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The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room temperature time-resolved serial crystallography. A covalent adduct forms between the carbon-4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined from the maidenhair fern Phy3 23 years ago. 

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. 

Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher-quality protein crystals yielding improved diffraction data and higher-resolution structures. Typically, counter-diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X-ray beamline. Despite the advantages of counter-diffusion protein crystallization, there are few fixed-target devices that utilize counter diffusion for crystallization. In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propylene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed-target utilization by crystallographers. 

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. 

The field of time-resolved macromolecular crystallography has been expanding rapidly after free electron lasers for hard x rays (XFELs) became available. Techniques to collect and process data from XFELs spread to synchrotron light sources. Although time-scales and data collection modalities can differ substantially between these types of light sources, the analysis of the resulting x-ray data proceeds essentially along the same pathway. At the base of a successful time-resolved experiment is a difference electron density (DED) map that contains chemically meaningful signal. If such a difference map cannot be obtained, the experiment has failed. Here, a practical approach is presented to calculate DED maps and use them to determine structural models. 

Inspired by recent progress in time-resolved x-ray crystallography and the adoption of time-resolution by cryo-electronmicroscopy, this article enumerates several approaches developed to become bigger/smaller, faster, and better to gain new insight into the molecular mechanisms of life. This is illustrated by examples where chemical and physical stimuli spawn biological responses on various length and time-scales, from fractions of Ångströms to micro-meters and from femtoseconds to hours. 

Abstract Global warming increases ecosystem respiration (ER), creating a positive carbon-climate feedback. Thermal acclimation, the direct responses of biological communities to reduce the effects of temperature changes on respiration rates, is a critical mechanism that compensates for warming-induced ER increases and dampens this positive feedback. However, the extent and effects of this mechanism across diverse ecosystems remain unclear. By analyzing CO2 flux data from 93 eddy covariance sites worldwide, we observed thermal acclimation at 84 % of the sites. If sustained, thermal acclimation could reduce projected warming-induced nighttime ER increases by at least 25 % across most climate zones by 2041-2060. Strong thermal acclimation is particularly evident in ecosystems at high elevation, with low-carbon-content soils, and within tundra, semi-arid, and warm-summer Mediterranean climates, supporting the hypothesis that extreme environments favor the evolution of greater acclimation potential. Moreover, ecosystems with dense vegetation and high productivity such as humid tropical and subtropical forests generally exhibit strong thermal acclimation, suggesting that regions with substantial CO2 uptake may continue to serve as strong carbon sinks. Conversely, some ecosystems in cold continental climates show signs of enhancing thermal responses, the opposite of thermal acclimation, which could exacerbate carbon losses as climate warms. Our study underscores the widespread yet climate-specific patterns of thermal acclimation in global terrestrial ER, emphasizing the need to incorporate these patterns into Earth System Models for more accurate carbon-climate feedback projections.