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For additional Rizzo Lab tutorials see DOCK Tutorials. Use this link Wiki Markup as a reference for editing the wiki. This tutorial was developed collaboratively by the AMS 536 class of 2012.

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.


Streptavidin & Biotin

Streptavidin is a tetrameric prokaryote protein that binds the co-enzyme biotin with an extremely high affinity. The streptavidin monomer is composed of eight antiparallel beta-strands which folds to give a beta barrel tertiary structure. A biotin binding-site is located at one end of each β-barrel, which has a high affinity as well as a high avidity for biotin. Four identical streptavidin monomers associate to give streptavidin’s tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighboring subunit. In this way, each subunit contributes to the binding site on the neighboring subunit, and so the tetramer can also be considered a dimer of functional dimers.

Biotin is a water soluble B-vitamin complex which is composed of an ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring. It is a co-enzyme that is required in the metabolism of fatty acids and leucine. It is also involved in gluconeogenesis.


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-original-files/
                              /01-dockprep/
                              /02-surface-spheres/
                              /03-box-grid/
                              /04-dock/
                              /05-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, '1DF8'. The following sections in this tutorial will adhere to this directory structure / naming scheme.

II. Preparing the Receptor and Ligand

Downloading the PDB Structure (1DF8)

The Protein Data Bank contains atomic coordinates for more than 79,000 molecules and is accessible via the internet from PDB. The target (protein or nucleic acid) is accessible by PDB ID, molecular name, or author. After entering the PDB ID, molecule name, or author, click Download Files, then select PDB File (text). A window will open; click on Save File → Downloads.

Preparing the Ligand and Enzyme in Chimera

If you are on "herbie" you can access Chimera directly by typing in Chimera at the prompt. Otherwise, download Chimera to your desktop and obtain your protein/nucleic acid complex of interest using Fetch (by ID).

Open Your Protein of Interest and Delete Unwanted Molecules:

Click on Open under the File menu and find where you saved your pdb file.

You only need one of the monomers to perform docking. Remove chain B by carrying out the following:

  1. SelectChainBAll.
  2. ActionsAtomsDelete.

To delete water molecules and/or other ligands, go to ToolsStructure EditDock Prep. Check all boxes and click okay. This will walk you through the steps needed to prepare the complex for docking, and will also assign partial charges to the protein and the ligand. Choose am 1-bcc for charges.

Save the file as 1DF8.dockprep.mol2.

dockprep image

This file contains a conformation of the complex with hydrogen atoms. The grid calculation is based on the receptor with its hydrogen atoms. The grid score is an energy calculation that is based on the following equation:

E GRID = E VDW + E ES. The score is an approximation of the molecular mechanics' energy function and it considers only through space interactions.

Create a Receptor File:

Go to SelectResidueBTN. Then go to ActionsAtomsDelete.

Save the file as 1DF8.receptor.mol2.

Create a Receptor File with No Hydrogen Atoms:

Go to SelectChemistryElementHDelete.

Save a PDB file as 1DF8.receptor.noH.pdb.

Create a Ligand File:

Open only the 1DF8.dockprep.mol2.

Go to SelectChainDelete. This will allow you to have only the ligand.

Save the file as 1DF8.ligand.mol2.

These are the files that you will need to continue with rigid or flexible docking.

III. Generating Receptor Surface and Spheres

Receptor Surface

To generate an enzyme surface, first open the receptor pdb file with the hydrogen atoms removed (1DF8.receptor.noH.pdb). Next, go to Actions -> Surface -> Show. Note that for DOCK calculation hydrogen atoms are considered, but for generating enzyme surface and spheres, it is necessary to use the protein without hydrogens.

2012_DOCK_Tutorial_1DF8_surface

Recent versions of Chimera include a Write DMS tool that facilitates calculation of the molecular surface. Go to Tools -> Structure Editing -> Write DMS. Save the surface as 1DF8.receptor.dms.

2012_DOCK_Tutorial_1DF8_dms

The Write DMS tool will "roll" a small probe (default radius = 1.4 Angstroms = Size of a water molecule) over the surface of the enzyme and calculate the surface normal at each point. DMS (distributed molecular surface) file is subsequently used as input file for sphgen.

Spheres

To generate docking spheres, we need to use a command line program called sphgen. To run the sphgen, we need a input file named INSPH.

1DF8.receptor.dms            #molecular surface file that we got from previous step
R                            #sphere outside of surface (R) or inside surface (L)
X                            #specifies 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) 
1DF8.receptor.sph            #clustered spheres file that we want to generate

Save the INSPH file. Then use this command to generate the spheres file:

sphgen -i INSPH -o OUTSPH

-i means input; -o means output.

You should get two output files: OUTSPH and 1DF8.receptor.sph. The OUTSPH file should similar to this:

density type = X
reading  1DF8.receptor.dms                                                     type   R
# of atoms =    881   # of surf pts =  10771
finding spheres for   1DF8.receptor.dms                                         
dotlim =     0.000
radmax =    4.000
Minimum radius of acceptable spheres?
  1.4000000
output to  1DF8.receptor.sph                                                    
clustering is complete     28  clusters

You can also open the spheres file that we generated in this step (1DF8_receptor.sph). This file contains detailed information about the spheres, which are divided into 28 clusters. Cluster 0 in the end of the spheres file is a combination of all the clusters.

