Portal

www.dockthor.lncc.br

User Guide
Version 1.0

Contributors

Diogo A. Marinho, Isabella A. Guedes, Eduardo Krempser, Camila S. de

Magalhães, Hélio J. C. Barbosa and Laurent E. Dardenne

www.gmmsb.lncc.br

Laboratório Nacional de Computação Científica – LNCC/MCTI

14 July 2013

INPI Software Registration Number 13318-3

DocktThor® registered mark
 Portal DockThor

1. Introduction

The DockThor Portal, developed by the group GMMSB/LNCC, is a free receptor-

ligand docking server idealized to facilitate and enable the use of the docking
methodology by the academic community. The implemented DockThor® program is a
flexible-ligand and rigid-receptor grid based method that employs a multiple solution
genetic algorithm and the MMFF94S molecular force field scoring function. The major
steps of the ligand and protein preparation are available on the DockThor Portal, being
possible to change the protonation states of the amino acid residues and include
cofactors (e.g. structural water molecules, metals, organic molecules) as rigid entities.
The user can also customize the main parameters of the energy grid and the genetic
algorithm.
The results of the docking process are analyzed and ordered automatically. The
parameters of the analysis can also be customized by the user. The DockThor Portal
employs the computational facilities provided by the Brazilian SINAPAD (Sistema
Nacional de Alto Desempenho) high performance platform.

2. Submiting a Docking Job

In the present version 1.0 of the DockThor Portal only protein receptors and
only .pdb filetype are accepted (or pre-prepared DockThor input files *.in). Non-proteic
receptors (e.g. DNA, RNA, another ligand) will be allowed in the next portal version.

2.1 Protein Preparation

1. Click on the Docking tab. After, click on the Protein tab to open the protein
preparation page.
 2. To submit your protein file click on the Upload button. So far, it is only
possible to upload Protein Data Bank (.pdb) type or DockThor input (.in)
protein files. The structure of the catalytic domain of human stromelysin
complexed with a non-peptide inhibitor (PDB code 1CAQ) is available as a
test case. To use this structure, click on Upload test file.
3. Prepare your protein file with the basic options clicking on Prepare. In this
step, all the missing amino acid residue side chain atoms will be
reconstructed. The protein atoms are recognized by the initial .pdb label
'ATOM', Atoms associated with the initial .pdb label 'HETATM' are ignored.
All the atoms are also recognized by their .pdb atom label (e.g. CA, CB etc).
If the atom label nomenclature is not correct the atom will be
reconstructed. For this reason, all the hydrogen atom positions will probably
be reconstructed1. If two side chain conformations are present for the same
residue in the .pdb file, only the first one will be considered by the program.

1
To maintain the original H's positions you have to use the same H atom .pdb nomenclature
used by the DockThor program (download and examine the generated *.in file).
4. Set the protonation states of the residues (Asp, Cys, Glu and His are set to
the default values - see appendix A). In the protein test file it is important to
change the protonation states of some histidine residues, which interact
with metals: His-123, His-119 and His-129 must be of type HISD.
5. Reprepare your protein file with the new protonation states clicking on
Reprepare.
6. View the prepared protein file with JSmol clicking on (View 3D). Useful
information about the use of this plugin can be found in
http://www.chem.uwec.edu/JmolTut/.
7. Click on Download prepared files to download the respective protein files.
protein.in – DockThor receptor input file (contains the MMFF94 atomic type
number, the atomic coordinates, the bond connectivity, the atomic partial
charges, the pdb type atom, residue and chain labels and the residue and chain
numbers).
resumo.out – contains the information of the protein preparation process.
protein_prep.pdb – prepared protein file in the Protein Data Bank format
(equivalent to the protein.in file for visualization).
protein.X - configuration file for each protein chain X. Contains the amino acid
residue list and the respective protonation state label.

8. Click on Next to submit your prepared protein file to docking. It is important

to notice that only the protein file in the format .in is necessary to docking.
 2.2 Ligand Preparation
1. To submit a single small molecule2 to docking, click Upload. If you want to
use the ligand test file (non-peptide inhibitor, PDB identifier DPS) click on
Upload test file).

