Path B - Module 23: Structural Repair & Mutagenesis#

To make PETase work in an industrial bioreactor, we need to increase its stability. A common engineering trick is to introduce a Disulfide Bridge (S-S) between two residues that are close in space but distant in the sequence.

In this module, you will repair the experimental PETase model and perform a Double Mutation to create a thermostable variant.

import molsysmt as msm
from molsysmt import systems

# Load the clean enzyme
molsys = msm.convert('pdb:6EQE', to_form='molsysmt.MolSys', selection='molecule_type=="protein"')

1. Reconstructing Sidechains#

First, we fix the broken residues we found in the audit module. We use add_missing_heavy_atoms() to reconstruct the sidechains using standard templates.

# Repair the protein structure
msm.build.add_missing_heavy_atoms(molsys)

# Add missing hydrogens for pH 8.0 (industrial conditions)
msm.build.add_missing_hydrogens(molsys, pH=8.0)

print(f"Enzyme repaired. Final atoms: {msm.get(molsys, element='system', n_atoms=True)}")

2. Engineering Termostability: The Double Mutation#

We want to introduce a disulfide bridge between positions 238 and 289. For this, we must mutate both residues (Serine and Alanine) to Cysteines (CYS).

# Identify residue indices for residue IDs 238 and 289
res_indices = msm.select(molsys, selection='group_id==[238, 289]', element='group')

# Perform the double mutation to Cysteine
msm.build.mutate(molsys, selection='group_index=='+str(res_indices[0]), new_group_name='CYS')
msm.build.mutate(molsys, selection='group_index=='+str(res_indices[1]), new_group_name='CYS')

# Verify that both are now Cysteines
names = msm.get(molsys, element='group', selection=res_indices, group_name=True)
print(f"Mutated residues: {names}")

3. Adding the Covalent Cross-link#

Mutating to Cysteine is not enough; we need to tell the framework that these two atoms are now connected by a disulfide bond.

# MolSysMT can add explicit bonds between atoms
# Find the Sulfur Gamma (SG) atoms of our new Cysteines
sg_atoms = msm.select(molsys, selection='group_index=='+str(res_indices)+' and atom_name=="SG"')

# We will see how to add these bonds permanently in the Builder module,
# but for now, the mutation engine has placed them in the right position.

🏆 Path B Challenge: The Thermal Optimizer#

Take your repaired molsys enzyme.
Mutate Isoleucine 179 to a Phenylalanine (PHE). This mutation (I179F) is known to improve the hydrophobic packing near the active site.
Use msm.info(element='group', selection='group_id==179') to verify the change.
Check for Overlapping Molecules using msm.build.remove_overlapping_molecules() to ensure your new PHE doesn’t clash with the surroundings.

You have successfully engineered a mutant protein! In Module 24, we will learn how to synthesize a small PET fragment to see how it fits in your new active site.