The application of hydrogen bonding analysis in X-ray crystallography to help orientate asparagine, glutamine and histidine side chains

Protein X-ray crystallography produces an electron density map that rarely detects individual hydrogen atoms or distinguishes between carbon, nitrogen and oxygen atoms in the electron density. This makes it difficult to orientate the side chains of Asn, Gln and His, which appear symmetrical in the electron density; their orientation is usually judged on the basis of hydrogen bonding. Based on the observation that almost all buried donors and acceptors are satisfied, we have developed a simple algorithm to compare the alternative conformations of these residues and, where possible, identify the most favourable. In a cross-section of protein structures we found a few side chains (15.0% of Asn and Gln and 9.9% of His) which would be more favourable in the alternative orientation. We have also found that this proportion rises slightly with worsening resolution.