The secondary structure elements of the vaccine construct can be divided into the following order: alpha helix (56.08 %), 310 helix (0 %), Pi helix (0 %), beta bridge (0 %), extended strand (16.40 %), beta-turn (7.94 %), bend region (0 %), and random coil (19.58 %). Compared to the I-tasser, phyre2, and Swiss-model, the 3Dpro predicted structure was determined as the most suitable structure based on the complete modeling of the given length of the amino acid sequence. Loop modeling was done at Leu29-Gln37, Ser51-Gln70, Glu72-Gln77, Glu57-Ser76, Val103-Thr113, and Glu167-Asp186. The model was refined to minimum RMSD of 1.734 Å and molprobity score of 1.134 that is quite low compared to the original structure score of 3.312, reflecting good quality of the modelled structure. Similarly, the clash score in contrast to the original structure is 94.7 times lower demonstrating steric clash free structure. The galaxy energy of the structure is very stable (-3784.31) and Ramachandran favored distribution increased from 93.6 to 95.7 %. The top 10 refined models of the MEPVC are tabulated in Table 2. The 3D models of the vaccine construct after loop modelling and refinement is presented in Figure 4.A. The overall Z-score of the modelled structure is -4 and the score is within the range of same size proteins in the pdb illustrating good quality as depicted in Figure 4.B. Refinement of the structure Ramachandran plot demonstrated the construct to contain 93.2 % of its residues in the most favored regions, while 5.7%, 0.0%, 1.1% residues are in additional allowed region, generously allowed region, and disallowed regions, respectively (Figure 4.C). The overall average G-factor of the construct is 0.05.
Fig. 4 A. The tertiary structure of the designed MEPVC. The red, green, purple, and yellow colors represent AAY linkers, epitopes, adjuvant, and EAAK linkers, respectively. B. The z-score plot indicating the overall good quality of the MEPVC. The z-score of the input MEPVC is shown by a black dot, validating the score within the range revealed for similar size proteins. C. Ramachandran plot for the MEPVC illustrating distribution of torsion angles (blue squares) comparative to the core (shown in red) and allowed (shown in brown) regions. Residues in the generously allowed region are shown in dark yellow whereas disallowed regions are depicted in pale yellow.