A common challenge in covalent docking is that the ligand in a crystal structure is already attached to the protein. Its chemistry in that complex may not match the free ligand you want to dock. If you treat the bound representation as an ordinary input—or let automatic preparation overwrite deliberate edits—you can lose important bond-order and hybridization information.
The 5MAJ example in the FITTED Suite tutorial offers a practical way to think about this setup. It uses human cathepsin L with a covalently bound 7KH ligand and walks through the details that are easy to overlook before starting a calculation.
1. Check the ligand’s chemistry first
In this example, the C8–N7 bond in 7KH is changed to a triple bond because the ligand is a nitrile when unbound. The hybridization of both C8 and N7 is then set to SP. These edits can be made with Edit > Edit bonds or by selecting a bond and changing its parameters in the Inspector; atom hybridization is also set through the Inspector.
The key practical point is to treat these as intentional input edits, not as cosmetic adjustments. The tutorial’s ligand has a modified bond order and hybridization, so its setup explicitly turns off the option to prepare the ligand—otherwise, the preparation step could conflict with the chemistry you just specified.
2. Define the receptor and binding site
Open the 5MAJ structure, then use the FITTED Suite app to set the 5MAJ structural model as the receptor. For the tutorial, the macromolecule type remains Protein, and the water-molecule setting is left at its default.
Because this is self-docking—the ligand is being docked back into the receptor structure it came from—the existing 7KH 301 ligand can define the binding site. Select it in the document, choose From bound ligand in the binding-site controls, and click Set. If no bound ligand is available, the tutorial describes using From position and positioning the binding-site sphere in the viewport.
3. Specify the covalent residue—and optionally a basic atom
Set the docking mode to Covalent only. In the example, the ligand reacts with the sulfur atom (SG) in the CYS 25 residue. Select that residue in the document and click Set for Covalent residue. You can specify the residue itself or an atom within it; the whole residue is then set automatically.
A Basic atom is optional. If you use one, the tutorial says it should be in a residue adjacent to the covalent residue. For this system, the example selects the ND1 nitrogen in HIS 163. Set it in the app after selecting the atom; omit this setting if you do not need to specify one.
Before clicking Dock, check the essentials: the receptor is set, the binding site is defined, the ligand is selected with the appropriate preparation option, the docking mode is covalent, and the covalent residue is specified. The tutorial uses two runs and notes that docking may take a few minutes. Results appear in the table and are loaded into the document; in a covalent run, the resulting pose includes part of the residue to which the ligand is docked.
This checklist helps focus attention on the setup decisions that matter most in this example: represent the free ligand’s chemistry deliberately, avoid re-preparing those edits, and identify the intended covalent partner. For the complete walkthrough, including non-covalent docking and further analysis, see the FITTED Suite tutorial.
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