In order to visualize the generated spheres, you can use a program called showsphere. Showsphere is an interactive program. In the command line, simply type

showsphere

You will be asked the following questions:

Enter name of sphere cluster file:
  1DF8.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:
  1DF8.output.
Process cluster 0 (contains ALL spheres) (<N>/Y)? N

In this case, 28 pdb files should be generated.

An alternative way to do this is to create an input file, showsphere.in, as follows:

1DF8.receptor.sph
-1
N
1DF8.output
N

Then type the command showsphere

showshpere < showsphere.in

After generating the pdb files by showsphere, you can visualize each cluster by UCSF Chimera. For example, open 1DF8.output_1.pdb by Chimera, then choose Actions->Atoms/Bonds->sphere, you will be able to see the spheres in cluster 1. You can also open the receptor file (1DF8.receptor.mol2) at the same time, then choose Presets->Interactive 3(hydrophobicity surface), then again choose Actions->Atoms/Bonds->sphere, you will be able to see what the spheres in cluster 1 look like on the enzyme surface.

2012_DOCK_Tutorial_1DF8_spheres

There are over 500 spheres in the spheres file(1DF8.receptor.sph). However, we're only interested in docking the ligand into the active site. Therefore we need to select only those spheres which are inside the active site, using sphere_selector program.

sphere_selector 1DF8.receptor.sph ../01-dockprep/1DF8.ligand.mol2 10.0

Sphere_selector filters the output from sphgen, selecting all spheres within a user-defined radius of a target molecule. In this case, we selected the spheres within 10 angstroms of our ligand. A file called selected_spheres.sph should be created, showing the spheres that are selected. You can again visualize it by showsphere. You can also change the radius (to 8 angstroms or 6 angstroms) or manually edit the file selected_spheres.sph so that you can select the spheres you want. In this tutorial, spheres within 6 angstroms of the ligand are used for the next step (with one sphere which does not belong to the active site being manually deleted).

2012_DOCK_Tutorial_1DF8_surface_spheres_10 angstroms
2012_DOCK_Tutorial_1DF8_surface_spheres_6 angstroms

IV. Generating Box and Grid

Box

In order to speed up docking calculations, DOCK generates a fine grid. At each point in the grid, electrostatic and the VDW probes' energies are precomputed. The energies are computed using a molecular force field. To determine the dimensions of the grid, however, we first generate a box that contains the outer boundaries for the grid calculation. The dimensions and location of the box can be determined using a program called showbox.

First create a directory where you will place the grid files.

$mkdir 03-box-grid
$cd 03-box-grid

showbox can be used interactively or a file with predetermined answers can be fed into the program.

The program asks the questions depicted in the diagram the right:

Error creating thumbnail: Unable to save thumbnail to destination
Flow Chart of Questions for Showbox (Red path is followed in this tutorial)

To run the program in the interactive mode, run

$showbox

To feed the answers to the questions, run

$showbox < showbox.in

For example, showbox.in can contain:

Y
5
../02-surface-spheres/selected_spheres.sph
1
1DF8.box.pdb

Y means we use automatic box construction, 5 is the extra margin to be enclosed around our ligand (in Angstroms), selected_spheres.sph is the sphere file we generated, 1 corresponds to the cluster number in the selected_spheres.sph file, and 1DF8.box.pdb is the output file. We can open the output box file in chimera to make sure the box is in the right place.

1DF8 receptor along with our ligand and the box we generated using showbox

Grid

Now let's generate a grid within our box. We will use the energy scoring method to generate a grid, resulting in three additional files with extensions *.nrg, *.bmp, and *.out. The *.nrg file contains the energy scoring, *.bmp contains the size, position and grid spacing and determines whether there are any overlaps with receptor atoms.

To generate the grid we will use the grid program. This program can either be used interactively, or an input file can be fed in, just like the showbox program.

Usage: grid [-i [input_file]] [-o [output_file]] ...
 [-standard_i/o] [-terse] [-verbose]
 
 -i: read from grid.in or input_file, standard_in otherwise
 -o: write to grid.out or output_file (-i required), 
     standard_out otherwise
 -s: read from and write to standard streams (-i and/or -o illegal)
 -t: terse program output
 -v: verbose program output

For our grid.in file, we will use the following answers:

compute_grids                  yes
grid_spacing                   0.3
output_molecule                no
contact_score                  no
energy_score                   yes
energy_cutoff_distance         9999
atom_model                     a
attractive_exponent            6
repulsive_exponent             9
distance_dielectric            yes
dielectric_factor              4
bump_filter                    yes
bump_overlap                   0.75
receptor_file                  ../01-dockprep/1DF8.receptor.mol2
box_file                       1DF8.box.pdb
vdw_definition_file            /opt/software/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
score_grid_prefix              grid