2. The recent uploaded ligand must be prepared to generate the respective

topology file (.top) for docking. This step comprises the correct MMFF94S
force field atom type assignment, the atomic partial charges calculation and
the assignment of the rotatable chemical bonds. If you want to add
hydrogen atoms automatically, just check the Add hydrogens box and
Reprepare the molecule again. The hydrogen atoms will be added by
OpenBabel tool using pH = 7.0.
3. To view the most recent structure of your ligand, click (View 3D).

2
In the present portal version, it is possible to dock only one ligand at time. A virtual screening version of
the DockThor Portal will be available in the future.
4. You can view the selected rotatable bonds just clicking on the “Rotatable
bonds...” button. If you uncheck some flexible rotatable bonds click on
Reprepare.
 Attention: If you click on Reprepare twice without check/uncheck one or more
rotatable bonds, all boxes will be automatically checked.
5. To download the prepared ligand files click on Download prepared files.
ligand.top – DockThor ligand input file. It contains the atom name, the atom
number, the MMFF94S atomic type number, the atomic partial charges, the
atomic coordinates and the atom valence. It contains also: (i) the atom
connectivity; (ii) force field torsional parameters; (iii) selected flexible bonds; (iv)
non-bonded intramoleular atom interactions.
new_ligand.pdb – prepared ligand in the Protein Data Bank format (for
visualization). This file is generated only when the Add hydrogens option is chosen.

6. Click Next to send the most recent ligand file to docking.

2.3 Cofactor and Water Preparation

1. For some protein-ligand complexes it is important to consider cofactor (e.g.

NAD, ATP, FAD, Mg, Zn etc.) and/or water molecules. The DockThor Portal
allows one or more cofactor and water molecules to be included; they are
kept fixed during the docking simulation. Upload each file at a time clicking
on Upload.
 2. As well as ligand, the cofactor and water files (one or more water molecules
per file) need to be converted to the topology file. Check the respective
boxes to add hydrogen atoms3 and click Prepare.
3. It is also possible to download the prepared files correspondent to each
cofactor/water clicking on Download prepared files. Each cofactor/water
file will generate one link for download. In the case of our test file, all the
five cofactors (two zinc and three calcium atoms) are into the same file.
4. Send the cofactor and/or water to docking clicking Next.

2.4 Docking Configuration

1. The next tab consists of the main options to run the docking job. It is
possible to see the original files for the ligand and the protein in Files
uploaded. Check if are correct.

2. Fill the Email field to receipt the link for the results page when the docking
job is finished. This field is mandatory.

3
Since MMFFLigand do not optimize the hydrogen atoms position, it is recommended to do it
previously and do not chose to include hydrogen atoms to these water files.
3. Insert the center coordinates of the grid energy in Grid center (Xc, Yc, Zc).

4. Insert the Grid Dimensions (±Δx, ±Δy, ±Δz). These values correspond to the
half of the grid size in each dimension, e.g. (Xc-Δx) ≤ X Dimension ≤ (Xc+Δx)

5. Select the spatial discretization of the energy grid. This value corresponds to
the spacing between the points of the grid (the default value is 0.25Å).
Check if the number of grid points do not exceed the limit allowed
(1,000,000) in the Grid points box.

6. To view the grid dimensions and the receptor click (View grid in 3D).
7. Choose a label to facilitate the identification of your docking job.
8. Some genetic algorithm (GA) parameters can be modified.
- The GA multisolution algorithm was optimized to deal with highly flexible
ligands and we do not recommend changing the standard number of
evaluations per run (i.e. 1,000,000) or the GA population size (i.e. 1,000).
Change these values at your own risk.
- Each submission job corresponds to 30 independent docking runs. A maximum
of 50 docking runs per job is permitted. It is also possible to run more
 independent docking runs submitting more than one job and changing the
initial seed. The seed must to have a negative value and for each run its value is
decreased by one. You should take this into account if you want to submit more
than one job in order to obtain more than 50 independent docking runs.

9. Run your docking job clicking on Dock!

10. When your docking is finished an email will be sent with a link to the results
page.

3. Analyzing the Docking Job

Once the docking job is finished, the user receives an email with the link to the
Results page as below:
 Your docking result is available at
http://www.dockthor.lncc.br/index.php?pg=submission&pgs=results&id=docking_gmmsb_1
caq_ligand_9l84mb

Follow the link in the email to go to the Results and Analyses page.

