Using PMX for ΔΔG on a glycosylated Fc (P238D back-mutation; PDB 3WJJ): viable workflows?

pmx
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Hi all,

I’d like to use PMX to compute ΔΔG of binding for an Fc that is N-glycosylated (structure based on PDB: 3WJJ) by reversing the mutation P238D. I know PMX supports Amber and CHARMM family protein force fields out of the box, but I’m unclear how best to proceed when linked glycans are present.

Concretely:

Amber/GLYCAM → GROMACS → PMX?
If I build the glycoprotein in tleap with Amber+GLYCAM, then convert to GROMACS formats (e.g., via ACPYPE or similar), is that topology/coordinate set acceptable input for pmx mutate? Key requirement: the covalent N-glycosidic bond (Asn–GlcNAc) must survive the conversion and be represented as a bonded connection in GROMACS. Has anyone run PMX successfully on such converted glycoprotein systems? (Background: GLYCAM→GROMACS conversion with ACPYPE is documented, but I’m not sure about PMX compatibility and preserving cross-residue bonds. )

All-CHARMM36 route with pdb2gmx + PMX CHARMM-mut ff?
Alternatively, prepare the glycoprotein entirely with CHARMM36 (jul2022) carbohydrate parameters in GROMACS (i.e., using carb.rtp/carb.r2b so mid-chain vs terminal sugar names map correctly), then use PMX’s CHARMM mutate workflow. Is there a recommended recipe (naming, r2b mappings for mid-chain GlcNAc, Man, Gal, Fuc, specbond.dat for linkages, etc.) known to work end-to-end with PMX? (GROMACS handles CHARMM36 glycans via carb.rtp + carb.r2b; correct residue naming and r2b mapping are critical. )

Any proven alternatives?
If the above aren’t practical, are there known workarounds (e.g., splitting protein/glycans, then merging with manual bonds in topology; or specific PMX mapping files for common N-glycans) that people have used successfully for ΔΔG with glycoproteins?

Motivation / constraints:
We prefer to avoid web services that require uploading proprietary coordinates (e.g., CHARMM-GUI), and we’re fine with some manual editing if that’s the current state-of-the-art. (We know CHARMM-GUI’s Glycan Reader/Modeler can produce GROMACS-ready glycoproteins; just checking for an offline/PMX-friendly path. )

Any pointers, example repos, or do/don’t tips (especially around PMX’s mutate step with carbohydrate-bearing topologies) would be hugely appreciated!

Thanks!

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The easiest way is to create a new residue entry in .rtp for the glycosylated aa and insert that into the currently supported force field, e.g. amber99sb-star-ildn-mut.ff/aminoacids.rtp

Glycosylation probably will introduce new atom types: those you will also need to add to atomtypes.atp and ffnonbonded.itp. The bonded params can be listed explicitly in the aminoacids.rtp

After this force field update, pdb2gmx will be able to parse your structure and you can use the standard pmx amino acid mutation protocol.

For the P2D mutation, you should not use Charmm ff, because proline has a different type of CMAP than aspartate, and CMAP alchemical perturbation is not supported by gromacs.

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Thank you very much for the reply. One more question, please. How to handle charge changing mutations? Is there a way to neutralize state B charge? does PMX have this feature?

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I would add a co-alchemical ion. For a P2D mutation, it could be one CL ion that has a charge of -1 in stateA and charge of 0 in stateB. This way the charge of the simulation box will remain constant during the transition. Make sure to restrain the ion at some distance larger than the cutoff from the protein/peptide.

Another way is to place both branches of the thermodynamic cycle in one box: protein P2D mutation and peptide D2P mutation. There is a script src/pmx/scripts/make_double_box.py that can create a box with the positioned structures.