Comparison of the RMSD values for AlphaFold biomphalysin predictions.

For the two sets of predictions (non-mature and Cter-mature), biomphalysins were compared two by two with ChimeraX matchmaker tool, for different regions in the proteins. When comparing Cα RMSD from ChimeraX matchmaker tool, 89% of the values for small lobe comparison are under 2.0 Å, while only 8% of the values when large lobes are compared (S11 Fig). This indicates very similar/conserved structure organization of all domains I/ small lobes, despite very relatively low sequence identity (15% of values are above 60% identity -S11B Fig). Large lobe organization is conserved, but its orientation changes between each prediction and affects global and large lobe RMSD. Removal of unfolded C-terminal residues also modify the orientation: RMSD are decreased, most of the biomphalysins seem more aligned along a vertical axis. This observation can be an artifact of AlphaFold predictions or a structural rearrangement necessary for the activity. Biomphalysin 20 is a particular case: it is the protein with the highest RMSDs, followed by biomphalysins 19 and 23. In the transmembrane region, there is a long α-helix instead of a loop and the insertion loop is positioned on the other side of the large lobe compared to other predictions. It is also the case for biomphalysin 19, but only when the prediction includes the unfolded C-terminal residues. Either biomphalysin 20 shape is a prediction artifact or a specific structural organization linked to its function. (XLSX)