A covalent docking setup can be easy to misconfigure for a simple reason: the ligand you start with may already be bonded to the protein. Its bond orders and hybridization can therefore describe the bound complex—not the reactive ligand you want the docking workflow to handle. If ligand preparation changes those details, the input may no longer represent the intended chemistry.
This walkthrough focuses on a practical example from the FITTED Suite tutorial: docking the covalent inhibitor 7KH 301 into human cathepsin L (5MAJ) in SAMSON. The key lesson is to check the ligand’s chemical representation before running the workflow, then explicitly identify the protein residue involved in the covalent reaction.
1. Start with the bound complex
Open 5MAJ.sam from the FITTED tutorial archive, or download the structure in SAMSON using the asset link provided in the original tutorial. The example contains cathepsin L and the covalently bound ligand 7KH 301.
For this tutorial system, no additional protein preparation is required: FITTED uses PREPARE to adjust bond order, add hydrogens, generate possible tautomers, and optimize the hydrogen-bond network. For other structures, check for alternate locations and missing residues or heavy atoms; the tutorial points to SAMSON’s Protein Preparation & Validation guide for that work.
2. Restore the ligand’s unbound chemistry
In the example, the ligand’s C8–N7 bond is changed to a triple bond because the ligand is a nitrile when unbound. The C8 and N7 atoms are also assigned SP hybridization. These adjustments are made in the structure before setting up docking.
You can edit the bond using Edit > Edit bonds, or select the bond and change its parameters in the Inspector. To set hybridization, select both atoms and modify their corresponding Inspector parameters. The tutorial’s supplied example already includes these changes, but it demonstrates how to make them.
Once the ligand has a deliberately modified bond order and hybridization, turn off the FITTED option to prepare the ligand (perceive bond order and add hydrogens). This avoids asking the workflow to re-prepare the ligand after you have manually set those properties.
3. Define the receptor, site, and reaction partner
Open the FITTED Suite from Home > Apps > Biology, or find it with SAMSON’s search box. Select the 5MAJ structural model and set it as the receptor. For this protein example, leave the macromolecule setting as Protein and keep the water setting at its default.
Because this is self-docking, use the already-bound 7KH 301 ligand to define the binding site: select it in the Document view and choose From bound ligand. Then set the same ligand as the docking ligand. If a bound ligand is unavailable, the interface also offers a position-based binding-site option.
Set the docking mode to Covalent only. In this complex, the reacting residue is CYS 25; select that residue (or an atom in it) and set it as the covalent residue. The tutorial identifies the sulfur atom SG as the protein atom involved in the covalent bond. A basic atom is optional. In this example, ND1 in the adjacent HIS 163 residue is specified as the basic atom.
4. 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. Click Dock; the interface shows the current stage and displays logs below the results table. When the calculation finishes, results are listed and loaded into the document. In the covalent result, the pose includes part of the residue to which it is docked.
Inspect the resulting pose rather than treating a completed docking run as the end of the analysis. The tutorial shows how to add a Ribbons visual model for the processed receptor, hide the original structure, and zoom in on the ligand to check its connection to the sulfur atom in CYS 25. You can then use SAMSON’s interaction-analysis tools to examine the complex further.
The practical checklist is short: correct the ligand’s bond order and hybridization, avoid re-preparing that edited ligand, set the covalent residue explicitly, and inspect the resulting pose. For the complete workflow and non-covalent docking example, read the FITTED Suite tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.