A covalent inhibitor is not just a ligand positioned in a binding pocket: it forms a bond with a receptor residue. That difference creates a practical modeling challenge. If the ligand is prepared as though it were an ordinary, unbound molecule, its bond orders and hybridization may no longer represent the reactive form you intend to dock.
This walkthrough focuses on a useful safeguard: preserve the ligand’s intended chemistry, then explicitly identify the receptor residue involved in the covalent interaction. The example uses the cathepsin L structure 5MAJ and its bound ligand, 7KH 301, in SAMSON’s FITTED Suite.
Start with a bound complex
Open 5MAJ.sam from the FITTED tutorial archive, or download the structure in SAMSON using its SAMSON Connect asset. The structure contains the 7KH 301 ligand covalently bound to the protein. Using a bound ligand to define the binding site makes this a self-docking setup: the search region is based on the ligand’s known location.
The tutorial structure is already prepared for the example. For other systems, check for issues such as alternate locations, missing residues, or missing heavy atoms. Protein preparation can affect what the docking workflow receives, so resolve relevant input problems before interpreting the result.
Preserve the ligand’s chemistry
In the 7KH 301 example, the C8–N7 bond is set to a triple bond because that portion of the ligand is nitrile when unbound. The C8 and N7 atoms are also assigned SP hybridization. These edits are made in the structural model before setting up the docking run.
This matters because ligand preparation can perceive bond orders and add hydrogens. For this example, after making the bond-order and hybridization edits, leave the FITTED Suite option to prepare the ligand unchecked. Otherwise, the preparation step could overwrite the chemistry you have deliberately specified. This is a case-specific instruction from the tutorial, not a universal rule for every ligand: use the preparation option according to how your ligand was prepared and what edits it needs to retain.
Set the receptor, site, and ligand
- Open the FITTED Suite from Home > Apps > Biology, or find it with SAMSON’s search box.
- Select the 5MAJ structural model, choose From document under Set receptor, and click Set. The tutorial leaves the water and macromolecule settings at their defaults.
- Select the bound
7KH 301ligand and set the binding site using From bound ligand. - Set the ligand from the document. Because its bond order and hybridization have been edited, uncheck the option to prepare it.
Identify the covalent partner
Choose Covalent only as the docking mode and set the number of runs to 2, as in the tutorial. Then specify the covalent residue. In this complex, the ligand reacts with the sulfur atom, SG, in CYS 25 of chain A. You can select the residue itself; the application accepts either the residue or an atom in it and sets the residue accordingly.
A basic atom is optional. The tutorial specifies ND1 in the adjacent HIS 163 residue. Select that atom and click Set for Basic atom if you want to provide it. Choose an output folder if needed, then click Dock. Progress appears on the button, with logs below the results table.
Check what the result contains
When calculations finish, poses appear in the results table and are loaded into the document. Selecting a table row selects its corresponding pose; the table can also be exported as CSV. In the document, look for the processed receptor, 5MAJ_pro, and the resulting ligand pose. A covalent docking pose also contains part of the residue to which the ligand is docked.
Hide the initial structure and its labels to make the result easier to inspect. You can add a ribbons visual model for the processed receptor, hide the structural model, and zoom in on the ligand. In this example, the resulting pose should show the ligand attached to the sulfur of CYS 25. Treat that as a visual check of the modeled attachment, alongside the pose and interaction analysis—not as a substitute for reviewing the input chemistry and the scientific context.
The central lesson is simple: for covalent docking, verify both sides of the setup. Preserve the ligand’s intended bond order and hybridization, and identify the receptor residue that forms the covalent link. Those checks make the workflow’s assumptions explicit and help you interpret the pose that comes back.
Learn more in the FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON at samson-connect.net.