          1
grid_score_grid_prefix                                       ../03.box-grid/1BJU.grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
ph4_score_secondary                                          no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_score_secondary                              no
amber_score_secondary                                        no
minimize_ligand                                              yes
simplex_max_iterations                                       1000
simplex_tors_premin_iterations                               0
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_random_seed                                          0
simplex_restraint_min                                        yes
simplex_coefficient_restraint                                10.0
atom_model                                                   all
vdw_defn_file                                                ../zzz.parameters/vdw.defn
flex_defn_file                                               ../zzz.parameters/flex.defn
flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
ligand_outfile_prefix                                        1BJU.rigid_restraint
write_orientations                                           no
num_scored_conformers                                        40
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Note that "conformer_search_type" is set to "rigid".

Note that simplex_restraint_min is set to "yes". This is a flag to perform RMSD tethering, which restrains the conformer sampling to not move too far away from the initial docking site.

The output file prefix is "1BJU.rigid_restraint". The output file "1BJU.rigid_restraint_scored.mol2" is saved in the directory where you ran dock6 (04.dock). This file contains the ligand coordinates, energy score, and RMSD values.

To view your results, you can open the pdb files of the receptor and ligand in CHIMERA from your 01.dockprep directory. The open your rigid docking results using viewdock: 

Tools > surface/binding analysis > viewdock > 1BJU.rigid_restraint_scored.mol2.

You can rank the conformers based on internal energy, grid score, or RMSD.

Best scored rigid docking result:

Rigid docking result;RMSD = 0.7946; blue: experiment; pink: predicted

The predicted pose and the experimental pose have an RMSD less than 2.000, meaning that we were able to reproduce the experimental ligand pose (crystal structure).

Flexible Docking

Flexible docking samples internal degrees of freedom.

The following should be performed in the 04.dock directory.

Create an empty file (flex.in) and run DOCK6: 

touch flex.in
dock6 -i flex.in

Flexible docking input file: flex.in 

conformer_search_type                                        flex
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
use_clash_overlap                                            no
write_growth_tree                                            no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
ligand_atom_file                                             ../01.dockprep/1BJU.lig.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       yes
rmsd_reference_filename                                      ../01.dockprep/1BJU.lig.mol2
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../02.surface-sphere/selected_spheres.sph
max_orientations                                             1000
critical_points                                              no
chemical_matching                                            no
use_ligand_spheres                                           no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           yes
grid_score_secondary                                         no
grid_score_rep_rad_scale                                     1
grid_score_vdw_scale                                         1
grid_score_es_scale                                          1
grid_score_grid_prefix                                       ../03.box-grid/1BJU.grid
multigrid_score_secondary                                    no
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
footprint_similarity_score_secondary                         no
ph4_score_secondary                                          no
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_score_secondary                                 no
SASA_descriptor_score_secondary                              no
amber_score_secondary                                        no
minimize_ligand                                              yes
minimize_anchor                                              yes
minimize_flexible_growth                                     yes
use_advanced_simplex_parameters                              no
simplex_max_cycles                                           1
simplex_score_converge                                       0.1
simplex_cycle_converge                                       1.0
simplex_trans_step                                           1.0
simplex_rot_step                                             0.1
simplex_tors_step                                            10.0
simplex_anchor_max_iterations                                500
simplex_grow_max_iterations                                  500
simplex_grow_tors_premin_iterations                          0
simplex_random_seed                                          0
simplex_restraint_min                                        no
atom_model                                                   all
vdw_defn_file                                                ../zzz.parameters/vdw.defn
flex_defn_file                                               ../zzz.parameters/flex.defn
flex_drive_file                                              ../zzz.parameters/flex_drive.tbl
ligand_outfile_prefix                                        1BJU.flex
write_orientations                                           no
num_scored_conformers                                        100
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       2.0
rank_ligands                                                 no

Note that "conformer_search_type" is set to "flex".

The output file prefix is "1BJU.flex" and the output file is saved as "1BJU.flex_scored.mol2" in the 04.dock directory. The file "1BJU.flex_scored.mol2" contains the ligand coordinates, energy score, and RMSD values. To view your results, you can open the pdb files of the receptor and ligand in CHIMERA from your 01.dockprep directory. Then open your flexible docking results using viewdock: 

Tools > surface/binding analysis > viewdock > 1BJU.flex_scored.mol2.

You can rank the conformers based on internal energy, grid score, or RMSD.

