The final disulfide bonding pattern of snakin-1 was represented by CysI-CysIX, CysII-CysVII, CysIII-CysIV, CysV-CysXI, CysVI-CysXII and CysVIII-CysX. This disulfide pattern could be extrapolated to other members of the snakin/GASA family

through sequence alignment (Fig. 1A). After the inclusion of remaining disulfide bonds through MODELLER, the best model showed a DOPE score of −5036.17432. The final model was obtained after energy minimization on SPDBV. The final model shows a minimum and a maximum 1D–3D average score of 0.3 and 0.55 in Verify 3D. In the Ramachandran plot, 72.2% of the residues are in favored regions; 14.8% are in HTS assay additional allowed regions and 11.1% in generously allowed regions; and an overall

G-factor of −0.23. The Z-score on ProSA was −5.85. The three-dimensional model was composed of two long α-helices composed of residues 2SSFCDSKCKLRCSKA16 and 20DRCLKYCGICCEE32 and one short 310-helix composed of 43NKH45, in addition, the structure was stabilized by see more six disulfide bonds ( Fig. 1B). Furthermore, the same structural scaffold could be observed for other members from this family through secondary structure predictions algorithms (data not shown). The snakin-1 final model is available as supplementary file 1. In the molecular dynamics simulation, a large displacement of two C-terminal segments, 37PSGTYGNK44 and 50YRDKKNSKGKS60, was observed. The root mean square deviation (RMSD) stabilization occurs after 30 ns of simulation with a variation of about 4.5 Å (Fig. 2A); removing the two C-terminal segments from RMSD calculation, a variation of about 3.5 Å was observed (Fig. 2A), reinforcing the supposition that the C-terminal segments are mainly responsible for the variation of 4.5 Å Dapagliflozin in the complete structure. The DSSP analyses indicated that the short 310-helix underwent a coil transition (Fig. 2B). However,

the overall structure is maintained, since it is knotted by six disulfide bonds (Fig. 2C). In addition, the root mean square deviation by residue also indicated that the C-terminal segments were responsible for the structural modification (Fig. 2D). The TM-Score with a value of 0.5023 indicated that the initial and final structures share the same fold. The snakin/GASA family has enormous biotechnological potential since it plays a defensive role against several plant pathogens, especially against the pathogens that attack potato, one of the most important crops worldwide [22]. However, the omission of structural information about this family hinders a deeper understanding about the class. The prediction of the disulfide bonding pattern of snakins is an enormous challenge, since there are 66 possible combinations of disulfide bonds.