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Alanine racemase (Alr) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent racemization between l- and d-alanine in bacteria. Owing to the potential interest in targeting Alr for antibacterial drug development, several studies have determined the structures of Alr from different species, proposing models for the reaction mechanism. Insights into its reaction dynamics may be conducive to a better understanding of the Alr reaction mechanism. In this study, we determined the structures of the apo and reaction states of Bacillus subtilis Alr (BsAlr) at room temperature using a fixed-target based X-ray free-electron laser. The 2.3 Å resolution structures revealed the alanine substrate or intermediate in various positions at the active site. Conformational change between the N- and C-terminal domains of BsAlr expanded the entryway for substrate binding. In the reaction state of BsAlr, two main alanine binding states were observed: one alanine molecule is positioned away from PLP, whereas the other alanine molecule is covalently bonded to PLP. These structures might represent the dynamic states of the substrate for entrance into, reaction with, or exit from the active site. Our approach provides a simple and rapid method for elucidating the intermediate structure of Alr, which can be expanded to other enzymes. [ABSTRACT FROM AUTHOR]

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) operates hard X-ray and soft X-ray beamlines for conducting scientific experiments providing intense ultrashort X-ray pulses based on the self-amplified spontaneous emission (SASE) process. The X-ray free-electron laser is characterized by strong pulse-to-pulse fluctuations resulting from the SASE process. Therefore, online photon diagnostics are very important for rigorous measurements. The concept of photo-absorption and emission using solid materials is seldom considered in soft X-ray beamline diagnostics. Instead, gas monitoring detectors, which utilize the photo-ionization of noble gas, are employed for monitoring the beam intensity. To track the beam position at the soft X-ray beamline in addition to those intensity monitors, an X-ray ionization beam position monitor (XIBPM) has been developed and characterized at the soft X-ray beamline of PAL-XFEL. The XIBPM utilizes ionization of either the residual gas in an ultra-high-vacuum environment or injected krypton gas, along with a microchannel plate with phosphor. The XIBPM was tested separately for monitoring horizontal and vertical beam positions, confirming the feasibility of tracking relative changes in beam position both on average and down to single-shot measurements. This paper presents the basic structure and test results of the newly developed non-invasive XIBPM.

The Pohang Accelerator Laboratory X‐ray Free‐Electron Laser (PAL‐XFEL) operates hard X‐ray and soft X‐ray beamlines for conducting scientific experiments providing intense ultrashort X‐ray pulses based on the self‐amplified spontaneous emission (SASE) process. The X‐ray free‐electron laser is characterized by strong pulse‐to‐pulse fluctuations resulting from the SASE process. Therefore, online photon diagnostics are very important for rigorous measurements. The concept of photo‐absorption and emission using solid materials is seldom considered in soft X‐ray beamline diagnostics. Instead, gas monitoring detectors, which utilize the photo‐ionization of noble gas, are employed for monitoring the beam intensity. To track the beam position at the soft X‐ray beamline in addition to those intensity monitors, an X‐ray ionization beam position monitor (XIBPM) has been developed and characterized at the soft X‐ray beamline of PAL‐XFEL. The XIBPM utilizes ionization of either the residual gas in an ultra‐high‐vacuum environment or injected krypton gas, along with a microchannel plate with phosphor. The XIBPM was tested separately for monitoring horizontal and vertical beam positions, confirming the feasibility of tracking relative changes in beam position both on average and down to single‐shot measurements. This paper presents the basic structure and test results of the newly developed non‐invasive XIBPM. [ABSTRACT FROM AUTHOR]

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. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Ever since the discovery that Mn was required for oxygen evolution in plants by Pirson in 1937 and the period-four oscillation in flash-induced oxygen evolution by Joliot and Kok in the 1970s, understanding of this process has advanced enormously using state-of-the-art methods. The most recent in this series of innovative techniques was the introduction of X-ray free-electron lasers (XFELs) a decade ago, which led to another quantum leap in the understanding in this field, by enabling operando X-ray structural and X-ray spectroscopy studies at room temperature. This review summarizes the current understanding of the structure of Photosystem II (PS II) and its catalytic centre, the Mn4 CaO5 complex, in the intermediate Si (i = 0-4)-states of the Kok cycle, obtained using XFELs.

