Your use of -DFLEXIBLE does nothing because your topology does not contain an #ifdef FLEXIBLE... block. It is not the standard TIP3P topology, likely because CHARMM-GUI inputs reflect conventional usage and TIP3P should always be treated as rigid. If you want to remove constraints from TIP3P, see the standard tip3p.itp approach that replaces SETTLE with normal bonds.
#ifdef FLEXIBLE #ifdef ORIGINAL_TIP3P [ bonds ] ; i j funct length force.c. 1 2 1 0.09572 502416.0 0.09572 502416.0 1 3 1 0.09572 502416.0 0.09572 502416.0 [ angles ] ; i j k funct angle force.c. 2 1 3 1 104.52 628.02 104.52 628.02 #else ;CHARMM TIP3p [ bonds ] ; i j funct length force.c. 1 2 1 0.09572 376560.0 0.09572 376560.0 1 3 1 0.09572 376560.0 0.09572 376560.0 [ angles ] ; i j k funct angle force.c. 2 1 3 1 104.52 460.24 104.52 460.24 #endif
I suspect the issue is more related to the force field being inadequate for a desolvated (vacuum) system, in which case you may want to minimize with dihedral or distance restraints to preserve the geometry, then perform NMA.