Contradiction between RFD3 design and AF3 prediction

[nmbiss747-netizen]

I used RFD3's hotspot-guided binder generation, but in the subsequent AlphaFold structure prediction, the binder did not appear near the expected binding site. Has anyone else encountered the same problem? I'm wondering if this means the RFD3 design is flawed, or if there's an issue with the AlphaFold prediction results.

[roccomoretti]

I'll mention that while RFD3 now does produce sequences, in practice it's observed they tend to be lower quality than dedicated design models like ProteinMPNN and LigandMPNN. If you haven't already, you may want to add a redesign step with one of those to your pipeline.

It's also observed that not all RFD/MPNN generated models successfully forward fold with AlphaFold. Forward folding actually is one of the main filtering steps used to pick designs. Generally you'll generate thousands of binder designs, and throw out the (substantial) fraction of designs which don't properly forward fold. If you've only tested a few designs with AlphaFold, you just may have gotten unlucky with which designs you obtained.

On the question of whether it's RFD3 or AlphaFold which has issues, one approach might be to use other prediction models, such as Boltz or Chai or RosettaFold. If they all dislike the structure, then it's probably RFD3 that is generating poor structures. If only AlphaFold thinks it bad, it might be that you've hit upon a weak spot of AlphaFold prediction. (This latter is probably unlikely. The most likely default assumption would be that RFD3 is generating backbones with issues.)

[nmbiss747-netizen]

I also considered the issue of the sequences generated by RFD3 itself, but I tried regenerating the sequences for the binder using ProteinMPNN, and the results were no different. It seems that AlphaFold believes the binder will inevitably bind at a specific location, regardless of which binding site I use in the RFD3 design stage. I tried designing the binder using the tail of the IgG Fc domain and the CH2-CH3 junction as hotspots, but AlphaFold's predictions almost always placed the binder in the central cavity of the Fc dimer.By the way, I also tried other structure prediction models, and the results were similar to AlphaFold's. Placing the binder in the central cavity of the Fc dimer, which is a clear pocket, does make sense, even though this wasn't the binding site I specified during the design stage. Does this mean that RFD, even with clear hotspot constraints, still cannot generate a binder specific to that particular site?