Avoid This Common Pitfall in Covalent Docking: Proper Ligand Bond Order and Hybridization

When setting up covalent docking simulations, one of the most frequent and frustrating sources of error is neglecting to properly define the chemical structure of the ligand—especially if the ligand comes from a crystal structure where it is already covalently bound to the receptor.

In covalent docking workflows, such as those available through the FITTED Suite SAMSON Extension, accurately representing the bond order and hybridization states of ligand atoms is critical for identifying the correct binding pose and interaction geometry.

Why It Matters

If the ligand is already covalently attached in the crystal structure, then its original, unbound electronic configuration is lost. Docking software, including FITTED, assumes the ligand starts unbound, so if you skip this step, you might end up with unrealistic results—or worse, failed simulations.

Real-Life Example

Let’s walk through how to handle this in SAMSON using the human cathepsin L protein-ligand structure (5MAJ) with a nitrile-containing inhibitor (7KH 301).

After opening the file 5MAJ.sam in SAMSON, locate the C8 atom in the 7KH 301 ligand. This atom is originally triple bonded to a nitrogen (N7) in the unbound form, but appears as a single bond due to its covalent attachment in the structure.

To fix this:

  • Select the bond between C8 and N7.
  • Open the Edit > Edit bonds toolbar, and set the bond order to Triple.
  • Next, select atoms C8 and N7, and open the Inspector.
  • Set their hybridization to SP appropriately. This reflects a nitrile group with linear geometry.

These changes ensure that FITTED sees the correct electronic structure of the ligand for generating accurate docking poses.

Changing bond order of C8-N7 bond to triple bond

Changing hybridization of C8, N7 atoms to SP

Don’t Rely Solely on Automation

While the FITTED Suite automates most of the system preparation, customizing the ligand description is one area where manual input can make a big difference—especially in covalent docking workflows. Think of it as the chemistry equivalent of proofreading before submitting a paper: essential.

Checklist for Ligand Structure Correction

  • 🔍 Inspect all bonds between the ligand and receptor.
  • 🔬 Set the original (pre-reaction) bond order and hybridization for ligand atoms.
  • ❌ Disable automatic ligand preparation if you’ve already customized the structure.

Following this process takes only a couple of minutes but saves a tremendous amount of time debugging docking results later.

To explore how FITTED handles other steps—such as setting covalent residues or running the docking itself—check out the rest of the documentation: Full FITTED Suite Tutorial.

SAMSON and all SAMSON Extensions are free for non-commercial use. You can download SAMSON at https://www.samson-connect.net.

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