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The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.

X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven atto-second X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser-, accelerator,- X-ray scientists as well as spectroscopists and biochemists optimizes X-ray pulse parameters, in tandem with sample delivery, crystal size, and advanced X-ray detectors. Ultimately, the new capability, attosecond serial X-ray crystallography and spectroscopy, will be applied to one of the most important problems in structural biology, which is to elucidate the dynamics of light reactions, electron transfer and protein structure in photosynthesis.

Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes., (© 2024. The Author(s).)

Nanoparticles, exhibiting functionally relevant structural heterogeneity, are at the forefront of cutting-edge research. Now, high-throughput single-particle imaging (SPI) with X-ray free-electron lasers (XFELs) creates opportunities for recovering the shape distributions of millions of particles that exhibit functionally relevant structural heterogeneity. To realize this potential, three challenges have to be overcome: (1) simultaneous parametrization of structural variability in real and reciprocal spaces; (2) efficiently inferring the latent parameters of each SPI measurement; (3) scaling up comparisons between 10 5 structural models and 10 6 XFEL-SPI measurements. Here, we describe how we overcame these three challenges to resolve the nonequilibrium shape distributions within millions of gold nanoparticles imaged at the European XFEL. These shape distributions allowed us to quantify the degree of asymmetry in these particles, discover a relatively stable "shape envelope" among nanoparticles, discern finite-size effects related to shape-controlling surfactants, and extrapolate nanoparticles' shapes to their idealized thermodynamic limit. Ultimately, these demonstrations show that XFEL SPI can help transform nanoparticle shape characterization from anecdotally interesting to statistically meaningful.

The main protease (M pro ) of SARS-CoV-2 is critical for viral function and a key drug target. M pro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized M pro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. M pro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized M pro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of M pro and the crystallization conditions necessary to study this structurally., (© 2024. The Author(s).)

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trp in /Met out transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60 nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13 MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility., (open access.)

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses., (© 2023. Springer Nature Limited.)

X-ray crystallography has witnessed a massive development over the past decade, driven by large increases in the intensity and brightness of X-ray sources and enabled by employing high-frame-rate X-ray detectors. The analysis of large data sets is done via automatic algorithms that are vulnerable to imperfections in the detector and noise inherent with the detection process. By improving the model of the behaviour of the detector, data can be analysed more reliably and data storage costs can be significantly reduced. One major requirement is a software mask that identifies defective pixels in diffraction frames. This paper introduces a methodology and program based upon concepts of machine learning, called robust mask maker (RMM), for the generation of bad-pixel masks for large-area X-ray pixel detectors based on modern robust statistics. It is proposed to discriminate normally behaving pixels from abnormal pixels by analysing routine measurements made with and without X-ray illumination. Analysis software typically uses a Bragg peak finder to detect Bragg peaks and an indexing method to detect crystal lattices among those peaks. Without proper masking of the bad pixels, peak finding methods often confuse the abnormal values of bad pixels in a pattern with true Bragg peaks and flag such patterns as useful regardless, leading to storage of enormous uninformative data sets. Also, it is computationally very expensive for indexing methods to search for crystal lattices among false peaks and the solution may be biased. This paper shows how RMM vastly improves peak finders and prevents them from labelling bad pixels as Bragg peaks, by demonstrating its effectiveness on several serial crystallography data sets., (© Alireza Sadri et al. 2022.)

Pump-probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump-probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented., (open access.)

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX., (© 2022. The Author(s).)

Liquid sample delivery systems are used extensively for serial femtosecond crystallography at X-ray free-electron lasers (XFELs). However, misalignment of the liquid jet and the XFEL beam leads to the X-rays either partially or completely missing the sample, resulting in sample wastage and a loss of experiment time. Implemented here is an algorithm to analyse optical images using machine vision to determine whether there is overlap of the X-ray beam and liquid jet. The long-term goal is to use the output from this algorithm to implement an automated feedback mechanism to maintain constant alignment of the X-ray beam and liquid jet. The key elements of this jet alignment algorithm are discussed and its performance is characterized by comparing the results with a manual analysis of the optical image data. The success rate of the algorithm for correctly identifying hits is quantified via a similarity metric, the Dice coefficient. In total four different nozzle designs were used in this study, yielding an overall Dice coefficient of 0.98., (© Jaydeep Patel et al. 2022.)

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control., (© 2022. The Author(s).)

Serial femtosecond crystallography is a rapidly developing method for determining the structure of biomolecules for samples which have proven challenging with conventional X-ray crystallography, such as for membrane proteins and microcrystals, or for time-resolved studies. The European XFEL, the first high repetition rate hard X-ray free electron laser, provides the ability to record diffraction data at more than an order of magnitude faster than previously achievable, putting increased demand on sample delivery and data processing. This work describes a publicly available serial femtosecond crystallography dataset collected at the SPB/SFX instrument at the European XFEL. This dataset contains information suitable for algorithmic development for detector calibration, image classification and structure determination, as well as testing and training for future users of the European XFEL and other XFELs., (© 2022. The Author(s).)

