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Phytochromes are red-light photoreceptors first identified in plants, with homologs found in bacteria and fungi, that regulate a variety of critical physiological processes. They undergo a reversible photocycle between two distinct states: a red-light-absorbing Pr form and a far-red light-absorbing Pfr form. This Pr/Pfr photoconversion controls the activity of a C-terminal enzymatic domain, typically a histidine kinase (HK). However, the molecular mechanisms underlying light-induced regulation of HK activity in bacteria remain poorly understood, as only a few structures of unmodified bacterial phytochromes with HK activity are known. Recently, cryo-EM structures of a wild-type bacterial phytochrome with HK activity are solved that reveal homodimers in both the Pr and Pfr states, as well as a heterodimer with individual monomers in distinct Pr and Pfr states. Cryo-EM structures of a truncated version of the same phytochrome—lacking the HK domain—also show a homodimer in the Pfr state and a Pr/Pfr heterodimer. Here, we describe in detail how structural information is obtained from cryo-EM data on a full-length intact bacteriophytochrome, and how the cryo-EM structure can contribute to the understanding of the function of the phytochrome. In addition, we compare the cryo-EM structure to an unusual x-ray structure that is obtained from a fragmented full-length phytochrome crystallized in the Pr-state. 

Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light–absorbing Pr state and a far-red light–absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo–electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.