A covalent inhibitor is not just a ligand that happens to sit in a binding pocket. It forms a bond with a specific protein residue, so the docking setup has to describe both the binding site and the intended reaction partner. Miss one of those details—or let ligand preparation overwrite a bond assignment you made—and the workflow may no longer represent the chemistry you want to investigate.
This walkthrough focuses on a concrete example from the FITTED Suite tutorial: docking the covalent inhibitor 7KH 301 into human cathepsin L (structure 5MAJ) using SAMSON. The example is useful because it highlights the decisions that differ from ordinary non-covalent docking.
1. Check the ligand chemistry before docking
In the example, 7KH 301 is shown bound to the protein, but part of its chemistry must represent the unbound ligand correctly for docking. The C8–N7 bond is changed to a triple bond, and the hybridization of both C8 and N7 is set to SP. These edits can be made with Edit > Edit bonds or by selecting the bond and changing its properties in the Inspector; hybridization is also set through the Inspector.
This is an important distinction: the tutorial’s bond and hybridization edits are already present in its supplied example, but they may need attention in other covalent complexes. Inspect the reactive group rather than assuming the structure file encodes the ligand in the form required for docking.
2. Avoid undoing those edits during ligand preparation
In the FITTED Suite, set the receptor from the 5MAJ structural model and define the binding site from the already bound 7KH 301 ligand. Then set that ligand from the document. Because its bond order and hybridization have been deliberately modified, uncheck the option to prepare the ligand (perceive bond order and add hydrogens). In this case, automatic preparation could conflict with the chemistry you have just specified.
This is a useful general check whenever you manually curate ligand chemistry: before running a preparation step, ask whether it might replace or reinterpret your edits. The tutorial notes that FITTED uses PREPARE for protein-side tasks such as adjusting bond order, adding hydrogens, generating possible tautomers, and optimizing the hydrogen-bond network. Still, the ligand option should remain off for this example because the ligand has a modified bond order and hybridization.
3. Tell the workflow which residue reacts
Choose Covalent only as the docking mode. For the 5MAJ example, the reacting residue is CYS 25 in chain A: its sulfur atom, SG, is the protein-side reaction partner. Select the CYS 25 residue (or an atom within it) and use the Set button for Covalent residue. The suite accepts either the residue or an atom in it and sets the whole residue.
The basic atom is optional. In this example, the tutorial also specifies ND1 in the adjacent HIS 163 residue. Select ND1 and use Set for Basic atom. If you do not have a reason to specify one, remember that this field is optional; do not confuse it with the covalent residue, which identifies the residue that reacts with the inhibitor.
4. Run, then verify the structural result
The tutorial uses two runs and allows you to choose whether to import only the best pose. Select an output folder if needed, then click Dock. The app shows the current stage on the button and displays logs below the results table. When calculations finish, results are loaded into the document; selecting a row in the table selects its corresponding pose, and the table can be exported as CSV.
For covalent docking, inspect more than the score or pose ranking. The resulting ligand pose includes part of the residue to which it is covalently docked. In this example, the displayed ligand should be connected to the sulfur atom of CYS 25. Hide the initial structure if it obscures the result, add a Ribbons visual model for the processed receptor (5MAJ_pro), and zoom in on the ligand to examine the bond in context. Visual inspection is a practical way to catch a setup mismatch before moving on to interaction analysis.
Quick checklist: confirm the reactive ligand bond order and hybridization; keep ligand preparation unchecked if you made those edits; select the correct covalent residue; add a basic atom only when appropriate; choose Covalent only; and inspect the returned pose at the reaction site.
For the complete workflow and the corresponding non-covalent docking example, see the FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON at samson-connect.net.