Check Restraints File

Ensure that your restraint file for the ligand is correct and matches the chemical properties of the ligand (bond lengths, angles, etc.). Sometimes, the default restraints may not be appropriate for the specific ligand, leading to issues during refinement.

You can also consider manually adjusting or creating a custom restraints file specific to your ligand using programs like JLigand or Coot's ligand builder, which allows you to generate the correct restraints based on the chemical structure.

Adjust the Refinement Parameters

During real-space refinement, you can adjust the parameters related to the refinement of ligands and RNA. Specifically, look into:

Weighting: The weighting between geometry (restraints) and electron density in the refinement. Too high a weight on the density can cause distortions to the ligand or RNA, leading to broken bonds. Try reducing the weight on density or increasing the weight on restraints.

Restraining Parameters: Make sure that the bond and angle restraints are sufficiently tight to prevent the geometry from straying too far from the expected values.

Review the Initial Model

Before refinement, check the geometry of the ligand and RNA in the starting model. If there are any unrealistic bond lengths or angles, they might exacerbate during the refinement process, especially under real-space conditions where there is less global energy minimization than in a typical least-squares refinement.

Use the Coot Ligand Fitting Tools

When fitting ligands into the electron density in Coot, make sure to use the appropriate ligand fitting tools that help preserve the chemistry of the molecule. Coot has a built-in feature for ligand fitting, which can help prevent large distortions to bond geometry.

Use TLS or Other Restraint Approaches for the RNA

If your RNA chain is large or flexible, consider using TLS (Translation-Libration-Screw) or additional geometric restraints on the RNA backbone to prevent significant changes in conformation during real-space refinement. This can help stabilize the structure of the RNA and prevent bond breaking.

Consider Refining Ligand and RNA Separately

You might want to try refining the RNA and the ligand separately, especially if you see that the RNA is stable but the ligands are not. This allows you to apply different refinement strategies to each component.

Inspect Electron Density

Carefully inspect the electron density map after the first refinement step to make sure that the ligands are still well-placed. If the electron density suggests a misalignment, try manually adjusting the ligand position before re-running the refinement.

Check the Force Field (Refinement Software)

If you’re using a refinement method other than Coot's real-space refinement (such as REFMAC or Phenix), make sure the force field is suitable for both RNA and the ligands. Some refinement methods may have trouble with ligands or small molecules during refinement, leading to broken bonds or incorrect geometry.

By addressing these points, you can prevent bonds from breaking during the refinement process in Coot and achieve a more stable and accurate final structure.