Fig. 4

Multi-stage design, comparison with native proteins and representation of experimental data for 1kx8-based epitope-scaffold. Analysis of the two-step design pipeline, followed by a comparison of the distributions obtained for native proteins and the designs and plotting of biochemical experimental data. With the exception of the panel identifiers, the image was directly created with the code presented in Table 3. a Comparison between the first (orange) and the second (blue) generation of designs. score – shows the Rosetta energy score; hbond_bb_sc – quantifies the hydrogen bonds between backbone and side chain atoms; hbond_sc - quantifies the hydrogen bonds occurring between side chain atoms; RMSD – root mean square deviation relative to the original template. Second-generation designs showed minor improvements on backbone hydrogen bonding and a substantial improvement in overall Rosetta Energy. b Score and cavity volume for the selected decoys in comparison with structures of CATH [31] domains of similar size. The vertical dashed black line represents the score and cavity volume of the original 1kx8 after minimization, highlighting the improvements relative to the original scaffold. c Circular Dichroism and Surface Plasmon Resonance data for the best design shows a well folded helical protein that binds with high affinity to the expected target