Structural based stability prediction does not correlate with the actual yeast display result.

<p>To determine the free energy change upon protein unfolding (ΔΔG), we employed software provided by Cyrus Biotechnology, which utilizes the Rosetta suite for structure prediction. We initiated ΔΔG calculations from the relaxed and energy-minimized structure of the 5JI4 scaffold to ensure the reference conformation was at the lowest possible energy state. We mutate each residue position, with the exception of cysteine residues due to their role in disulfide bond formation, to all amino acids except cysteine. The ΔΔG values were computed for each mutated residue per position. Furthermore, the mean ΔΔG across all mutations per position was calculated, providing an aggregate measure of stability changes attributable to each position. In our analyses, a ΔΔG value falling below the median for the entire scaffold was indicated with an ’X’ in the corresponding table, denoting a residue crucial to folding stability. Conversely, in the context of Yeast Surface Display (YSD), a residue’s Enrichment Score (ES) surpassing the median was marked as ’X’, highlighting its significance.</p> <p>(PPTX)</p>