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Pelatihan Penambatan Molekul (Molecular Docking)

Molecular Docking using Autodock 4.2


with AutoDockTools in Linux
Test Case: Neuraminidase Inhibitors

Introduction
Molecular docking is a method to predict the binding mode of interaction between two (or more)
molecules, including their interaction energy (scores). Here, we will concentrate on the docking
of small molecule: ligand, and its target: protein receptor.

Prior to performing docking simulation, we have to prepare the required input files. In general,
there are ligand file, receptor file, and configuration file.

In Autodock 4, ligand and receptor should be prepared in pdbqt format. The configuration files
needed are: grid parameter file (gpf) and docking parameter file (dpf). The python scripts for
these purposes have been written and ready to use.

Good news for GUI-lovers. Here, we will do the molecular docking work, from preparation to
results analysis, with the help of user friendly - Graphical User Interface called AutoDockTools
(ADT).

In this exercise, we are going to study the interaction between Neuraminidase Inhibitors (NaI)
and its target: obviously Neuraminidase. Currently, there are two FDA-approved drugs for NaI:
Zanamivir and Oseltamivir. Unfortunately, the mutations occurred at the network region of
binding site: H274Y (histidine mutates into tyrosine at position 274) has greatly reduces N1
subtype of Neuraminidase towards Oseltamivir. On the other hand, Zanamivir, which could not be
formulated in tablet form due to its polarity problem, still maintained its activity on
Neuraminidase H274Y mutant. For this reason, it is always challenging for drug designer to find
the alternative-better NaI than the current one.

AutoDockTools Interface
Autodocktools (ADT) has been installed in our Ubuntu image. We can call ADT by typing
“autodocktools” in terminal.

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$ autodocktools

First, we need to setup the working directory. Go to “File” > “Preferences” > “Set...”

In “Set User Preferences” window, fill up the “Startup Directory” form with
“/home/cadd/Documents/docking” followed by click “Set”. Press “Dismiss” to close the
window.

Menu Bar

ADT4.2 widget

Dashboard
And
Hierarchy
Window

Molecule
Window

Sequence
Window

Figure 1. The interface of ADT.

Exercise 1: Pre-processing the PDB file


Here, we already have chosen PDB ID, i.e. 3CKZ, 3CL0, and 2HU4.

To open the PDB file of 3CKZ, in the Menu Bar (MB), click “File” > “Read Molecule” > select
“3CKZ.pdb” > “Open”.

Click “Edit” in MB then press “Delete Water”.

In MB, click “Edit” > “Hydrogens” > “Add”. Select “All Hydrogens”, “noBondOrder”, and
“yes” then click “OK”.

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In MB, click “File” > “Save” > “Write PDB” and give a name of “3CKZ_H.pdb” then click “OK”.

Clear the molecule window by using “Edit” > “Delete” > “Delete All Molecules”.

In terminal, go to working directory by calling

$ cd ~/Documents/docking

Here, you will be able to differentiate the identity of receptor and ligand.

Separate the ligand and receptor using “grep” command.

$ grep ATOM < 3CKZ_H.pdb > 3CKZ_rec.pdb


$ grep ZMR < 3CKZ_H.pdb > 3CKZ_lig.pdb

Now, we already prepared the separated receptor and ligand.

Exercise 2: Ligand File Preparation for AutoDock


In ADT4.2 widget, click “Ligand” > “Input” > “Open”. Change the “Files of type” from “PDBQT
files” into “PDB files”. Then select “3CKZ_lig.pdb”, click “Open”.

Click “OK” to close summary


Click “Ligand” > “Torsion Tree” > “Choose Torsion”

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Save the ligand in PDBQT format using “Ligand” > “Output” > “Save as PDBQT”. Then give a
name of “3CKZ_lig.pdbqt” then click “Save”

Then we hide the visibility of ligand by clicking the red circle under “L” (lines) for 3CKZ_lig.