Line by line:

  1. compute scoring grids (yes)
  2. what is the distance between grid points along each axis (in Angstroms).
  3. write up coordinates of the receptor into a new file
  4. compute contact grid? default is no
  5. compute energy score? yes - we are using this method to compute force fields on probes
  6. the max distance between atoms for the energy contribution to be computed
  7. 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
  8. attractive component stands for exponent of the attractive LJ term in VDW potential
  9. repulsive component stands for exponent in the repulsive LJ term in VDW potential
  10. distance dielectric stands for the dielectric constant to be linearly dependent on distance
  11. distance dielectric factor is the coefficient of the dielectric
  12. bump filter flag determines if we want to screen orientation for clashes before scoring and minimization
  13. bump_overlap stands for the fraction of allowed overlap where 1 corresponds to no allowed overlap and 0 corresponds to full overlap being permitted.
  14. our receptor file
  15. the box file we generated in the Box section
  16. VDW parameters file
  17. Prefix for the grid file name. All the extensions will be generated automatically.

V. Docking a Single Molecule for Pose Reproduction

Docking and Results

Change directory into “04-dock” and create an empty input file called dock.in

$touch dock.in     #create a file called “dock.in”

Run dock6.4

$dock6 -i dock.in

Alternatively:

$dock6 -i dock -v # “-v” option allows you to print out the information regarding each growth step on terminal

Or:

$dock6 -i dock -o dock.bf.out -v # “-o” allows you to write the aforementioned information into an output file named “dock.bf.out”

1st run:

Notice that running dock6 with an empty input file will require you to answer a series of questions. For the first run we will deactivate most of the features by selecting “no”. Parameters requiring specification are listed below:

ligand_atom_file [database.mol2] ():   	../01-dockprep/1DF8.ligand.mol2
ligand_outfile_prefix [output] ():   		1DF8.output

What the program is doing in the 1st run is to take the ligand.mol2 file and directly generate an output file without any additional manipulations. You would expect it to be exactly the same as the original pose. You can open it in Chimera by using the “ViewDock” function under “Tools->Surface/Binding Analysis”. We will not show the result here.

2nd run:

The real experiment begins here. Notice that we selected “no” for most of the functions. This time we will try change some parameters in the dock.in file.

$vim dock.in

Parameters being changed are listed below:

orient_ligand                                               yes

The "orient_ligand" option tells the program whether to try orientations different from the pose in the original .mol2 file. Note that because of the change, additional questions are asked by the program. For simplicity we keep most of the answers as default. File paths that need to be specified are listed below:

receptor_site_file                          [receptor.sph] ():../02-surface-spheres/selected_spheres_06A.sph
vdw_defn_file                               [vdw.defn] ():../03-grid/vdw_AMBER_parm99.defn 
flex_defn_file                              [flex.defn] ():../03-grid/flex.defn 
flex_drive_file                             [flex_drive.tbl] ():../03-grid/flex_drive.tbl

The receptor_site_file should be the selected spheres file (.sph) generated in a previous step (02 surface and spheres), according to which the program orients the ligand.

The vdw_defn_file instructs the dock6 program to use the Van der Waals parameter sets from the AMBER force field.

The flex_defn_file and the flex_drive_file contain the information required by the program to sample conformations.

The result is shown below. As you may notice the result doesn't look very good.

Example alt text
DOCK second Run result

3rd Run:

Here we will further specify parameters to improve the result.

calculate_rmsd                                               yes
score_molecules                                              yes
num_scored_conformers                                        10

Again, further questions will be asked when you run the program. Keep most answers as the default values. Paths requiring specification are listed below:

grid_score_grid_prefix                                        [grid] ():../03-grid/1DF8.grid

Note that the above specification will tell the program to load the 1DF8.grid.nrg file you generated in the previous step (grid) for scoring.

simplex_max_iterations                                        [1000] ():20
write_conformations                                           [yes] (yes no):no
cluster_rmsd_threshold                                        [2.0] ():0.2

The simplex_max_iteration parameter specifies number of minimization cycles.

Note the program will cluster poses that are very close together (rmsd smaller than the threshold specified in the cluster_rmsd_threshold parameter) into a cluster.

This time the program returned the best 10 poses. The one with the best grid score (-52.8, shown blue) superimposed quite well with the original pose in the crystal structure (RMSD = 0.71). You can view the grid scores in ViewDock by selecting “Column->Show->Grid Score”

Example alt text
DOCK third Run result

4th Run:

This time we will set the ligand as flexible:

flexible_ligand                                              yes

Further questions will be asked during the run. Keep most of the answers as the default values except for:

simplex_grow_max_iterations                                   [20] ():500

The simplex_grow_max_iteration specifies the maximum number of iterations per cycle per growth step.

Notice that the run is significantly slower this time. Again the best pose generated (-64.8) is shown in blue. The grid score improved significantly but the RMSD did not change much (0.79)

Example alt text
DOCK fourth Run result

5th Run:

This time we will turn the bump filter on:

bump_filter                                                  yes

The bump_filter option filters out conformations that cause clashes between atoms.

