2.85 and 2.99 Å resolution structures of 110 kDa nitrite reductase determined by 200 kV cryogenic electron microscopy

Cu-containing nitrite reductases (NiRs) are 110 kDa enzymes that play central roles in denitrification. Although the NiRs have been well studied, with over 100 Protein Data Bank entries, such issues as crystal packing, photoreduction, and lack of high pH cases have impeded structural analysis of their catalytic mechanisms. Here we show the cryogenic electron microscopy (cryo-EM) structures of Achromobacter cycloclastes NiR (AcNiR) at pH 6.2 and 8.1. The optimization of 3D-reconstruction parameters achieved 2.99 and 2.85 angstrom resolution. Comprehensive comparisons with cryo-EM and 56 AcNiR crystal structures suggested crystallographic artifacts in residues 185-215 and His255 ' due to packing and photoreduction, respectively. We used a newly developed map comparison method to detect structural change around the type 2 Cu site. While the theoretical estimation of coordinate errors of cryo-EM structures remains difficult, combined analysis using X-ray and cryo-EM structures will allow deeper insight into the local structural changes of proteins.

Automated summary

Cu-containing nitrite reductases play central roles in denitrification, but structural analysis of their catalytic mechanisms has been impeded by issues such as crystal packing and photoreduction. Cryo-EM structures of Achromobacter cycloclastes NiR at different pH values were obtained, revealing structural changes and crystallographic artifacts in certain residues. The combination of X-ray and cryo-EM structures provides deeper insight into local structural changes of proteins, despite challenges in estimating coordinate errors in cryo-EM structures.