Opportunities and challenges for assigning cofactors in cryo-EM density maps of chlorophyll-containing proteins

The accurate assignment of cofactors in cryo-electron microscopy maps is crucial in determining protein function. This is particularly true for chlorophylls (Chls), for which small structural differences lead to important functional differences. Recent cryo-electron microscopy structures of Chl-containing protein complexes exemplify the difficulties in distinguishing Chl b and Chl f from Chl a. We use these structures as examples to discuss general issues arising from local resolution differences, properties of electrostatic potential maps, and the chemical environment which must be considered to make accurate assignments. We offer suggestions for how to improve the reliability of such assignments. In this Perspective, Christopher Gisriel et al. discuss the challenges in accurate assignment of co-factors in cryo-EM, particularly for chlorophylls. They explore the factors that lead to misassignment and offer suggestions for improving reliability of cryo-EM-based assignments.