We have created the design pipeline from Protein Hunter, Boltz-2, and custom ranking algorithm

• Extract the nanobody scaffold from the available structure (7TXZ) together with the contact residues of target protein G.
• Run iterative hallucination approach with Protein Hunter (Boltz-2 version) to refine CDR3 regions by fixing FR regions
• Estimate iPTM, iPSAE and Pyrosetta scores including dSASA and interface_hbonds and calculate the combined scores with the inverse weights •⁠ ⁠pTM: 1 •⁠ ⁠⁠ipTM: 1 •⁠ ⁠⁠iPAE: 1 •⁠ ⁠⁠dSASA: 2 •⁠ ⁠⁠hbonds: 2
• Rank the sequences based on custom scores to shortlist the designs
• Cluster the sequence similarity of CDR3 regions and choose the one with highest ipSAE from each distinct cluster
• Rank top 10 from custom scores + ipSAE