A covalent docking setup can feel deceptively simple: choose a protein, choose a ligand, and click Dock. But when the ligand is already attached to the protein in a crystal structure, its recorded chemistry may describe the bound form—not the free ligand the docking workflow needs to model. A bond order or hybridization mismatch can therefore become an important preparation step.
This walkthrough focuses on a practical case from the FITTED Suite tutorial: covalent self-docking of the 7KH 301 ligand in the human cathepsin L structure 5MAJ. The central lesson is to distinguish three tasks: prepare the ligand’s chemical representation, define where it binds, and identify the protein residue involved in the covalent reaction.
1. Check the ligand’s chemistry first
Open 5MAJ.sam in SAMSON and inspect the ligand before configuring docking. In the tutorial, the ligand is already covalently attached to the protein. The C8–N7 bond is changed to a triple bond because the ligand is a nitrile when unbound. The C8 and N7 atoms are also assigned SP hybridization.
These edits are specific to the example, not a general instruction to make every ligand bond triple. The useful habit is to verify whether the bound structure’s bond orders and atom properties represent the ligand form you intend to dock. In SAMSON, the tutorial makes these changes through Edit > Edit bonds or the Inspector; hybridization is set in the Inspector after selecting the atoms.
Because the ligand has been manually edited, leave its chemistry intact when passing it to FITTED Suite: select the ligand, choose From document under Set ligand, and uncheck the option to prepare it (perceive bond order and add hydrogens). In this workflow, automatic ligand preparation could overwrite the edits you just made.
2. Define the receptor and binding site
In the FITTED Suite app, select the 5MAJ structural model and set it as the receptor using From document. The tutorial leaves the water-molecule and macromolecule settings at their defaults (Protein).
For this self-docking example, use the existing bound 7KH 301 ligand to define the binding site. Find the ligand in chain A, select it, then choose From bound ligand in the binding-site controls and click Set. If no bound ligand is available, the documented alternative is to define the site from a position using the sphere in the Viewport.
This separation is useful when troubleshooting: the ligand used to define the search region and the ligand supplied for docking may be the same structure in a self-docking test, but they serve different setup roles.
3. Tell the workflow what reacts
Set the docking mode to Covalent only. The tutorial uses two runs; this is an example setting, not a universal recommendation. Then specify the covalent residue. In 5MAJ, the ligand reacts with the sulfur atom (SG) of CYS 25. Selecting the residue—or an atom in it—and clicking Set for Covalent residue identifies the reaction partner.
A basic atom can also be specified, but it is optional. The tutorial uses ND1 in the neighboring HIS 163 residue. This is a separate input from the covalent residue: the covalent residue is the one that reacts with the inhibitor; the basic atom is an optional atom in an adjacent residue.
4. Review the result in context
After docking, the results appear in the table and are loaded into the document. The processed receptor is named 5MAJ_pro; the covalent pose includes part of the residue to which the ligand is docked. You can select a result row to select its corresponding pose, hide the original structure in the Document view, and add a Ribbons visual model to inspect the complex more clearly.
When reviewing a pose, check that the intended covalent connection is represented and that you are examining the processed receptor and the corresponding ligand pose. For additional interaction analysis, the tutorial also describes the Protein-Ligand Interaction Analyzer and Hydrogen Bond Finder.
The key preparation check is straightforward: confirm the ligand’s intended chemistry, preserve deliberate edits by disabling automatic ligand preparation when appropriate, and explicitly identify the protein residue involved in the covalent reaction.
Read the full FITTED Suite tutorial for the complete non-covalent and covalent workflows.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.