Binder design workflow:

1. Initial length screening: First, use Bagel to perform exhaustive searches across different sequence lengths (80, 120, 160, and 200 amino acids) to determine the most suitable starting length range.

2. Region-specific design: Based on the selected optimal length, designs are carried out for different functional regions of RBX1:

   • Generate ~120 aa sequences targeting hotspot residues in the Cullin region;
   • Generate ~160 aa sequences targeting hotspot residues at the Cullin and Glomulin interface.

3. Backbone selection and sequence design: Select top-ranking backbone structures from Bagel outputs and perform sequence design and optimization using SoluMPNN.

4. Rule-based filtering and iterative optimization: Apply heuristic rules summarized from the Bits to Binders competition for initial filtering. Select sequences with top global scores and re-input them into Bagel for further optimization.

5. Orthogonal evaluation and final selection: Use AF2 for orthogonal validation of high-ranking designs generated by Bagel, and select the final candidate binder sequences.