3.1 Results and Analyses

For each docking run the final population is clustered using the total energy and
a RMSD = 1.0Å criteria. Only the leaders (top energy solution) of each cluster will be
used to the final clustering analysis step:
1. It is possible to cluster and order the docking solutions according to two
criterions: Total Energy (intermolecular ligand-receptor + intramolecular
ligand energies) or Interaction Energy (only intermolecular ligand-receptor
energy). The default is Analysis by total energy.

2. Click Analyze to perform the docking poses analyses.

3. You can change the clustering criterion (default = 2.0Å) and the number of
the best ligand binding modes (default = 20) selected to analyses and
visualization. Click on the Result Settings button.
4. It is possible to view the results interactively in the website. Click on View
results interactively.
5. To see each docking ligand binding mode (i.e. cluster leader) select the
corresponding one on the results table. For each solution is shown the
corresponding Run of the Genetic Algorithm, the Model (number of the
cluster leader) in the corresponding GA run, the Total Energy, the
Intermolecular Energy and the RMSD (root mean square deviation
calculated using the non H atoms) relative to the top ranked pose. This value
gives an idea of the conformational difference between the alternative
ligand binding modes.
Attention: If you have submitted a reference file, the last column refers to the
RMSD between each pose and the reference conformation.
6. It is also possible to upload a reference ligand conformation (.pdb type file)
to perform redocking analyses or to help the investigation of the distinct
binding modes. After this click Analyze to perform the docking poses
analyses and click on View results interactively. The RMSD (in the last
column) is now calculated relative to the uploaded reference ligand pose. It
is possible to hide the protein to facilitate the visualization. If necessary,
click on Reload Visualization to upload the JSMol scene.
7. To download the docking results, including the clustered conformations,
click Download Results.
dockthor.out – general information about each DockThor run.
parameters.txt – general information about the docking parameters used.
results.out – total number of clusters obtained (after analyzing all runs).
out.log – summary of the clustering analysis for the best cluster leaders (ranking,
energies, RMSD).
out.mol2 – contains the atomic coordinates of the best cluster leaders (multimodel
.mol2 type file, ordered according to the results described in out.log).
ligand_run_X.log – contains the information of the cluster leaders obtained in run X
(using a RMSD criterion of 1.0Å).
ligand_run_X.pdb – contains the atomic coordinates of the cluster leaders obtained in
run X (multimodel .pdb type file).
protein.in – DockThor receptor input file.
ligand.top – DockThor ligand input file.
 8. It is always possible to perform other analyses and download the respective
files.

4. Softwares

The Portal DockThor uses the following programs:

MMFFLigand: generates the topology file for the ligand and cofactor files
through MMFF94S force field and OpenBabel tools;
PdbThorBox: prepares the protein file (adds hydrogen atoms, changes amino
acid protonation states, completes missing side chains) with the MMFF94 force
field;
DockThor: the docking program is a flexible ligand rigid receptor grid based
method that employs a multiple solutions genetic algorithm as the search
method and the MMFF94 force field as the scoring function.
Dtstatistic: clusters and ranks the docking poses according to total or
interaction energy.
* All these programs were developed by the GMMSB/LNCC group.
Appendix A

Residue Protonation States

ASP - Negatively charged aspartic acid (default).

ASPN1 - Neutral aspartic acid with a H bonded to the Oδ1
ASPN2 - Neutral aspartic acid with a H bonded to the Oδ2

GLU - Negatively charged glutamic acid (default).

GLUN1 - Neutral glutamic acid with a H bonded to the Oε1.
GLUN2 - Neutral glutamic acid with a H bonded to the Oε2.

CYSH - Neutral cysteine with a H bonded to S (default).

CYS - Negatively charged cysteine.
CYSS - Neutral cysteine (disulfide bond).

HIS - Neutral histidine with a H bonded to N (default)

HISD - Neutral histidine with a H bonded to N.
HISP - Positively charged histidine.

ARG - Positively charged arginine (default).

ARGN1 - Neutral arginine at Nω1.
ARGN2 - Neutral arginine at Nω2.

LYS - Positively charged lysine (default).

LYSN - Neutral lysine.