Exercise 3: Receptor Preparation for AutoDock


“Grid” > “Macromolecule” > “Open”. Change the “Files of type” from “PDBQT files” into “all
files”. Then select “3CKZ_rec.pdb”, click “Open”.

Close contact between


side chains

The error might be resulted from improper protonation, incomplete structure, missing residues,
etc. The in-depth explanation will be given in the end of exercise 3. Nevertheless, since the
error is not part of our binding site of interest, we can proceed to the next step without the
need of amendment.

Then in “Modified Autodock4 Macromolecule File” window, give a name of “3CKZ_rec.pdbqt”,


click “save”.

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In this case, these atoms have zero charge because they are physically not existed, e.g.
additional hydrogens at N-terminal were given due to abnormal distance of peptide bond
between two residues (amino were created instead of amide). Again, since they are not part of
our binding site, we can ignore them now.

Exercise 4: Preparation of Grid Parameter File


In ADT4.2 widget, click “Grid” > “Set Map Types” > “Choose Ligand”. In “Choose Ligand”
windows, select “3CKZ_lig” then click “Select Ligand”.

Now, we will set up the searching space using grid box.

In ADT4.2 widget, click “Grid” > “Grid Box...”

In this case, the binding site of interest has been revealed from the co-crystallised ligand-protein
complex. Hence, we can define the center the box using the co-crystallised ligand: click
“Center” > “Center on Ligand”. Then you can adjust the number of points, spacing, and center
if you find it necessary.
This snapshot might give you another idea to define the more accurate binding site:

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In “Grid Options” window then click “File” > “Close saving current”

Lastly, we save the grid parameter file using “Grid” > “Output” > “Save GPF...”. Save the
parameter file as 3CKZ.gpf

What to do if the receptor doesn't have co-crystallised ligand? In that kind of situation, we can
rely on literature studies, binding site prediction, blind docking, etc.

Exercise 5: Preparation of AutoDock 4 Parameter File (dpf)


In ADT4.2 widget, click “Docking” > “Macromolecule” > “Set Rigid” then in “PDBQT
Macromolecule File” window, select “3CKZ_rec.pdbqt” > “Open”.

To assign the ligand for docking, again click “Docking”, then “Ligand” > “Choose...”. In
“Choose Ligand” windows, select “3CKZ_lig” then click “Select Ligand”.

In this exercise, no need to change anything here, and proceed with clicking “Accept”.
In AD4.2 widget, click “Docking” > “Search Parameters” > “Genetic Algorithm...”

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Click “Docking” > “Output” > “Lamarckian GA(4.2)...”> save as “3CKZ.dpf”.

Exercise 6: Running AutoGrid 4


In ADT4.2 widget, click “Run” > “Run AutoGrid...”

Make sure all the forms are filled up. In general, AutoGrid command should be:

autogrid4 –p “grid_parameter_file” -l “grid_log_file”

then click “Launch”.

Nice level might be adjusted to decide how “nice” autogrid4's “attitude” towards the other
working program at the same time. Value of -20 means autogrid4 will take the top priority of
CPU processing.

To monitor the progress of autogrid job, run to terminal and type:

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$ tail -f 3CKZ.glg

You can use <ctrl> + c to interrupt command above.

Exercise 7: Running AutoDock 4


In AD4.2 widget, click “Run” > “Run AutoDock...”

Again, make sure all forms are filled up, then click “Launch”

In general, AutoDrid command should be:

autodock4 –p “docking_parameter_file” -l “docking_log_file”

To monitor the docking progress, you can do the same thing with previous step using “tail”
command in terminal for “3CKZ.dlg”.

Exercise 8: Visual Inspection of AutoDock 4 Results


In AD4.2 widget, click “Analyze” > “Dockings” > “Open”. In “Docking Log File:” window, select
“3CKZ.dlg” then click “Open”.

