A covalent docking run can look like an ordinary docking setup until one detail changes everything: the ligand’s chemistry at the reactive site. If bond order or hybridization is inconsistent with the unbound ligand you intend to dock, automated ligand preparation may overwrite the chemistry you carefully set. The practical lesson is to treat covalent docking as a sequence of connected decisions: prepare the ligand correctly, identify the reacting residue, and only then launch the search.
This walkthrough uses the 5MAJ structure of human cathepsin L, where the 7KH ligand is covalently bound to CYS 25. It is a self-docking example, so the structure provides a known binding pose and a useful reference for setting up the workflow. The same setup logic can help when you are diagnosing why a covalent docking input needs more attention than a non-covalent one.
1. Check the ligand’s reactive-group chemistry
In the tutorial structure, the ligand’s C8–N7 bond is changed to a triple bond because the ligand is a nitrile when unbound. The hybridization of both C8 and N7 is set to SP. These edits are made in the ligand before it is supplied to FITTED Suite.
The key workflow detail is what happens next: because the tutorial ligand has manually modified bond order and hybridization, the option to prepare the ligand—perceive bond order and add hydrogens—is unchecked when setting the ligand in FITTED Suite. Otherwise, the preparation step could conflict with the edited input. This is not a universal instruction to skip preparation; it is specific to a ligand whose chemistry has already been deliberately modified.
2. Define the receptor and search site
Open the 5MAJ.sam tutorial structure and select the 5MAJ structural model as the receptor in FITTED Suite. For this example, leave the water-molecule setting at its default and keep the macromolecule type set to Protein. If working with a metalloprotein, DNA, or RNA, the macromolecule setting should instead match that system.
Because this is self-docking, use the already bound 7KH 301 ligand to define the binding site. In the app, choose From bound ligand and click Set. This gives the search site a clear structural reference. If there is no bound ligand, the alternative described in the tutorial is to set the site From position by positioning the sphere in the viewport.
3. Tell the workflow what reacts
For covalent docking, set the docking mode to Covalent only. The tutorial uses two runs and optionally imports only the best pose; the output folder can also be selected before starting. Most importantly, specify the covalent residue: in this example, the sulfur atom SG of CYS 25 is the reactive partner. You can select the residue itself or an atom in it; setting the residue identifies the reaction site for the workflow.
A basic atom is optional. In this example, ND1 in the adjacent HIS 163 residue is supplied as the basic atom. If you use this option, select the intended atom and click Set for Basic atom. Keeping the covalent residue and optional basic atom as separate inputs makes the setup easier to check: one identifies the residue that forms the covalent link, while the other provides the optional neighboring atom specified by the workflow.
4. Inspect what the calculation returns
Click Dock and follow the current stage shown on the button; the logs appear below the results table. When the calculation finishes, results are shown in the table and loaded into the document. Selecting a row selects its corresponding pose, and the table can be exported as CSV.
For the covalent example, the processed receptor is named 5MAJ_pro. The resulting ligand pose also contains part of the residue to which it is covalently docked. Hide the initial structure and its labels to make the processed result easier to inspect, then add a ribbons visual model for the receptor if desired. Zooming in on the ligand lets you inspect its connection to the sulfur atom of CYS 25.
After docking, compare poses and examine the ligand–receptor context rather than treating the score or pose alone as a complete assessment. SAMSON also provides tools for interaction analysis, including the Protein-Ligand Interaction Analyzer and Hydrogen Bond Finder described in the tutorial.
For the complete workflow and its non-covalent docking example, see the FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.