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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. 

The upgrade of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France to an Extremely Brilliant Source (EBS) is expected to enable time-resolved synchrotron serial crystallography (SSX) experiments with sub-millisecond time resolution. ID29 is a new beamline dedicated to SSX experiments at ESRF–EBS. Here, we report experiments emerging from the initial phase of user operation at ID29. We first used microcrystals of photoactive yellow protein as a model system to exploit the potential of microsecond pulses for SSX. Subsequently, we investigated microcrystals of cytochromecnitrite reductase (ccNiR) with microsecond X-ray pulses. CcNiR is a decaheme protein that is ideal for the investigation of radiation damage at the various heme-iron sites. Finally, we performed a proof-of-concept subsecond time-resolved SSX experiment by photoactivating microcrystals of a myxobacterial phytochrome. 

With time-resolved crystallography (TRX), it is possible to follow the reaction dynamics in biological macromolecules by investigating the structure of transient states along the reaction coordinate. X-ray free electron lasers (XFELs) have enabled TRX experiments on previously uncharted femtosecond timescales. Here, we review the recent developments, opportunities, and challenges of pump-probe TRX at XFELs. 

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to theMycobacterium tuberculosisβ-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.