This article provides a comprehensive guide to computational protein-ligand docking and virtual drug screening using the AutoDock suite of programs. The methods are widely used for studying biomolecular interactions and structure-based drug design. The protocol covers various aspects of docking and virtual screening, including basic docking, virtual screening with a small ligand library, docking with selective receptor flexibility, active site prediction, and docking with explicit hydration. The entire process is estimated to take approximately 5 hours. The AutoDock suite includes several complementary tools: - **AutoDock Vina**: A turnkey computational docking program with rapid gradient-optimization conformational search. - **AutoDock**: A computational docking program based on an empirical free energy force field and rapid Lamarckian genetic algorithm search. - **Raccoon2**: An interactive graphical tool for virtual screening and analysis. - **AutoDockTools**: An interactive graphical tool for coordinate preparation, docking, and analysis. - **AutoLigand**: A program for predicting optimal sites of ligand binding on receptors. The article also discusses the limitations of the default methods and advanced techniques to improve results, such as treating receptor sidechains explicitly and modeling explicit water molecules. The protocol is demonstrated using the kinase domain of the proto-oncogene tyrosine protein kinase c-Abl as a target, including redocking experiments, virtual screening, cross-docking, active site prediction, and docking with explicit water molecules. The article emphasizes the importance of careful coordinate preparation and provides detailed steps for each method, along with troubleshooting tips. It highlights the effectiveness of the AutoDock suite in solving specific problems and its widespread use in research and drug discovery.This article provides a comprehensive guide to computational protein-ligand docking and virtual drug screening using the AutoDock suite of programs. The methods are widely used for studying biomolecular interactions and structure-based drug design. The protocol covers various aspects of docking and virtual screening, including basic docking, virtual screening with a small ligand library, docking with selective receptor flexibility, active site prediction, and docking with explicit hydration. The entire process is estimated to take approximately 5 hours. The AutoDock suite includes several complementary tools: - **AutoDock Vina**: A turnkey computational docking program with rapid gradient-optimization conformational search. - **AutoDock**: A computational docking program based on an empirical free energy force field and rapid Lamarckian genetic algorithm search. - **Raccoon2**: An interactive graphical tool for virtual screening and analysis. - **AutoDockTools**: An interactive graphical tool for coordinate preparation, docking, and analysis. - **AutoLigand**: A program for predicting optimal sites of ligand binding on receptors. The article also discusses the limitations of the default methods and advanced techniques to improve results, such as treating receptor sidechains explicitly and modeling explicit water molecules. The protocol is demonstrated using the kinase domain of the proto-oncogene tyrosine protein kinase c-Abl as a target, including redocking experiments, virtual screening, cross-docking, active site prediction, and docking with explicit water molecules. The article emphasizes the importance of careful coordinate preparation and provides detailed steps for each method, along with troubleshooting tips. It highlights the effectiveness of the AutoDock suite in solving specific problems and its widespread use in research and drug discovery.