AutoDock Flexible Molecular Docking Tutorial

Autodock is a molecular docking software package, open source and free, the official website is, the latest version is AutoDock 4.2.6, including AutoDock and AutoGrid two modules, AutoDock software package download address It is, including Linux, Mac OS and Windows versions and source code. AutoDockTools is a visualization program for AutoDock docking. The latest version is MGLTools1.5.6, and the download address is This article will share with you the method of Autodock flexible docking (some amino acids of the protein are set to be flexible, and the docking ligand is flexible).


In this tutorial, the crystal structure of protease 1YT9 is used as the receptor for molecular docking, and the ligand IOS in the structure is used as the docking ligand molecule.

1. Download the crystal structure from the protein database, use the molecular viewing software to extract the structure of the ligand IOS (pymol, viewerpro, Discovery Studio and other software are available), and use other tools to confirm the hydrogenation, charge, and protonation status, and finally Save it as lig.pdb as the ligand structure for molecular docking.

2. Delete the ligand IOS and excess water molecules of the crystal structure 1YT9, and save it as protease.pdb as the receptor structure for molecular docking.

3. Determine the amino acids that need to be flexible (mainly amino acids near the binding pocket, which can be determined with many software). This tutorial uses pymol as an example. Open the 1yt9.pdb file with pymol, click A——preset——ligand sites——cartoon in turn, you can see the amino acids that interact with the ligand, and then click on the amino acid to see the amino acid serial number. the four amino acids D25 and D30 of the A chain and D25 and D30 of the B chain are used as flexible amino acids.

4. Open lig.pdb with AutoDockTools, click Ligand——Input——Choose.

5. Select Output as the lig.pdbqt file.

6. Open the protease.pdb file, first Input——Choose Micromolecule, and select protease. Then expand the triangle symbol on the left, select the previously determined flexible amino acids (D25, D30 of chain A and D25, D30 of chain B), and then click Flexible Residues——Choose Torsions in Currently Selected Residues, and set the above four amino acids For flexibility, click Flexible Residues-Output-Save Flexible PDBQT, and save as protease_flexible.pdbqt, and save rigid PDBQT, and save as protease_rigid.pdbqt.

7. Select Grid——Macromolecule——Open, click to select protease_rigid.pdbqt.

8. Set the grid box and output as the file protease_rigid.gpf. Note that you need to add the suffix gpf when saving under Windows.

9. Click Docking-Macromolecule-Set RigidFilename, select the file protease_rigid.pdbqt, and then select the file protease_flexible.pdbqt to set the flexible residue filename; the final output is protease_lig.dpf, you can modify the number of docking runs in the dpf file.

Finally, run the following command in the cmd window of Windows:

1. autogrid4.exe –p protein_ligand.gpf–l protein_ligand.glg
2. autodock4.exe –p protein_ligand.dpf–l protein_ligand.dlg

The calculation amount of flexible docking is much larger than that of semi-flexible docking, so the calculation time will be much longer.

* For Research Use Only.