BoltzGen + custom ranking metrics for selection. Configuration can be found in the submission overview, but in short: Design aim:

• We sought to design nanobody-based binders with NEUTRALISING activity against the Nipah Virus. Thus, we focused the designs from this submission on sites that would break the interaction between NiV-G and EFNB2.

Hotspot ID:

• We began with the 2vsm structure provided from Adaptyv, and used BioPython to identify residues in contact with EFNB2 (interaction interface residues were defined as those with carbon atoms within 10 angstroms of the other chain)
• In addition, we focused on a subset of positions shown to affect EFNB2 binding through DMS experiments published by Larson et al. (Larson BB et al, Cell, 2025)
• Finally, we used a PyMol script to identify 3-4 residues that formed a hydrophobic surface as an optimal target
• In the end, we selected the residues: I401, R402, W504 as our hotspots for generating binders

Design using BoltzGen:

• We used BoltzGen to create 10,000 designs using the hotspots defined, and the 4 default nanobody templates provided in the BoltzGen repo.
• However, we adjusted the length of CDRs to the following:
  • CDR1: 3-5 aa
  • CDR2: 3-5 aa
  • CDR3: 7-14 aa