How X-rays damage LPMO protein in diffraction experiments

What is it about?

X-ray diffraction is the most widely used tool used to determine protein structures at atomic resolution. Despite its importance to structural biologists, X-rays are known to damage proteins, particularly those containing metals or redox cofactors. Radiation damage often flies under the radar undetected, and when it occurs at a protein's functional site, misinterpreting radiation damage artifacts as biologically relevant insights is a real concern. We performed a systematic study on the effects of X-rays on a crystal of NcAA9D, a type of lytic polysaccharide monooxygenase (LPMO). LPMOs contain a copper atom in their active site, which, while important for their function, is especially prone to the effects of X-rays. After analyzing 72 structures of NcAA9D, we found that X-rays progressively reduce the active site copper from Cu(II) to Cu(I), forming an electron density feature that is easy to misinterpret. Along the way, we also report our findings on how complex distributions of dose throughout a crystal can complicate the analysis of radiation damage.

Why is it important?

Our findings help to more fully describe the effects of radiation damage in LPMOs. We also describe the complexities inherent to analyzing protein structures with conformational heterogeneity and demonstrate how rigorous statistical analysis can be used to overcome these complexities.