Best scored flexible docking result:

Flexible docking result;RMSD = 0.45; blue: experiment; pink: predicted

The predicted pose and the experimental pose have an RMSD less than 2.000, meaning that we were able to reproduce the experimental ligand pose (crystal structure). If your top scored conformer has a high RMSD, this is considered a DOCKing failure. In the event of a DOCKing failure, consider setting "simplex_random_seed" to a different value. This is a random number generator that determines the starting point for conformer sampling. Alternatively, you could increase sampling.

VI. Virtual Screening

Katie Maffucci

Virtual screening uses libraries of small molecules to identify novel chemical structures that are most likely to bind a drug target. The search is narrowed to a few "hit" compounds that can be synthesized or purchased and tested against the target. Structure-based virtual screening is computationally demanding, and requires a parallel computing infrastructure to handle the task. LIRED is a Linux-based computing cluster well suited for this purpose.

DOCK Preparation

To set up the virtual screen, make a directory using the command: "mkdir 05.largevirtualscreen" Print your working directory using the "pwd" command and copy this dock directory into your LIRED account: "scp -r /gfps/home/username/AMS536/docktutorial/05.largevirtualscreen ."

QSUB File

To submit your job to the cluster, a qsub c chell script must be made so that the job can be executed. Its contents should be as follows: 

   #! /bin/tcsh
   #PBS -l nodes=2:ppn=2
   #PBS -l walltime=24:00:00
   #PBS -o zzz.qsub.out
   #PBS -e zzz.qsub.err
   #PBS -N large_vs
   #PBS -M your.name@stonybrook.edu
   #PBS -V
cd /gfps/home/username/AMS536/docktutorial/ /gpfs/software/intel_2016_update1/impi/5.1.2.150/bin64/mpirun np 4 \ /home/tmcgee/local/AMS_536/dock6/bin/dock6.mpi \

Where -l = resources required for the job, -o = defines output,-e = defines error, ppn = processors per node, -N = name of job, walltime = how long the job can run, and -M = to set up an email alert when the job is complete

Input File

An input file must also be made. "vi large_vs.in": 

   ligand_atom_file                                             yourligandmol2file.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/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                                  500

   simplex_grow_tors_premin_iterations                          0

   simplex_random_seed                                          0

   simplex_restraint_min                                        no

   atom_model                                                   all

   vdw_defn_file                                                ../docktutorial/zzz.parameters/vdw_AMBER_parm99.defn

   flex_defn_file                                               ../docktutorial/zzz.parameters/flex.defn

   flex_drive_file                                              ../docktutorial/zzz.parameters/flex_drive.tbl

   ligand_outfile_prefix                                        large_vs

   write_orientations                                           no

   num_scored_conformers                                        1

   rank_ligands                                                 no

Post Processing

After the virtual screen, the top hits and poses will be rank ordered according to the scoring function(s) chosen in previous input files. Depending on the size of the database, the top 1% of molecules can still number in the hundreds. To further refine the screen, cartesian minimization should be performed. To do this, create an input file 1BJU.cart_min.ligs.in: 

  conformer_search_type                                        rigid 

  use_internal_energy                                          yes 

  internal_energy_rep_exp                                      12 

  ligand_atom_file                                             ../06.large-virtual-screen/1BJU.output_scored.mol2 

  limit_max_ligands                                            no 

  skip_molecule                                                no 

  read_mol_solvation                                           no

  calculate_rmsd                                               no

  use_database_filter                                          no

  orient_ligand                                                no

  automated_matching                                           no

  critical_points                                              no

  chemical_matching                                            no

  use_ligand_spheres                                           no

  bump_filter                                                  no

  score_molecules                                              yes

  contact_score_primary                                        no

  contact_score_secondary                                      no

  grid_score_primary                                           no

  grid_score_secondary                                         no

  multigrid_score_primary                                      no

  multigrid_score_secondary                                    no

  dock3.5_score_primary                                        no

  dock3.5_score_secondary                                      no

  continuous_score_primary                                     yes

  continuous_score_secondary                                   no

  cont_score_rec_filename                                      ../01.dockprep/1BJU.rec.mol2

