Docking small molecules with Autodocktm

Instructor: Elon YarivAutodock Tutorial

Protein DataBank (PDB) . .www rcsb org

1971 – PDB founded with 7 X-ray structures.2015 – 102,318 X-ray, 11,256 NMR & 933 EM structures.

PDB HomepageSequence

Ligand structureDrug Target

New StructuresType ALK

• 90 search results.• Scroll down to PDB entry 4CNH.

Click to enter!!

Click!!

Visualizing structures in Autodock Tools

File->Preferences->Set…

Type working directory here

3D Viewer

DashBoard

Menu

File->Read molecule

Or Click

New molecule appears

Hold to rotate around Axis

Hold to move

Hold/Scroll to zoom + Shift

Hold to move in/out

Select

You can change selection level through this

menu to atoms, residues, chains

or molecules.

Select 4CNH.pdb you’ve just

downloaded

+ Shift

+ Shift

Left Click rectangleto select chain B

Edit->Delete->Delete selected AtomsEdit->Delete Water

Two identical proteins in crystal symmetry unit.The protein is monomeric.We will delete chain B and all water molecules.

Left Click to expand

Step1: Expand chain A and select residue 3U92402.

Step2: create new set, and call it “ligand”

Step3: Select the protein, subtract the ligand and create

another set: “receptor”

Representations• L – line• B – Ball & Sticks• C – Space fill• R – Cartoon• MS – Molecular surface• L – Labels• CL - Colouring

Step1: Select ligand (green dot)

Step2: Select->Spherical region

Step3: left click

Step4: Click

Step5: select from protein

Step6: click select, then closeBefore closing make sure the selection level is at residues.

Add the selection to a new set: “protein near ligand”

● Hide everything.● Change “protein” representation to cartoon.● Colour by rainbow.● Show ligand and “protein near ligand” as ball&stick.● Right click the show ball&stick button next to “ligand”.

You can change the representation of “ligand” and “protein near ligand”Individually.

To render high quality images go to:3D graphics->Render large image

File->Save->Current session

Save to a folder of your liking.

Session file ends with .psf

To load an old session:

File-> Read session

Saving Your Progress:

File->Save->Write PDB

Call the new file – 4CNH_monomer.pdb

Confirm

Saving structure files:

Break

PDB structures aren’t perfect• Missing residues.• Missing sidechains.

Hydrogens cannot be resolved in most X-ray structures

• Hydrogens aren’t present in PDB structures.• Histidine protonation.• Asp/Gln orientation.

Adding explicit hydrogensOpen ‘Edit’ menu

Press to add hydrogens

Quick solution but not the best.

We will not do this in this workshop.

Molprobity - molprobity.biochem.duke.edu/• Adds explicit hydrogens.• Asp/Gln flips.• Changes Histidine protonation.

type ‘4CNH’

Or upload 4CNH_monomer.pdb

Select this option

Fix Asn/Gln/His residues

Hydrogen placement for X-ray structures

Click to add hydrogens

Review Results

Confirm results and download .pdb file

Choose which residues you want to flip.

Steric Clashing in PDB structures

Structures must be minimized to relax protein strain.

Chimera built-in minimization: www.cgl.ucsf.edu/chimera/3Drefine (webserver): sysbio.rnet.missouri.edu/3Drefine/Chiron (webserver): troll.med.unc.edu/chiron/login.phpPDB-Redo (webserver): www.cmbi.ru.nl/pdb_redo/

Select the ligand, and then remove it

Don’t forget to set selection level to residues.

• Read the molprobity output file.• Delete the ligand

residue within the structure.

Preparing the ligand• Automatically detects

rotatable bonds, rings, assigns charges and merges non-polar hydrogens.

• After loading the ligand, in the torsion tree menu, select “detect root…”.

Change file type to .mol2 then

choose 3U9.mol2 from the tutorial

folder.

Open the ligand menu

Select “Choose Torsions…” option.

