When (and How) to Remove Crystal Waters Before Simulation

Removing water molecules may seem like a straightforward preprocessing step before running a molecular dynamics simulation. However, when dealing with experimental structures such as those from the Protein Data Bank (PDB), some of the water molecules—especially those near the active site—play pivotal roles in protein-ligand interactions or overall stability of the system. The challenge is in distinguishing between structural (or functional) waters and bulk solvent that should be removed prior to solvation.

This blog post offers a practical approach using the GROMACS Wizard in SAMSON to selectively remove non-essential waters while keeping those that matter most.

Why it matters

Removing all water molecules by default might compromise the underlying biochemistry of your system. Certain tightly bound waters are part of hydrogen bond bridges or coordinate with metal ions, and their removal could disrupt interaction networks in the binding site.

Fortunately, SAMSON’s GROMACS Wizard provides a straightforward method to identify and remove only those waters located outside of your system’s active site.

Selective crystal water deletion: a step-by-step guide

  1. Select the region you want to protect—typically residues, atoms or ligands within the active site.
  2. Right-click on the current selection either from the Document view or directly in the Viewport.
  3. From the context menu, choose Expand selection > Advanced.
  4. In the pop-up dialog:
    • Set the Node type to Water.
    • Set the selection criterion to beyond a certain distance (e.g., 5 Å) around your selection.
    • Check auto-update to preview selected waters in real time.
  5. Click OK and verify that only the distant waters are highlighted.
  6. Right-click the selection and choose Erase selection to remove those water molecules only.

Expand selection: select water beyond 5A

When to preserve water molecules

If your active site contains tightly bound water molecules that act as hydrogen bonding bridges or are coordinated with metal ions or ligands, removing them could lead to significant deviations from experimentally validated poses. These water molecules should usually be preserved during preprocessing.

If you’re unsure whether a crystal water is functional, reviewing the literature associated with the PDB ID (e.g., 1AKI) can provide some context. Another strategy involves performing energetic analysis after simulation setup to see how their presence impacts binding interactions or protein structure.

Use cases in GROMACS

Once you’ve removed only the non-essential crystal waters, the system is ready for solvation with appropriate water models (e.g., TIP3P) and additional ions if desired. Since GROMACS can add general solvent molecules during the preparation stage, filtering the original PDB waters to include only the crucial ones creates a cleaner starting point, potentially accelerating simulation convergence and improving accuracy.

To see this process in its original context and for more in-depth instructions, visit the full documentation page: https://documentation.samson-connect.net/tutorials/gromacs-wizard/preprocess/.

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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