Further questions will be asked during the run. Keep answers as the default values. Specify the following path:

bump_grid_prefix                                              [grid] ():../03-grid/1DF8.grid

Note that the path tells the program to access the .bmp file generated in the previous grid step.

The best pose (grid score -65.3) is again shown in blue. Notice that turning on the bump filter does not alter either the grid score or the RMSD (0.78).

Example alt text
DOCK fifth Run result

Remember you can tweak any parameter in the dock.in file instead of keeping them as default.

Following is the final dock.in file used during the fifth run:

ligand_atom_file                                             ../01-dockprep/1DF8.ligand.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       no
use_database_filter                                          no
orient_ligand                                                yes
automated_matching                                           yes
receptor_site_file                                           ../02-surface-spheres/selected_spheres_06A.sph
max_orientations                                             500
critical_points                                              no
chemical_matching                                            no
use_ligand_spheres                                           no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
flexible_ligand                                              yes
min_anchor_size                                              40
pruning_use_clustering                                       yes
pruning_max_orients                                          100
pruning_clustering_cutoff                                    100
pruning_conformer_score_cutoff                               25
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-grid/1DF8.grid
dock3.5_score_secondary                                      no
continuous_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_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                                                ../03-grid/vdw_AMBER_parm99.defn
flex_defn_file                                               ../03-grid/flex.defn
flex_drive_file                                              ../03-grid/flex_drive.tbl
ligand_outfile_prefix                                        1DF8.output
write_orientations                                           no
num_scored_conformers                                        10
write_conformations                                          no
cluster_conformations                                        yes
cluster_rmsd_threshold                                       0.2
rank_ligands                                                 no
                                                                 

Now we will write up a script for submitting your dock job to Seawulf. Create a script called “sub.dock.csh”

$vim sub.dock.csh

wherein you write:

#! /bin/tcsh 
#PBS -l nodes=1:ppn=1 
#PBS -l walltime=01:00:00 
#PBS -o zzz.qsub.out 
#PBS -j oe 

cd ~/AMS536/DOCK_tutorial/05-dock_qsub 

/nfs/user03/sudipto/dock6/bin/dock6 -i dock.in -o dock.out 

This will request 1 processor from the cluster for your job. When you are submitting the job:

$qsub sub.dock.csh

Note that you might have to make the script executable before running it:

$chmod +x sub.dock.csh

Results

VI. Virtual Screening

Virtual Screening Preparation

Virtual screening is a widely used method in computational drug design. It searches large libraries of chemical compounds to identify favorable structures that bind to a target molecule. To perform virtual screening, we use ligands.mol2, a mol2 file which contains 101 small molecules to be the virtual library. The computational cost is reasonable for a quick search. Usually we use larger molecule set from chemical database, such as ZINC(http://zinc.docking.org/).

Since the computational cost of virtual screening is much higher than single-ligand docking, it is better for us to run it on Seawulf. We need to compress the whole DOCK-Tutorial directory, copy it to Seawulf and compress it.

tar -cvf DOCK-Tutorial.tar DOCK-Tutorial/
scp DOCK-Tutorial sw:/nfs/user03/usrname/AMS536
tar -xvf DOCK-Tutorial.tar DOCK-Tutorial

Virtual Screening Protocol

The purpose of virtual screening is different from single molecule docking, so we need to modify our previous docking script dock.in to vs.in. We can see the difference between the two files.

1c1 
< ligand_atom_file                                             ligands.mol2 
--- 
> ligand_atom_file                                             ../01-dockprep/1DF8.ligand.mol2 
56,59c56,59 
< vdw_defn_file                                                /nfs/user03/sudipto/dock6/parameters/vdw_AMBER_parm99.defn 
< flex_defn_file                                               /nfs/user03/sudipto/dock6/parameters/flex.defn 
< flex_drive_file                                              /nfs/user03/sudipto/dock6/parameters/flex_drive.tbl 
< ligand_outfile_prefix                                        1DF8.vs.output 
--- 
> vdw_defn_file                                                ../03-box-grid/vdw_AMBER_parm99.defn 
> flex_defn_file                                               ../03-box-grid/flex.defn 
> flex_drive_file                                              ../03-box-grid/flex_drive.tbl 
> ligand_outfile_prefix                                        1DF8.output 
61c61 
< num_scored_conformers                                        1 
--- 
> num_scored_conformers                                        10 
63c63,64 
< cluster_conformations                                        no 
--- 
> cluster_conformations                                        yes 
> cluster_rmsd_threshold                                       0.2 

num_scored_conformers: 1 -> 10 In virtual screening, we only need the most favorable pose of each candidate molecule and compare them.
cluster_conformations: yes -> no Slightly different conformations are not clustered together. They are treated as different conformations in the docking process.

In order to generate different search spaces, we can modify some other parameters.

max_orientations: The maximal number of anchor orientations that will be generated.
min_anchor_size: The minimum number of atoms for an anchor.
pruning_use_clustering: Pruning conformations during the clustering process.
use_internal_energy: Using repulsive VDM to avoid internal clashes.