Rotatable bonds and rings are highlighted.

You can make bond non-rotatable by selecting it.

Number of torsions in molecule.

Confirm selection

Important: Sampling more then 9-10 torsions is inefficient!!!

• Save the ligand.• Hide root marker.• Hide it and display the

receptor – 4CNH_monomerFH_reg

PDPQT format:Contains atom coordinates of structure. This file also

contains partial charges and autodock atom types.

Both ligand and receptor must be converted to PDBQT.

Recognized atom types:H, C, N, O, F, Mg, P, S, Cl, Ca,

Mn, Fe, Zn, Br and I.

Open the ligand menu

Preparing the Grid input files

Open Grid menuSelect this molecule

Click

After dismissing the warning message,

select the directory and the name of the protein .PDBQT file.

Determining grid dimensionsOpen menu through

Grid->Gridbox…

Set box size to

50x50x50

Set centroid to:(45,-4.5,-49)

The Grid box appears at origin

of axis (0,0,0).

The position and size of the box must be altered

When finished press the file tab in the Grid Box window.

Choose: “Close saving current”

Select the Ligand

Autogrid will use the atom types found in

the ligand.It will place them at

each grid point

Open Grid menu

Confirm

• Save the grid file as “4CNH.gpf”.

This file includes the grid box dimensions and the atom types found in the ligand molecule.

Open Grid menu

Save the grid parameters file

Choose the .gpf you’ve created

Creating the Grid

Name of the log file (.glg extension)

Filled in automaticall

y

Once finished press “Launch”.

Open Run -> AutoGrid…

Select autogrid4.exe

Monitoring results• Job last about 15 seconds.• Can be monitored by opening 4CNH.glg with Notepad++.• Or with the ‘tail’ command in Unix.• Here you will find the error messages.

• Output: Grid map file for each atom type in the ligand..map file: List of the scores of every point in the grid, most values are zero or large positive numbers.

Initializing the docking simulations

(But first we must prepare the parameter files)

Open Docking menu and select Open Docking menu

and select

Choose the receptor structure file:4CNH_monomerFH_reg.pdpqt

Choose ligand “3U9”.

A new window will open that allows you to review ligand properties, set the initial

location of the ligand and the initial number of torsions.

Press “Accept” without changing anything.

Docking -> Search Parameters -> Genetic Algorithm

New window opensNumber of output results

Change duration to short

Confirm to exit

Short simulation: about 1 minMedium simulation: about 10-20 min

Long simulation: Can only guess…

Always generate more then 1 ligand pose.

“Docking Parameters…” window• For advanced users.• You shouldn’t mess with these!

• Add the crystal ligand conformation, i.e. 3U9.pdb, as a reference for RMS calculations.• This will give you an indication

how well the results were.Write the directory of

3U9.pdb

Select ‘no output’

“Accept” to Exit

Click to expand

• This file contains all the sampling parameters you’ve just set, the receptor file, ligand file and grid file location.

• Call it “3U9.dpf” do not forget to write the file extension.

Docking -> Output-> Lamarckian GA(4.2)…

Running Autodock (finally!)

Choose the .dpf you’ve created

Select Autodock4.exe

Press to start simulation

Select autodock4.exe

Loading results• To load results into open the Analyze menu->Dockings->Open…• Select 3U9.dlg – the output file.• To view the different poses:Analyze->Conformations->Play, Ranked by Energy

Pose Rank

Next pose

Play/PausePose Player

optionsMove to first pose

Exit

Displaying Hydrogen bonds• Open the analyze menu->Macromolecule->Choose…• Select 4CNH_monomerFH_reg and confirm• Open the pose player options menu

Select to build Hydrogen bonds

Show energy components for

docked pose

Final docked pose

• 2 H-Bonds• 1.34Å RMSD from

crystal coordinates.• Maximal deviation 4.3Å

This window is opened by ticking the show info box shown in the

previous slide

Break