Exploiting fourth-generation synchrotron radiation for enzyme and photoreceptor characterization.

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.

The structure of Chlamydomonas LOV1 as revealed by time-resolved serial synchrotron crystallography.

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 C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

Photoreception and signaling in bacterial phytochrome revealed by single-particle cryo-EM.

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).

KINNTREX: a neural network to unveil protein mechanisms from time-resolved X-ray crystallography.

Here, a machine-learning method based on a kinetically informed neural network (NN) is introduced. The proposed method is designed to analyze a time series of difference electron-density maps from a time-resolved X-ray crystallographic experiment. The method is named KINNTREX (kinetics-informed NN for time-resolved X-ray crystallography).

Blue and red in the protein world: Photoactive yellow protein and phytochromes as revealed by time-resolved crystallography.

Time-resolved crystallography (TRX) is a method designed to investigate functional motions of biological macromolecules on all time scales. Originally a synchrotron-based method, TRX is enabled by the development of TR Laue crystallography (TRLX). TR serial crystallography (TR-SX) is an extension of TRLX.

Directed ultrafast conformational changes accompany electron transfer in a photolyase as resolved by serial crystallography.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules.

"Multisystem Inflammatory Syndrome in Children"-Like Disease after COVID-19 Vaccination (MIS-V) with Potential Significance of Functional Active Autoantibodies Targeting G-Protein-Coupled Receptors (GPCR-fAAb) for Pathophysiology and Therapy.

Background: An infection with SARS-CoV-2 can trigger a systemic disorder by pathological autoimmune processes. A certain type of this dysregulation is known as Multisystemic inflammatory syndrome in children (MIS-C). However, similar symptoms may occur and have been described as Multisystemic inflammatory syndrome after SARS-CoV-2 Vaccination (MIS-V) following vaccination against SARS-CoV-2.

Signal Transduction in an Enzymatic Photoreceptor Revealed by Cryo-Electron Microscopy.

Phytochromes are essential photoreceptor proteins in plants with homologs in bacteria and fungi that regulate a variety of important environmental responses. They display a reversible photocycle between two distinct states, the red-light absorbing Pr and the far-red light absorbing Pfr, each with its own structure. The reversible Pr to Pfr photoconversion requires covalently bound bilin chromophore and regulates the activity of a C-terminal enzymatic domain, which is usually a histidine kinase (HK).

Monitoring of carbon-water fluxes at Eurasian meteorological stations using random forest and remote sensing.

Simulating the carbon-water fluxes at more widely distributed meteorological stations based on the sparsely and unevenly distributed eddy covariance flux stations is needed to accurately understand the carbon-water cycle of terrestrial ecosystems. We established a new framework consisting of machine learning, determination coefficient (R), Euclidean distance, and remote sensing (RS), to simulate the daily net ecosystem carbon dioxide exchange (NEE) and water flux (WF) of the Eurasian meteorological stations using a random forest model or/and RS. The daily NEE and WF datasets with RS-based information (NEE-RS and WF-RS) for 3774 and 4427 meteorological stations during 2002-2020 were produced, respectively.

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals.

Practical considerations for the analysis of time-resolved x-ray data.

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.

Detection and attribution of an anomaly in terrestrial photosynthesis in Europe during the COVID-19 lockdown.

Carbon dioxide (CO) uptake by plant photosynthesis, referred to as gross primary production (GPP) at the ecosystem level, is sensitive to environmental factors, including pollutant exposure, pollutant uptake, and changes in the scattering of solar shortwave irradiance (SW) - the energy source for photosynthesis. The 2020 spring lockdown due to COVID-19 resulted in improved air quality and atmospheric transparency, providing a unique opportunity to assess the impact of air pollutants on terrestrial ecosystem functioning. However, detecting these effects can be challenging as GPP is influenced by other meteorological drivers and management practices.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters.

Biological function investigated by time-resolved structure determination.

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.

Heterogeneity in the M. tuberculosis β-Lactamase Inhibition by Sulbactam.

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals .

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution.

Joint optimization of land carbon uptake and albedo can help achieve moderate instantaneous and long-term cooling effects.

Both carbon dioxide uptake and albedo of the land surface affect global climate. However, climate change mitigation by increasing carbon uptake can cause a warming trade-off by decreasing albedo, with most research focusing on afforestation and its interaction with snow. Here, we present carbon uptake and albedo observations from 176 globally distributed flux stations.

Protein motions visualized by femtosecond time-resolved crystallography: The case of photosensory vs photosynthetic proteins.

Proteins are dynamic objects and undergo conformational changes when functioning. These changes range from interconversion between states in equilibrium to ultrafast and coherent structural motions within one perturbed state. Time-resolved serial femtosecond crystallography at free-electron X-ray lasers can unravel structural changes with atomic resolution and down to femtosecond time scales.

Transient state measurements on proteins by time-resolved crystallography.

After decades of diligent development at synchrotron light sources, the field of time-resolved crystallography (TRX) is elevating to new heights. Driven by the appearance of X-ray free electron lasers and supported by serial crystallography, a large number of biological systems can now be investigated with TRX. This short review cannot comprehensively cover all of them, but it will shine light on, and provide a connection between, some of the recent and past results.

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection.

Macromolecular movies, storybooks written by nature.

Twelve years ago, the first free electron laser for hard X-rays (XFEL), the Linac Coherent Light Source (LCLS), came online. Due to the extreme brilliance and the ultrashort pulse duration of their X-ray radiation, XFELs are exceptionally well positioned to conduct time-resolved studies on biological macromolecules. Here, some of the pioneering experiments and recent results are summarized.