Figure 2.
When accounting for length, CDR-H3 loop flexibility analysis of the crystallographic antibody set reveals naïve (blue) antibodies to be slightly more flexible than mature (red). Floppy Inclusions and Rigid Substructure Topography-Pebble Game was used to determine the degrees of freedoms (DOFs) as a function of hydrogen-bonding energy cutoffs in crystal structure ensembles created by Rosetta FastRelax. Results were split, depending on whether the antibody was naïve or mature, as determined by BLAST alignment to its germline V-genes, and the mean DOFs were calculated along with the SD, shown in a lighter shade of the respective color. Subplots, below each main plot, show the p-value computed by a Kolmogorov–Smirnov (KS) test comparison of the naïve and mature DOF distributions for each hydrogen-bonding energy cutoff, with null hypothesis being that the distributions are the same. A dashed line indicates a p-value of 0.05. (A) To permit comparison across loops of multiple lengths, the DOFs were scaled to a theoretical maximum for each length (a value of 1 indicates all DOFs are available whereas a value of 0 indicates not DOFs are available). We found the scaled DOFs to be similar for both naïve and mature antibodies, quantified by KS test p-values and the areas under the curve (AUCs) ± SD: −4.70 ± 0.46 and −4.70 ± 0.48, respectively. (B) To exclude length effects on flexibility calculations, we compared DOFs for the most popular length (10 residues). We found the naïve AUC ± SD at −128.82 ± 8.99 was greater than the mature AUC ± SD at −121.85 ± 10.09, but still within a SD. The distributions appear similar at cutoffs between 0 and −6.0 kcal/mol, according to the KS test p-values.