Genesis Predicts Optimal Insertion Sites for Allosteric Protein Switch Design: A B-factor Z-score Framework Validated Across 244 Experimental Sites

We present the Genesis Insertion Site Predictor, a zero-cost computational method that uses crystallographic B-factor z-scores from PDB structures to predict optimal domain insertion sites for allosteric protein switch engineering.

Key results: Validated on 244 experimentally characterized insertion sites across four structurally unrelated proteins (CAT, Cas12a, TEM-1, Kir2.1). AUC = 0.706 (95% CI: 0.639-0.770), permutation p < 0.001. Across three proteins with direct experimental data, AUC = 0.762 (p = 0.002). The top-ranked prediction in TEM-1 (Asp214) is independently confirmed by Baker lab (Nature Biotechnology, 2026) as the optimal insertion site.

Web server: https://app-ebcd5b50.base44.app/functions/predictInsertionSites

The method requires only a PDB structure and completes in under 1 second, offering ~10,000x speedup over experimental screening.