How I redesigned a thermostable enzyme using ProteinMPNN inverse folding - and validated every design with AlphaFold2

E155 and E215 at 6.03 Å - exactly the nucleophile/acid-base separation expected for a GH5 retaining endoglucanase. This step matters. If I had accepted the literature numbering without checking, I would have fixed the wrong residues and the constrained run would be biologically meaningless. Step 2: B-factor profile Before running ProteinMPNN, I computed per-residue B-factors to identify flexibility hotspots - regions that might benefit most from redesign: High-flexibility regions (B > 30 Ų):…