Parallel computing can reduce the running time of virtual screening. Here is our job submission script sub.virtual_screen.csh.

#! /bin/tcsh
#PBS -l nodes=4:ppn=2
#PBS -l walltime=01:00:00
#PBS -o zzz.qsub.out
#PBS -j oe
#PBS -V
set nprocs = `wc -l $PBS_NODEFILE | awk '{print $1}'`
echo "Running on ${nprocs} processors"
echo ""
echo "processor list are:"
cat $PBS_NODEFILE
cd ~/AMS536/DOCK_Tutorial/06-virtualscreen
mpirun -np $nprocs dock6.mpi -i vs.in -o vs.out

Finally, we can submit the job and perform virtual screening.

qsub sub.virtual_screen.csh

Virtual Screening Results

After performing virtual screening on Seawulf or any other computer, if you are using a parallel computer, you should get a multi-mol2 file 1DF8.vs.output_scored.mol2 which contains the mol2 files of all succesfully docked ligands, a vs.out file which contains the dock results of successfully docked ligands, and vs.out.1 through vs.out.7 which contain dock results from different nodes you use (the number will vary according to the number of nodes you use, here we use 8 nodes as mentioned before).

The vs.out file is returned by the leading node and contains the information of each succesfully docked ligands, looks like this:

Molecule: ZINC33171556

Anchors:               1
Orientations:          500
Conformations:         116

   Grid Score:          -52.373383
     Grid_vdw:          -51.643253
      Grid_es:           -0.730129
   Int_energy:            6.125062

The vs.out.1 through vs.out.2 files are returned by other nodes processing those ligands, separately. For those succesfully docked ligands, the file will return the elapsed time for docking, and for those not succesfully docked, it will return an error massege like this:

Molecule: ZINC20605433

Elapsed time:  0 seconds

ERROR:  Could not complete growth.
        Confirm that the grid box is large enough to contain the ligand,
        and try increasing max_orientations.

If you download the multi-mol2 file from seawulf using:

scp sw:~/AMS536/DOCK_tutorial/06-virtual-screen/1DF8.vs.output_scored.mol2 ./

Now you have this multi-mol2 file 1DF8.vs.output_scored.mol2 on your local machine, you can actually open this file in Chimera to visually check you docking results and do some visual analysis. But it's not a good idea to directly open your multi-mol2 file because it contains information of all 47 succesfully docked ligands, if you just open this file, it will be pretty messy. So what you will do is, first you open the receptor file 1DF8.receptor.mol2 and the ligand 1DF8.ligands.mol2 as a reference. And then use the ViewDock function of Chimera to look at your 47 ligands one or however many you want at a time. You can find ViewDock via Tools -> Surface/Binding Analysis -> ViewDock, and then find your 1DF8.vs.output_scored.mol2 file in your own directory and click on open. Now a new window of ViewDock will pop out and there will be an extra ligands in Chimera main window which is the highlighted ligand in ViewDock window. You can look at the ligands one at a time, you can also hold ctrl and click on different ligands to view them at the same time, this will give you a direct idea of how good these ligands dock. The other thing you can do is, the multi-mol2 file 1DF8.vs.output_scored.mol2 contains the energy score of every ligand, so in ViewDock window, you can go to Column -> show -> Grid Score to show the grid score, and then you can click on the head of the column to rank order all the ligands by their grid scores.The picture showing here is the best and worst scored ligands of out calculation, the best scroed one in cyan and the worst scored on in magenta, and the reference ligand is colored according to elements. As you can see, the best scored ligand fits in the binding pocket very well, but the worst scored one almost sticks out of the binding pocket.

Example of Virtual Screening Result

VII. Running DOCK in Serial and in Parallel on Seawulf

The Seawulf Cluster has 235 dual processor nodes (2 processors per node), for a total of 470 individual processors. These are 3.4Ghz Intel Pentium IV Xeon processors from Dell. Each node has 2GB attached RAM and a 40GB hard disk.

Typically you will use one processor on a single node to dock one ligand. If you are docking multiple ligands, you can use more than processor in parallel mode, but you should never use more processors than you have ligands.

Running DOCK in Serial on a Single Processor

The following sample code can be used to run DOCK on one processor on a single node:

#!/bin/tcsh                 
#PBS -l nodes=1:ppn=1        
#PBS -l walltime=01:00:00   
#PBS -N dock6           
#PBS -M user@ic.sunysb.edu 
#PBS -j oe                   
#PBS -o pbs.out            

cd /nfs/user03/sudipto/DOCK_Tutorial                     
/nfs/user03/sudipto/dock6/bin/dock6 -i dock.in -o dock.out

Here is an explanation of the code and format:

#!/bin/tcsh                                                 #Execute script with tcsh
#PBS -l nodes=1:ppn=1                                       #Use one node, and one processor per node, so one single processor 
#PBS -l walltime=01:00:00                                   #Allow 1 hour for your job run
#PBS -N dock6                                               #Name of your job
#PBS -M user@ic.sunysb.edu                                  #Get an email notifying you when your job is completed
#PBS -j oe                                                  #Combine the output and error streams into a single output file
#PBS -o pbs.out                                             #Name of your output file

cd /path-to-your-home-directory-on-seawulf/DOCK_Tutorial    #Change to your home directory and folder with dock files                  
/nfs/user03/sudipto/dock6/bin/dock6 -i dock.in -o dock.out  #Specifies path to dock executable and provide input and output filenames

Running DOCK in Parallel using MPI

The following sample code can be used to run DOCK on 4 nodes, using both processors on each node, for a total of 8 processors.

