The planar peptide model has guided our understanding and interpretation of protein crystal structures since its origin in the 1950s. It is well understood that deviations from this model occur, but the majority of peptides are planar, as measured by the standard omega torsion angle. Here, we report the first analysis in proteins of the contribution of pyramidalization of the peptide nitrogen to peptide non-planarity in proteins. We do this by considering peptide bonds before proline residues, as in such prolyl-peptides the peptide hydrogen is replaced by a carbon atom in the proline side chain – allowing its pyramidalization to be accurately assessed in ultra-high resolution structures. Our results show that peptides deviate more from planarity than previous studies have detected, and in fact, are not even planar on average. Additionally, our data show, in agreement with early small molecule and peptide research, that the nitrogen atom in prolyl peptides is generally pyramidalized, and plays an almost equal role in generating deviations from planarity as does pure rotation. Correlation analyses show that local conformational features only explain a small fraction of the variability in planarity in real structures. We conclude that the tertiary environment exerts a dominant influence on non-planarity
