Covalent docking has an easy-to-miss setup challenge: the ligand in a crystal structure may already be attached to the protein, even though the docking calculation needs a representation of the ligand before it formed that bond. If bond order, hybridization, or preparation settings are inconsistent, the input may no longer describe the reactive ligand you intend to dock.
The 5MAJ example in SAMSON’s FITTED Suite tutorial offers a practical way to think through this setup. It uses a ligand covalently bound to cathepsin L and walks through the details that differ from a non-covalent docking workflow. The key is to treat ligand chemistry, receptor site, and reacting residue as connected parts of one setup—not as interchangeable selections.
Start with the ligand’s intended chemistry
In the example, the ligand is 7KH 301. Before setting it in the FITTED Suite, the tutorial modifies the C8-N7 bond to a triple bond and sets the hybridization of both C8 and N7 to SP. These changes represent the nitrile form of the ligand when unbound.
This is why the ligand-preparation checkbox matters. For the non-covalent example, ligand preparation is left enabled to perceive bond orders and add hydrogens. For this covalent example, preparation is turned off after the bond order and hybridization have been deliberately modified. Applying automatic preparation after making those edits could conflict with the chemistry you have specified.
Before proceeding, check that the intended bond order and atom hybridizations are present, and make a note of whether ligand preparation should be enabled or disabled. This small check helps prevent a later step from undoing the chemical representation you meant to use.
Define the receptor and binding site
Open the 5MAJ structure and set its structural model as the receptor in the FITTED Suite. The tutorial leaves the water-molecule setting at its default and keeps the macromolecule type set to Protein. For the binding site, it uses the already bound 7KH 301 ligand. Selecting the bound ligand and choosing From bound ligand gives the workflow a site reference tied to the structure.
If a suitable bound ligand is not available, the FITTED Suite also allows the binding-site center to be specified by position. In that case, the center is adjusted using the sphere in the viewport. Whichever method you use, make sure the site is set before starting the docking run.
Specify the covalent partner
Set the docking mode to Covalent only, then identify the covalent residue. In this example, the ligand reacts with the sulfur atom SG in CYS 25 of chain A. The interface accepts either the residue or an atom in it; specifying the residue is sufficient for the workflow described here.
A basic atom can also be provided, but it is optional. The tutorial selects ND1 in the adjacent HIS 163 residue. If you use this option, verify that the selected atom is the intended neighboring atom rather than just another atom with a similar name.
Run, then inspect what was imported
The tutorial sets the number of runs to two and makes importing only the best pose optional. Choose an output folder if you want to control where results are saved, then start docking. The app displays the current stage on the Dock button, with logs below the results table.
When the calculation finishes, results appear in the table and are loaded into the document. In the document, the processed receptor is named 5MAJ_pro. A covalent result pose also contains part of the residue to which the ligand is attached. Hide the original structure and its labels if they obscure the result; adding a ribbons visual model to the processed receptor can make the complex easier to view. Zooming in on the ligand can help you inspect the bond to the sulfur of CYS 25.
A useful final check is to confirm the selected pose, the reacting residue, and the visible covalent attachment in the document. The results table can be exported as CSV, and selecting a row selects its corresponding pose.
For the full walkthrough, including non-covalent docking and further analysis, see the FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.