#! /bin/tcsh
#PBS -l nodes=4:ppn=2
#PBS -l walltime=24:00:00
#PBS -o zzz.qsub.out
#PBS -j oe
#PBS -V

set nprocs = `wc -l $PBS_NODEFILE | awk '{print $1}'`

echo "Running on ${nprocs} processors"

cd /nfs/user03/sudipto/DOCK_Tutorial
cp /nfs/user03/sudipto/dock6/parameters/vdw_AMBER_parm99.defn .
cp /nfs/user03/sudipto/dock6/parameters/flex* .

mpirun -np $nprocs /nfs/user03/sudipto/dock6/bin/dock6.mpi -i dock.in -o dock.out

For more information, see PBS Queue

Serial Calculation for Pose Reproduction

Parallel Virtual Screen

VIII. Molecular Footprints

Preparation (minimization) of Reference Molecule

The footprint of the reference molecule guides the selection of molecules/poses in the footprint rescoring method, therefore careful preparation of this reference molecule is necessary. Typically the reference molecule will be an endogenous substrate or known inhibitor from an x-ray crystallographic structure.

Preferably, the molecule should be prepared with hydrogens and charges using AMBER and subsequently converted to mol2 format using the 'top2mol2' command. This will provide you with a 'monotonic' mol2 file -- meaning that all the hydrogens will be listed with the corresponding protein residue, rather than separately at the end of the file (like Chimera's 'DockPrep' tool does).

An alternative way to generate the "monotonic" receptor file is to open the receptor.mol2 file with Chimera, save as pdb file, reopen the pdb file and go through DockPrep, and again save as a mol2 file. In this case, the new mol2 file will be monotonic.

Once the molecule is prepared, it should be minimized in the receptor using a tethered minimization. This is especially important if the crystal structure was refined without hydrogens (typically hydrogens are not used in refinement unless the resolution is ~1 Angstrom or better). Minimization is needed to help alleviate any steric clashes involving the newly added hydrogens (which would otherwise disrupt your footprint results).

An example input file for use on seawulf (using dock6.5):

ligand_atom_file                                             ligand.mol2
limit_max_ligands                                            no
skip_molecule                                                no
read_mol_solvation                                           no
calculate_rmsd                                               yes
use_rmsd_reference_mol                                       no
use_database_filter                                          no
orient_ligand                                                no
use_internal_energy                                          yes
internal_energy_rep_exp                                      12
flexible_ligand                                              no
bump_filter                                                  no
score_molecules                                              yes
contact_score_primary                                        no
contact_score_secondary                                      no
grid_score_primary                                           no
grid_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                                      receptor.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
descriptor_score_secondary                                   no
gbsa_zou_score_secondary                                     no
gbsa_hawkins_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                                                /nfs/user03/tbalius/zzz.programs/dock6.5/parameters/vdw_AMBER_parm99.defn
flex_defn_file                                               /nfs/user03/tbalius/zzz.programs/dock6.5/parameters/flex.defn
flex_drive_file                                              /nfs/user03/tbalius/zzz.programs/dock6.5/parameters/flex_drive.tbl
ligand_outfile_prefix                                        xtalmin
write_orientations                                           no
num_scored_conformers                                        1
rank_ligands                                                 no

Molecular footprints comparisons

After rescoring of the molecules by footprint-based score, typically three output files will be generated: a mol2 file showing the ligands information, a footprint.txt file showing the footprints infomation, and a hbond.txt file showing the hydrogen bond information. The footprint.txt file contains per residue interaction of the receptor with the ligand, for both reference molecule and the docked molecule. Shown below is an example of VDW footprints comparison.

2012_DOCK_Tutorial_Footprints_1
2012_DOCK_Tutorial_Footprints_2

Actually the upper picture and the bottom picture are comparing the same molecule to a same reference molecule. The difference is that the reference molecule for the upper case is the original ligand.mol2 without energy minimization! Obviously, the reference molecule is not correct in this case. As you can see, several very disfavored VDW interactions were observed for the reference molecule. In contrast, the bottom picture shows the correct comparison.

IX. Frequently Encountered Problems

Barbara

How to Open Files

If you want to view a file in chimera, first transfer the file from "herbie" or "seawulf" to your laptop using WinSCP. Then open Chimera on your laptop to open the files.