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL., (open access.)

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. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX., (© Diandra Doppler et al. 2022.)

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand-maximize-compress ( EMC ) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered., (© Yulong Zhuang et al. 2022.)

A peak-finding algorithm for serial crystallography (SX) data analysis based on the principle of 'robust statistics' has been developed. Methods which are statistically robust are generally more insensitive to any departures from model assumptions and are particularly effective when analysing mixtures of probability distributions. For example, these methods enable the discretization of data into a group comprising inliers ( i.e. the background noise) and another group comprising outliers ( i.e. Bragg peaks). Our robust statistics algorithm has two key advantages, which are demonstrated through testing using multiple SX data sets. First, it is relatively insensitive to the exact value of the input parameters and hence requires minimal optimization. This is critical for the algorithm to be able to run unsupervised, allowing for automated selection or 'vetoing' of SX diffraction data. Secondly, the processing of individual diffraction patterns can be easily parallelized. This means that it can analyse data from multiple detector modules simultaneously, making it ideally suited to real-time data processing. These characteristics mean that the robust peak finder (RPF) algorithm will be particularly beneficial for the new class of MHz X-ray free-electron laser sources, which generate large amounts of data in a short period of time., (© Marjan Hadian-Jazi et al. 2021.)

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described., (open access.)

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.

Intense, ultrashort, and high-repetition-rate X-ray pulses, combined with a femtosecond optical laser, allow pump-probe experiments with fast data acquisition and femtosecond time resolution. However, the relative timing of the X-ray pulses and the optical laser pulses can be controlled only to a level of the intrinsic error of the instrument which, without characterization, limits the time resolution of experiments. This limitation inevitably calls for a precise determination of the relative arrival time, which can be used after measurement for sorting and tagging the experimental data to a much finer resolution than it can be controlled to. The observed root-mean-square timing jitter between the X-ray and the optical laser at the SPB/SFX instrument at European XFEL was 308 fs. This first measurement of timing jitter at the European XFEL provides an important step in realizing ultrafast experiments at this novel X-ray source. A method for determining the change in the complex refractive index of samples is also presented.

The excited-state dynamics of a T-shaped bichromophoric molecule, consisting of two strong fluorophores, diphenyloxazole and diphenylpyrazoline, directly linked in an orthogonal geometry, was investigated. Despite the weak coupling ensured by this geometry and confirmed by the electronic absorption spectra, this dyad exhibits only weak fluorescence in both apolar and polar solvents, with fluorescence lifetimes ranging from 200 ps in CHX to 10 ps in ACN. Ultrafast spectroscopic measurements reveal that the fluorescence quenching in polar solvents is due to the population of a charge-separated state. In non-polar solvents, this process is energetically not feasible, and a quenching due to an efficient intersystem crossing (ISC) to the triplet manifold is proposed, based on quantum-chemical calculations. This process occurs via the spin-orbit charge-transfer (SOCT) ISC mechanism, which is enabled by the charge-transfer character acquired by the S1 state of the dyad upon structural relaxation and by the orthogonal arrangement of the molecular orbitals involved in the transition. The same mechanism is proposed to explain why the recombination of the charge-separated state is faster in medium than in highly polar solvents, as well as to account for the fast decay of the lowest triplet state to the ground state.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 μm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.

The excited-state dynamics of the push-pull azobenzene Methyl Orange (MO) were investigated in several solvents and water/glycerol mixtures using a combination of ultrafast time-resolved fluorescence and transient absorption in both the UV-visible and the IR regions, as well as quantum chemical calculations. Optical excitation of MO in its trans form results in the population of the S 2 ππ* state and is followed by internal conversion to the S 1 nπ* state in ∼50 fs. The population of this state decays on the sub-picosecond timescale by both internal conversion to the trans ground state and isomerisation to the cis ground state. Finally, the cis form converts thermally to the trans form on a timescale ranging from less than 50 ms to several minutes. Significant differences depending on the hydrogen-bond donor strength of the solvents, quantified by the Kamlet Taft parameter α, were observed: compared to the other solvents, in highly protic solvents (α > 1), (i) the viscosity dependence of the S 1 state lifetime is less pronounced, (ii) the S 1 state lifetime is shorter by a factor of ≈1.5 for the same viscosity, (iii) the trans-to-cis photoisomerisation efficiency is smaller, and (iv) the thermal cis-to-trans isomerisation is faster by a factor of ≥10 3 . These differences are explained in terms of hydrogen-bond interactions between the solvent and the azo nitrogen atoms of MO, which not only change the nature of the S 1 state but also have an impact on the shape of ground- and excited-state potentials, and, thus, affect the deactivation pathways from the excited state.