  cont_score_att_exp                                           6

  cont_score_rep_exp                                           12

  cont_score_rep_rad_scale                                     1

  cont_score_use_dist_dep_dielectric                           yes

  cont_score_dielectric                                        4.0

  cont_score_vdw_scale                                         1

  cont_score_es_scale                                          1

  footprint_similarity_score_secondary                         no

  ph4_score_secondary                                          no

  descriptor_score_secondary                                   no

  gbsa_zou_score_secondary                                     no

  gbsa_hawkins_score_secondary                                 no

  SASA_descriptor_score_secondary                              no

  amber_score_secondary                                        no

  minimize_ligand                                              yes

  simplex_max_iterations                                       1000

  simplex_tors_premin_iterations                               0

  simplex_max_cycles                                           1

  simplex_score_converge                                       0.1

  simplex_cycle_converge                                       1.0

  simplex_trans_step                                           1.0

  simplex_rot_step                                             0.1

  simplex_tors_step                                            10.0

  simplex_random_seed                                          0

  simplex_restraint_min                                        yes

  simplex_coefficient_restraint                                10.0

  atom_model                                                   all

  vdw_defn_file                                                ../zzz.parameters/vdw.defn

  flex_defn_file                                               ../zzz.parameters/flex.defn

  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl

  ligand_outfile_prefix                                        1BJU.cartmin

  write_orientations                                           no

  num_scored_conformers                                        1

  rank_ligands                                                 no

You'll need to make another minimization input file with similar contents: 

  conformer_search_type                                        rigid 

  use_internal_energy                                          yes

  internal_energy_rep_exp                                      12

  ligand_atom_file                                             ../01.dockprep/1BJU.lig.mol2

  limit_max_ligands                                            no

  skip_molecule                                                no

  read_mol_solvation                                           no

  calculate_rmsd                                               no

  use_database_filter                                          no

  orient_ligand                                                no

  bump_filter                                                  no

  score_molecules                                              yes

  contact_score_primary                                        no

  contact_score_secondary                                      no

  grid_score_primary                                           no

  grid_score_secondary                                         no

  multigrid_score_primary                                      no

  multigrid_score_secondary                                    no

  dock3.5_score_primary                                        no

  dock3.5_score_secondary                                      no

  continuous_score_primary                                     yes

  continuous_score_secondary                                   no

  cont_score_rec_filename                                      ../01.dockprep/1BJU.rec.mol2

  cont_score_att_exp                                           6

  cont_score_rep_exp                                           12

  cont_score_rep_rad_scale                                     1

  cont_score_use_dist_dep_dielectric                           yes

  cont_score_dielectric                                        4.0

  cont_score_vdw_scale                                         1

  cont_score_es_scale                                          1

  footprint_similarity_score_secondary                         no

  ph4_score_secondary                                          no

  descriptor_score_secondary                                   no

  gbsa_zou_score_secondary                                     no

  gbsa_hawkins_score_secondary                                 no

  SASA_descriptor_score_secondary                              no

  amber_score_secondary                                        no

  minimize_ligand                                              yes

  simplex_max_iterations                                       1000

  simplex_tors_premin_iterations                               0

  simplex_max_cycles                                           1

  simplex_score_converge                                       0.1

  simplex_cycle_converge                                       1.0

  simplex_trans_step                                           1.0

  simplex_rot_step                                             0.1

  simplex_tors_step                                            10.0

  simplex_random_seed                                          0

  simplex_restraint_min                                        yes

  simplex_coefficient_restraint                                10.0

  atom_model                                                   all

  vdw_defn_file                                                ../zzz.parameters/vdw.defn

  flex_defn_file                                               ../zzz.parameters/flex.defn

  flex_drive_file                                              ../zzz.parameters/flex_drive.tbl

  ligand_outfile_prefix                                        1BJU.cartmin_ref

  write_orientations                                           no

  num_scored_conformers                                        1

  rank_ligands                                                 no

To execute the job, you'll need to make a cartminligs.qsub.tcsh script similar to the previous one: 

   #!/bin/tcsh
   #PBS -l walltime=09:00:00
   #PBS -l nodes=1:ppn=24
   #PBS -q medium
   #PBS -N 1BJU
   #PBS -V
   #PBS -M youremail@stonybrook.edu
   #PBS -j oe
   cd /gpfs/home/username/AMS536/docktutorial/07.cartesian_min
   /gpfs/software/intel_2016_update1/impi/5.1.2.150/bin64/mpirun -np 24 \
  /home/tmcgee/local/AMS_536/dock6/bin/dock6.mpi \
  -i 1BJU.cart_min_ligs.in \
  -o 1BJU.cart_min_ligs.out

VIII. Frequently Encountered Problems

EVERYONE

You may see that your vdw scores are zero - this is highly unlikely to actually be the case. If you are using an older version of Chimera and used the "dockprep" button to add charges and hydrogens to your receptor, the charges were likely not saved. You must add the charges and hydrogens manually.

When submitting the qsub script, make sure that there are no spaces after the " \ " on the mpirun line - the job will not run in parallel if there are spaces after the backslash.

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