Woo Suk

Distinguishing overlapped spheres

Sometimes your 1DF8.output_1.pdb can have some overlapped spheres. In this case, you cannot find them before you delete one sphere and you have to repeat this one more time to delete another one.
In order to avoid the situation, you can adjust a parameter in INSPH file like following:

1DF8.receptor.dms            
R                            
X                            
0.001                          
4.0                          
1.4                          
1DF8.receptor.sph            

You can change the fourth parameter from 0.0 to other values such as 0.1, 0.01, or 0.001. Because what you want is just to distinguish the very close spheres, you don't need large numbers.

Longfei

Installation of DOCK

If you want to install DOCK on your own desktop or laptop, and you are a beginner of Unix/Linux, you might encounter some problems during installation. A good reference for the installation is the DOCK User Manual(http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm). The problem I encountered was: after I used gnu as configuration file to configure the Makefile, I successfully made the config.h file, but when I was trying to build the DOCK executables, an error message " g77 command not found " appeared. And it is quite inconvenient to install the g77 command. A way to solve this problem is to open the config.h file, and manually change the g77 in that file to gfortran.

Use the Correct Path of Your File

One frequently encountered problem is using the wrong path of your file. For example, when you use sphere_selector to generate spheres around the ligand, it requires two files--one is the spheres file .sph and another is your ligand file, but your ligand file is probably not in your working directory! So you need either copy your ligand file to the current directory or specify the correct path of your ligand file like below:

sphere_selector 1DF8.receptor.sph ../01-dockprep/1DF8.ligand.mol2 10.0

This is also important when you run DOCK. Remember to use the correct path and name of the your file.

Notice the Shell That You are Using

This tutorial is based on the shell tcsh, and maybe you are using a different shell. This will not cause any trouble in most of the time, but you may want to notice this in some circumstances, for example when you want to execute some .csh file. One way to solve this problem is that simply type "tcsh" in the command line, and you will be running tcsh in another shell.

Roman

One of the problems one may encounter with grid creation is the sampling location for ligand binding. It is important to create a grid and dock the ligand to the correct location in the protein. If the grid is created in the incorrect location, the ligand binding sites will be sampled within the grid, and the best binding site will not be sampled. In our case, we are docking it to an area where a ligand is known to bind to streptavidin. However, this may not always be the case.

One solution is to dock to the center of mass of the spheres clusters created with spheregen and assess the center of mass as viable or not based on your chemical intuition.

Another way is to assess the protein for buried sites where there may also be a number of spheres. Both are options in the showbox program.

Finally, one can attempt to find proteins with similar sequence and determine where their ligand binding sites are, and then pick the binding site in the protein you are docking to using that information.

Quan

Hui

Keep the previous dock6 output .mol2 files

When we run dock6 program, the output file would be "1DF8.output_scored.mol2", with the prefix "1DF8.output" that you specified in dock.in file. In the case that you would have several runs of dock6 program with the output file of the same path (in the same directory), and you want to keep the result for each run, you need to rename "1DF8.output_scored.mol2" file to another one, otherwise the old .mol2 file will be overwritten by the new .mol2 file generated in the next run.

For example, you can rename the old .mol2 file generated by the first run and then perform the second run.

$mv 1DF8.output_scored.mol2 first_1DF8.output_scored.mol2

Tuoling

Some recommended parameters we can change when running dock

max_orientations                                             500
min_anchor_size                                              40
pruning_conformer_score_cutoff                               25
pruning_clustering_cutoff                                    100

Explanations of these parameters can be found on the website provided by Long Fei. Increase the number of these parameters can lead to increased running time. But it enables more thorough cycling and may give you better results.

Yuchen

Always Remember Your Path

One situation often appears is that the program will return an error saying "Could not open 1DF8.ligands.mol2 for reading." or something like that indicating that the program couldn't find the input file you specified in you dock.in or vs.in or some other files. In this case, the most often reason is that the path you specified in your input file is wrong. So the first thing you might want to do is to check those paths, pay special attention to those paths where you use "." to indicate the same folder and ".." to indicate the folder up one level because this is the place where you are most likely to make mistakes. If you are not sure of the usage of "." and "..", it will be safer for you to use the absolute paths of the files. And you can use the command pwd to ask the system where you are if you don't know that.

Yunting

Delete Spheres Manually

We need to play some tricks if we want to manually select the spheres in selected_spheres.sph.

1. Transfer sph file to pbd file by using showsphere.
2. Open the pdb file in Chimera and choose the sphere we want to delete.
3. Identify the number of that sphere by Actions->Label->residue->specifier.
4. Open sph file, delete that sphere and modify the number of spheres in that cluster.

