GJOses Lab

1. Target Preparation The receptor structure was obtained from PDB entry 2LGV. Given the intrinsically disordered protein (IDP) nature of the receptor's tail regions, hotspot residues were selected from surface-exposed regions less occluded by the IDP tail, prioritizing accessible binding interfaces.

2. Backbone Generation with RFDiffusion3 (RFD3) A total of 1,000 miniprotein backbone folds were generated using RFD3, targeting the identified hotspot residues on the receptor. Selected hotspots residues: A76 (CG), A99 (CA), A100 (CA)

3. Sequence Design with ProteinMPNN Each of the 1,000 RFD3-generated backbones was subjected to sequence design using ProteinMPNN. Fifty sequences were generated per backbone fold. The top 10 sequences per fold were selected based on LigandMPNN score, yielding a total of 10,000 candidate sequences.

4. Structure Prediction with AlphaFold3 (AF3) Each of the 10,000 candidate sequences was predicted in complex with the 2LGV receptor using AlphaFold3. Predictions were run using 1 seed and 5 samples per sequence, for 5 predictions per sequence, resulting in a total of 50,000 predicted structures.

5. Filtering and Evaluation Predicted complexes were evaluated by: (i) superimposing the ordered region of the receptor between the RFD3-designed backbone and the AF3-predicted structure, (ii) computing the RMSD of the binder between the RFD3 and AF3 models, and (iii) extracting AF3 confidence metrics including pTM and PAE scores.

The top 100 design models with the lowest binder RMSD were selected as primary candidates. We acknowledge that an RMSD cut-off of up to 20 Å is unusually permissive; however, since the remaining metrics (pTM, PAE, etc.) were consistently favorable for these designs, they were retained and appended to the candidate list as low-priority entries for potential follow-up. After removing redundant sequences, a final set of 47 binder candidates was obtained for experimental consideration.

https://drive.google.com/file/d/1tUYSm4KhcDVtWJHdMtDwjSe77XlvNHPH/view?usp=drive_link