Crystallization and initial X-ray crystallographic analysis of a de novo-designed protein with left-handed βαβ units.

A newly designed protein featuring a rare left-handed βαβ motif has successfully been crystallized and characterized by preliminary X-ray diffraction. The computational design was conducted using a combination of Rosetta BluePrintBDR, ProteinMPNN and AlphaFold2, generating eight candidates based on predicted stability and folding accuracy. The final construct was expressed, purified and crystallized in space group P2. Complete X-ray diffraction data were collected on the BL2S1 beamline at the Aichi Synchrotron and processed to 1.95 Å resolution. Despite multiple attempts, molecular replacement using the AlphaFold2 model did not yield a conclusive solution, suggesting that alternative phasing methods or refined modeling approaches will be needed. This work highlights both the promise and the challenges of pushing protein biodesign into underexplored structural motifs and provides a foundation for future structural and functional investigations.