DOCK spheres within 6.0 ang of ligands
cluster     1   number of spheres in cluster    22
  60  28.86000  11.09173   4.97943   2.337  586 0  0
  67  28.67840   9.74620  12.13940   1.400   60 0  0
 161  27.11509  13.42709  13.48051   1.401  385 0  0
 174  27.79940  12.82468  12.04078   2.475  480 0  0
 183  34.13903  14.01393   7.31591   3.590  691 0  0
 187  36.78641  17.02434   9.54436   3.481  691 0  0
 385  30.01753  14.79655  14.46633   1.402  174 0  0
 463  25.74631  14.83304   9.98248   1.400  589 0  0

Kip

Molecule preparation for molecular footprinting

A sample script:

#!/bin/tcsh
 
#check programs
foreach prog (antechamber parmchk tleap)
        set prog_version = `which $prog`
        if ( $prog_version == /nfs/user03/tbalius/amber10_ompi/bin/$prog ) then
                echo $prog OK, current path is: `which $prog` 
        else
                echo please double-check $prog path, current path is: `which $prog`
        endif 
end  

#set your home directory on seawulf
set mydir = "$HOME"

# set up your ligands
foreach lig (ligand_name1 ligand_name2 ligand_name3)

        #set working dir and go there
        echo setting up directory for: $lig
        mkdir  "$mydir/DOCK_project2/10-ligandprep-top2mol2/$lig"
        set workdir = "$mydir/DOCK_project2/10-ligandprep-top2mol2/$lig"
        cd $workdir
        cp ../$lig.mol2 .
        echo 'Done!'

        #convert mol2 to pdb and prep files
        echo converting mol2 to pdb and prep files
        antechamber -i $lig.mol2 -fi mol2  -o $lig.ante.pdb  -fo pdb
        antechamber -i $lig.mol2 -fi mol2  -o $lig.ante.prep -fo prepi
        echo 'Done!'

        #check for missing parameters
        echo checking parameters for ligand: $lig
        parmchk -i $lig.ante.prep -f  prepi -o $lig.ante.frcmod
        echo 'Done!' 

        #kludgey way to make input files
        echo making input file for ligand: $lig
        echo "set default PBradii mbondi2" > tleap.$lig.in                                          
        echo "source leaprc.ff99SB" >> tleap.$lig.in        
        echo "loadoff ../rizzo_amber7.ionparms/ions.lib" >> tleap.$lig.in
        echo "loadamberparams ../rizzo_amber7.ionparms/ions.frcmod" >> tleap.$lig.in             
        echo "source leaprc.gaff" >> tleap.$lig.in
        echo "loadamberparams ../rizzo_amber7.ionparms/gaff.dat.rizzo" >> tleap.$lig.in
        echo "loadamberparams $lig.ante.frcmod" >> tleap.$lig.in
        echo "loadamberprep $lig.ante.prep" >> tleap.$lig.in     
        echo "lig = loadpdb $lig.ante.pdb" >> tleap.$lig.in      
        echo "saveamberparm lig $lig.gas.leap.prm7 $lig.gas.leap.rst7" >> tleap.$lig.in  
        echo "solvateBox lig TIP3PBOX 10.0" >> tleap.$lig.in                           
        echo "saveamberparm lig $lig.wat.leap.prm7 $lig.wat.leap.rst7" >> tleap.$lig.in  
        echo "charge lig" >> tleap.$lig.in                                                      
        echo "quit" >> tleap.$lig.in
        echo 'Done!' 

        #run tleap
        echo running tleap for ligand: $lig
        tleap -s -f tleap.$lig.in > tleap.$lig.out
        echo 'Done!' 

        #make a new mol2 file
        echo creating final mol2 file for ligand: $lig
        top2mol2 -p $lig.gas.leap.prm7 -c $lig.gas.leap.rst7 -o $lig.gas.leap.mol2
        echo 'Done!'

        #make a new pdb file
        echo creating final pdb file for ligand: $lig
        ambpdb -p $lig.gas.leap.prm7 -tit "pdb" < $lig.gas.leap.rst7 > $lig.gas.leap.pdb
        echo 'Done!'
end

Keeping your data in sync

One problem you may encounter is that you want to run your jobs on Seawulf, but you also want the same files on a mathlab computer (or your home computer) for analysis. One option is to use RSYNC to keep your files on Seawulf and Herbie (or any mathlab computer) in sync. For example, if you want to move your DOCK Tutorial files to Seawulf, do the following from Herbie or any mathlab computer:

rsync -arv /home/username/DOCK_Tutorial/ sw:/nfs/user03/username/DOCK_Tutorial
  • Note: The trailing slash on /home/username/DOCK_Tutorial/ means that rsync will only copy the contents of your DOCK_Tutorial folders. If you want to copy the DOCK_Tutorial folder itself, as well as its contents, then remove the trailing /.

To copy newer files from Seawulf back to Herbie, do the following from Seawulf:

rsync -arv /nfs/user03/username/DOCK_Tutorial/ username@compute.mathlab.sunysb.edu:/home/username/DOCK_Tutorial

You can apply this same strategy to then sync files with your home computer as well. Use the following format:

rsync -arv source target
  • Note: this is easiest if you are using a Linux or Mac computer at home.

Deleting jobs

If you make a mistake and need to delete a job from the queue, first list all your queued or running jobs:

qstat -u username
  • IMPORTANT: If you get no output from qstat, it means that whatever jobs you have submitted are done!

If you do have jobs running or waiting in the queue, qstat will output a list that includes their job id(s). Find the job id for the one you want to delete, and do:

qdel jobid