Cryo-EM structure of Escherichia coli HPII catalase serendipitously co-purified with a recombinant adhesin construct.

Catalases are key antioxidant enzymes that protect aerobic organisms from reactive oxygen species generated through metabolic activities and host immune responses. In Escherichia coli, the monofunctional HPII catalase (encoded by katE) forms a stable tetrameric enzyme that contributes to oxidative stress resistance. During the purification of a recombinantly produced construct of the Vibrio cholerae FrhA adhesin, we unexpectedly observed a co-purifying protein species which peptide mass spectrometry identified as E. coli HPII catalase. Initial reconstruction was limited by severe preferred orientation, with ~98% particles adopting a dominant view that produced anisotropic reconstructions. We evaluated multiple processing strategies to mitigate this bias and found that the recently reported high-resolution ab initio reconstruction workflow provided the most effective improvement in angular sampling, yielding a 3.2 Å reconstruction with D2 symmetry. The resulting map enabled atomic model building, and comparison with existing crystallographic models showed strong overall agreement while revealing modest differences in tetrameric dimensions and surface-exposed regions that may reflect reduced lattice constraints in the cryo-EM structure. Together, this work reports the first cryo-EM structure of the E. coli HPII catalase serendipitously co-purified with an adhesin and provides a practical workflow for overcoming severe preferred orientation in single-particle cryo-EM analysis.