Quantitative assessment of chlorophyll types in cryo-EM maps of photosystem I acclimated to far-red light

Chlorophyll cofactors are vital for the metabolism of photosynthetic organisms. Cryo-electron microscopy (cryo-EM) has been used to elucidate molecular structures of pigment-protein complexes, but the minor structural differences between multiple types of chlorophylls make them difficult to distinguish in cryo-EM maps. This is exemplified by inconsistencies in the assignments of chlorophyll f molecules in structures of photosystem I acclimated to far-red light (FRL-PSI). A quantitative assessment of chlorophyll substituents in cryo-EM maps was used to identify chlorophyll f-binding sites in structures of FRL-PSI from two cyanobacteria. The two cryo-EM maps provide direct evidence for chlorophyll f-binding at two and three binding sites, respectively, and three more sites in each structure exhibit strong indirect evidence for chlorophyll f-binding. Common themes in chlorophyll f-binding are described that clarify the current understanding of the molecular basis for FRL photoacclimation in photosystems.