Then “Analyze” > “Conformations” > “Load”. You can select and double click each “3CKZ_lig
x_x” to see their conformations. To save the individual pose of your ligand, click “Write Current
Coords” then give a name of “3CKZ_lig_1_1.docked.pdbq

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As previously described, you can open the macromolecule here by “Analyze” >
“Macromolecule” > “Open...”, and show it in molecular surface representation. You will be
able to see how the ligand fit inside the binding site of receptor.
To see the distribution of all docked conformations by clustering, click “Analyze” >
“Clusterings” > “Show...”

To visually see the distribution of all docked conformations' center, click “Analyze” >
“Dockings” > “Show as Spheres...”

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Grid Maps were calculated in order to speeding up the calculation of interaction energy during
docking. Luckily, this concept provides additional advantages in ligand optimization.
In AD4.2 widget, click “Analyze” > “Grids” > “Open”. In “Load Grid” window, select
“3CKZ_rec.HD.map” and “3CKZ_rec.OA.map” then click “OK”.

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Then, you will see the affinity map of HD (hydrogen donor atom) and OA (oxygen acceptor atom)
like this:

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Exercise 9: Inspection of AutoDock 4 DLG Files


Open the “3CKZ.dlg” file in our working directory using “geany” (or any text editor).

cadd@ubuntu:~/CADD$ geany 3CKZ.dlg

Scroll down on the file until we find “CLUSTERING HISTOGRAM”

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In this case, the best docked conformation has RMSD value of 0.68 compared to the co-
crystallized ligand (reference) hence indicate that the docking parameter used is successfully
reproduce the experimental finding.
Each solution (conformation) can be determined from lines started with “MODEL” and ended
with “ENDMDL”.
MODEL 5
USER Run = 5
USER Cluster Rank = 1
USER Number of conformations in this cluster = 6
USER
USER RMSD from reference structure = 0.679 A
USER
USER Estimated Free Energy of Binding = -9.51 kcal/mol [=(1)+(2)+(3)-(4)]
USER Estimated Inhibition Constant, Ki = 107.03 nM (nanomolar) [Temperature = 298.15 K]
USER
USER (1) Final Intermolecular Energy = -12.19 kcal/mol
USER vdW + Hbond + desolv Energy = -8.77 kcal/mol
USER Electrostatic Energy = -3.42 kcal/mol
USER (2) Final Total Internal Energy = -3.46 kcal/mol
USER (3) Torsional Free Energy = +2.68 kcal/mol
USER (4) Unbound System's Energy [=(2)] = -3.46 kcal/mol
USER
USER
USER
USER DPF = ./3CKZ.dpf
USER NEWDPF move 3CKZ_lig.pdbqt
USER NEWDPF about -30.744801 -56.660000 8.905000
USER NEWDPF tran0 -30.804361 -56.776331 8.903641
USER NEWDPF axisangle0 -0.746572 0.543220 -0.384112 3.803745
USER NEWDPF quaternion0 -0.024777 0.018028 -0.012748 0.999449
USER NEWDPF dihe0 -180.00 -147.60 30.11 176.23 66.95 10.95 -61.58 -3.22 -29.60
USER
USER x y z vdW Elec q RMS
ATOM 1 C2 ZMR A 469 -30.318 -54.417 7.533 -0.16 -0.06 +0.144 0.679
ATOM 2 C3 ZMR A 469 -29.191 -55.081 7.890 -0.34 -0.04 +0.045 0.679
ATOM 3 C4 ZMR A 469 -29.243 -56.202 8.923 -0.28 -0.16 +0.150 0.679

