SplitSeek-Pro: accurate prediction of splittable sites on protein structures

Understanding protein architecture and predicting its structural tolerance to profound remodeling is pivotal for engineering functional proteins. We present SplitSeek-Pro, a deep learning model that evaluates amino acid-level splittability in folded proteins, a property critical for protein engineering tasks such as circular permutation and split reconstitution. By integrating primary sequences with 3D structural features, SplitSeek-Pro achieves residue-resolution predictions through a two-stage training process: large-scale pre-training followed by high-quality fine-tuning. Experimental validation on three distinct proteins confirms its superior predictive power over existing methods. Notably, SplitSeek-Pro identifies characteristic segments that function as cohesive, integral fragments analogous to super-secondary structural motifs. These results establish SplitSeek-Pro as a robust tool for rational protein engineering and offer insights into the fundamental structural building blocks of protein folding. To facilitate community access, we provide an automated web server at http://splitseek.topo.bio. Predicting protein splitability is pivotal for engineering functional variants. Here the authors present SplitSeek-Pro, a deep learning model integrating sequence and 3D features to achieve accurate residue-resolution split site prediction for protein design.