Troubleshooting

1. Using .mol2 and .frcmod Files to Generate Libraries

When using .mol2 and .frcmod files in Tleap, metals may not have their connectivity and atomic numbers correctly assigned. To avoid this issue, it is recommended to use the pre-generated .lib and .frcmod files, which include all the necessary data for proper parameterization.

Your Tleap script should include the following commands:

loadamberparams COMPLEX.frcmod
loadoff COMPLEX.lib

After generating the prmtop file, check the %FLAG ATOMIC_NUMBER section to ensure that the atomic numbers of metals are correctly assigned. This is particularly important for metalloproteins, as the atomic number for metals may sometimes be incorrectly recorded as -1.

2. Handling Metals with High Coordination Numbers

If your system includes a metal with a coordination number greater than 8, you may encounter the following warning when generating the prmtop file in Tleap:

Bond: Maximum coordination exceeded on A<Co1 23>
-- setting atoms pert=true overrides default limits

This warning indicates that the metal exceeds the default bond limit of 8 in AmberTools, causing some metal bonds to be omitted in the generated prmtop file, leading to incorrect results.

Solution: To resolve this issue, increase the maximum bond limit in the AmberTools source code:

Navigate to the AmberTools source directory:

cd $AMBER_HOME/AmberTools/src/leap/src/leap

Edit the atom.h file to increase the bond limit. By default, the maximum number of bonds is set to 8:
```
/* maximum 8 bonds out of each atom */
#define MAXBONDS 8
```
Change 8 to the desired number of bonds.
Recompile Amber to apply the changes, allowing Tleap to recognize the new MAXBONDS limit.

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3. Resolving Atom Mismatch Errors in Metalloprotein Parameterization

If you encounter the following error while generating parameters for a metalloprotein system:

Error processing files: Number of atoms mismatch: Structure has 91, MOL2 has 63

This error occurs because the structure used during optimization, frequency, and charge calculations is incorrect—it lacks the necessary capping atom.

Solution:

To fix this issue, use Option 2 in the easyPARM menu to generate the correct structure. Alternatively, you can manually add the capping atom, though Option 2 is strongly recommended for accuracy.

4. Error: Bad Substitution in Bash Script

When running the ./easyPARM script, you may encounter errors like the following:

./01_easyPARM.sh: line 782: ${metalloprotein_choice,,}: bad substitution
./01_easyPARM.sh: line 811: ${charmm_FF,,}: bad substitution

This error occurs because the script is using the Bash-specific lowercase conversion operator ${variable,,}, which is not supported in older Bash versions or other shells.

Solution: Updating the Shebang Line

The most effective solution is to modify the shebang line at the beginning of the script to ensure it uses a compatible Bash version:

# Change this:
#!/bin/bash

# To this:
#!/usr/bin/env bash

This change ensures that the system will use the most appropriate version of Bash available in the environment, rather than the one specifically located at /bin/bash, which may be outdated.

Alternative Solutions

If changing the shebang doesn't resolve the issue, you can try:

Upgrading your Bash version to 4.0 or newer

Verification

After implementing the fix, the script should properly handle the case conversion for metalloprotein_choice and charmm_FF variables, allowing it to generate the CHARMM files correctly.

Confirm the fix worked by ensuring that all subsequent functionalities, such as residue name changes and parameter file generation, complete without errors.

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