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ATOM 4 C5 ZMR A 469 -30.576 -56.937 8.750 -0.18 -0.11 +0.143 0.679
ATOM 5 C6 ZMR A 469 -31.743 -55.945 8.831 -0.15 -0.11 +0.185 0.679
ATOM 6 O6 ZMR A 469 -31.637 -54.953 7.808 -0.13 +0.11 -0.335 0.679
ATOM 7 NE ZMR A 469 -28.236 -57.231 8.682 -0.24 +0.27 -0.217 0.679
ATOM 8 HE ZMR A 469 -28.043 -57.441 7.703 -0.26 -0.33 +0.178 0.679
ATOM 9 CZ ZMR A 469 -27.567 -57.902 9.528 +0.01 -0.72 +0.665 0.679
ATOM 10 NH2 ZMR A 469 -26.709 -58.827 9.074 -0.38 +0.17 -0.235 0.679
ATOM 11 NH1 ZMR A 469 -27.692 -57.724 10.841 -0.32 +0.34 -0.235 0.679
ATOM 12 2HH2 ZMR A 469 -26.614 -58.964 8.068 -0.39 -0.07 +0.174 0.679
ATOM 13 1HH2 ZMR A 469 -26.171 -59.366 9.754 -0.41 -0.14 +0.174 0.679
ATOM 14 1HH1 ZMR A 469 -27.154 -58.262 11.520 -0.28 -0.31 +0.174 0.679
ATOM 15 2HH1 ZMR A 469 -28.344 -57.020 11.185 +0.06 -0.29 +0.174 0.679
ATOM 16 N5 ZMR A 469 -30.726 -58.010 9.723 -0.02 +0.18 -0.352 0.679
ATOM 17 H5 ZMR A 469 -31.085 -57.765 10.646 +0.13 -0.10 +0.163 0.679
ATOM 18 C10 ZMR A 469 -30.428 -59.297 9.494 -0.29 -0.03 +0.214 0.679
ATOM 19 C11 ZMR A 469 -30.531 -60.214 10.681 -0.47 -0.01 +0.117 0.679
ATOM 20 O10 ZMR A 469 -30.071 -59.736 8.414 -0.80 -0.08 -0.274 0.679
ATOM 21 C1 ZMR A 469 -30.314 -53.127 6.843 -0.25 +0.12 +0.233 0.679
ATOM 22 O1A ZMR A 469 -29.204 -52.638 6.517 -1.06 -0.82 -0.642 0.679
ATOM 23 O1B ZMR A 469 -31.381 -52.541 6.591 -1.14 -0.82 -0.642 0.679
ATOM 24 C7 ZMR A 469 -33.104 -56.584 8.604 -0.10 -0.06 +0.180 0.679
ATOM 25 O7 ZMR A 469 -33.445 -56.419 7.227 +0.01 +0.07 -0.390 0.679
ATOM 26 H7 ZMR A 469 -32.735 -55.953 6.803 +0.09 -0.06 +0.210 0.679
ATOM 27 C8 ZMR A 469 -34.174 -55.898 9.464 -0.25 -0.11 +0.173 0.679
ATOM 28 C9 ZMR A 469 -35.270 -55.276 8.599 -0.30 -0.08 +0.198 0.679
ATOM 29 O9 ZMR A 469 -36.555 -55.534 9.184 -0.26 +0.30 -0.398 0.679
ATOM 30 H9 ZMR A 469 -36.478 -55.561 10.130 -0.31 -0.43 +0.209 0.679
ATOM 31 O8 ZMR A 469 -34.749 -56.865 10.355 -0.15 +0.26 -0.391 0.679
ATOM 32 H8 ZMR A 469 -35.651 -56.625 10.533 -0.16 -0.29 +0.210 0.679
TER
ENDMDL

Real Exercise:
1. Repeat the docking using PDB code 3TI6 (H1N1 2009 complexed with Oseltamivir). You are
expected to give low RMSD value for best docked conformations compared to the co-
crystallized ligand.
2. Do cross docking between Oseltamivir with PDB code 3TI6, 3CKZ. Analyze properly in terms
of binding mode interaction and energy component. Explain the differences of binding
between Oseltamivir against wild type and H274Y mutant.

Happy Docking! ☺

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