Secondary structure experimental validation. To confirm the proper folding of the studied variants, circular dichroism (CD) spectra were collected for all soluble variants: AncDhaA1, AncDhaA10-11, AncDhaA13-16, AncDhaA18, and AncDhaA20 (Fig. ). Overall, CD spectra of most variants highly resembled those of the templates (typical α/β-hydrolase fold), confirming proper folding. On the contrary, the spectra of AncDhaA1, AncDhaA11, AncDhaA15, and AncDhaA18 (Fig. ) deviated from the templates. To further understand the secondary structure of the variants, BeStSel server was used for fitting experimental data and analysis of PDB structures, and PDBMD2CD was used for predicting CD spectra from experimental structures of templates and AlphaFold models of selected variants. Fig. shows that the prediction of CD spectra based on AlphaFold structures did not match the experiments in all the variants. This highlights a limitation in AlphaFold's ability to accurately predict changes in folding and emphasizes the need for further improvements in computational methods. Experimental validation remains essential to address this challenge.