A covalent docking workflow has an extra challenge that can be easy to overlook: the ligand’s representation must make sense both as a molecule and as a covalent partner to the protein. A bond order or hybridization that describes the bound structure may not describe the ligand’s unbound form. If those details are inconsistent, preparing the ligand automatically could overwrite edits you intended to preserve.
This walkthrough uses the 5MAJ cathepsin L complex in SAMSON and the FITTED Suite to show which setup decisions are specific to covalent docking—and how to keep track of them.
Start with the bound complex
Open 5MAJ.sam from the FITTED tutorial archive. The structure contains the 7KH 301 ligand covalently bound to the protein. The tutorial example already includes the bond-order and hybridization edits described below, but understanding them is useful when preparing a similar system yourself.
Check the ligand’s bond representation
In this example, the ligand’s C8-N7 bond is changed to a triple bond because the ligand is nitrile when unbound. The C8 and N7 atoms are also assigned SP hybridization. These edits can be made with Edit > Edit bonds or by selecting the bond and changing its parameters in the Inspector; select the atoms to set their hybridization in the Inspector.
The practical takeaway is to distinguish edits that are part of your intended ligand representation from the preparation the docking workflow can perform. For this edited example, when you set the ligand in the FITTED Suite, uncheck the option to prepare it (perceive bond order and add hydrogens). Otherwise, automatic preparation could conflict with the bond-order and hybridization settings you have just specified. For an ordinary non-bound ligand without such edits, the non-covalent tutorial leaves ligand preparation enabled; the right choice depends on the input and the changes you need to preserve.
Define the receptor, site, and ligand
Open the FITTED Suite from Home > Apps > Biology, or find it with SAMSON’s search box. Set the 5MAJ structural model as the receptor. For this protein example, leave the macromolecule type set to Protein and the water-molecule parameter at its default.
Because this is self-docking—the ligand is already present in the structure—use 7KH 301 to define the binding site. Select the ligand in the Document view, then choose From bound ligand in the binding-site section and click Set. Set the same ligand as the docking ligand using From document.
Specify the covalent partner
Choose Covalent only as the docking mode. The workflow also asks for a covalent residue: the residue with which the inhibitor reacts. In the 5MAJ example, this is CYS 25, specifically its sulfur atom, SG. You can select the residue—or an atom in it—and set it as the covalent residue.
A basic atom is optional. The example uses the ND1 nitrogen in the adjacent HIS 163 residue. If you provide one, select that atom and click Set for Basic atom. The tutorial suggests an atom in a residue adjacent to the covalent residue, such as a nitrogen in a neighboring histidine.
Run and inspect the result
Set the number of runs to 2, choose whether to import only the best pose, and select an output folder if needed. Then click Dock. The app displays the current stage on the button and shows logs below the results table.
When the calculation finishes, results appear in the table and are loaded into the document. The processed receptor is named 5MAJ_pro. For covalent docking, the resulting ligand pose also contains part of the residue to which it is docked. In this example, hide the initial structure and add a Ribbons visual model for the processed receptor to make the complex easier to inspect. Selecting the ligand and pressing Shift+Space zooms in on it.
These steps help separate three decisions that are easy to conflate: how the ligand is represented, where the search takes place, and which protein residue is designated as the covalent partner. For interaction analysis and the non-covalent workflow, see the full FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.