Single bubble sonoluminescence (SBSL) is the phenomenon of synchronous light emission due to the violent collapse of a single spherical bubble in a liquid, driven by an ultrasonic field. During the bubble collapse, matter inside the bubble reaches extreme conditions of several gigapascals and temperatures on the order of 10000 K, leading to picosecond flashes of visible light. To this day, details regarding the energy focusing mechanism rely on simulations due to the fast dynamics of the bubble collapse and spatial scales below the optical resolution limit. In this work we present phase-contrast holographic imaging with single x-ray free-electron laser (XFEL) pulses of a SBSL cavitation bubble in water. X-rays probe the electron density structure and by that provide a uniquely new view on the bubble interior and its collapse dynamics. The involved fast time-scales are accessed by sub-100 fs XFEL pulses and a custom synchronization scheme for the bubble oscillator. We find that during the whole oscillation cycle the bubble's density profile can be well described by a simple step-like structure, with the radius R following the dynamics of the Gilmore model. The quantitatively measured internal density and width of the boundary layer exhibit a large variance. Smallest reconstructed bubble sizes reach down to R ≃ 0.8 μ m , and are consistent with spherical symmetry. While we here achieved a spatial resolution of a few 100 nm, the visibility of the bubble and its internal structure is limited by the total x-ray phase shift which can be scaled with experimental parameters. [ABSTRACT FROM AUTHOR]

There is a growing interest in designing and building compact X-ray Free Electron Lasers (FELs) for scientific and industry applications. In this paper, we report an X-ray Regenerative Amplifier FEL (XRAFEL) design based on a proposed Ultra Compact X-ray FEL configuration. Our results show that an XRAFEL can dramatically enhance the temporal coherence and increase the spectral brightness of the radiation in the hard X-ray regime without increasing the footprint of the FEL configuration. The proposed compact, fully coherent, and high-flux hard X-ray source holds promise as a valuable candidate for a wide range of high-impact applications in both academia and industry. [ABSTRACT FROM AUTHOR]

In X-ray diffraction imaging (XDI), electron density maps of a targeted particle are reconstructed computationally from the diffraction pattern alone using phase-retrieval (PR) algorithms. However, the PR calculations sometimes fail to yield realistic electron density maps that approximate the structure of the particle. This occurs due to the absence of structure amplitudes at and near the zero-scattering angle and the presence of Poisson noise in weak diffraction patterns. Consequently, the PR calculation becomes a bottleneck for XDI structure analyses. Here, a protocol to efficiently yield realistic maps is proposed. The protocol is based on the empirical observation that realistic maps tend to yield low similarity scores, as suggested in our prior study [Sekiguchi et al. (2017), J. Synchrotron Rad. 24, 1024–1038]. Among independently and concurrently executed PR calculations, the protocol modifies all maps using the electron-density maps exhibiting low similarity scores. This approach, along with a new protocol for estimating particle shape, improved the probability of obtaining realistic maps for diffraction patterns from various aggregates of colloidal gold particles, as compared with PR calculations performed without the protocol. Consequently, the protocol has the potential to reduce computational costs in PR calculations and enable efficient XDI structure analysis of non-crystalline particles using synchrotron X-rays and X-ray free-electron laser pulses.

Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances. While recent publications have proposed a radical-based catalytic mechanism as well as novel sulfonate-based binding modes of MCR for its native substrates, the structure of the active state of MCR, as well as a complete characterization of the reaction, remain elusive. Previous attempts to structurally characterize the active MCR-Ni(I) state have been unsuccessful due to oxidation of the redox- sensitive catalytic Ni center. Further, while many cryo structures of the inactive Ni(II)-enzyme in various substrates-bound forms have been published, no room temperature structures have been reported, and the structure and mechanism of MCR under physiologically relevant conditions is not known. In this study, we report the first room temperature structure of the MCRred1-silent Ni(II) form using an X-ray Free-Electron Laser (XFEL), with simultaneous X-ray Emission Spectroscopy (XES) and X-ray Diffraction (XRD) data collection. In celebration of the seminal contributions of inorganic chemist Dick Holm to our understanding of nickel-based catalysis, we are honored to announce our findings in this special issue dedicated to this remarkable pioneer of bioinorganic chemistry.

The Femtosecond X-ray Experiments (FXE) instrument at the European X-ray Free-Electron Laser (EuXFEL) provides an optimized platform for investigations of ultrafast physical, chemical and biological processes. It operates in the energy range 4.7–20 keV accommodating flexible and versatile environments for a wide range of samples using diverse ultrafast X-ray spectroscopic, scattering and diffraction techniques. FXE is particularly suitable for experiments taking advantage of the sub-MHz repetition rates provided by the EuXFEL. In this paper a dedicated setup for studies on ultrafast biological and chemical dynamics in solution phase at sub-MHz rates at FXE is presented. Particular emphasis on the different liquid jet sample delivery options and their performance is given. Our portfolio of high-speed jets compatible with sub-MHz experiments includes cylindrical jets, gas dynamic virtual nozzles and flat jets. The capability to perform multi-color X-ray emission spectroscopy (XES) experiments is illustrated by a set of measurements using the dispersive X-ray spectrometer in von Hamos geometry. Static XES data collected using a multi-crystal scanning Johann-type spectrometer are also presented. A few examples of experimental results on ultrafast time-resolved X-ray emission spectroscopy and wide-angle X-ray scattering at sub-MHz pulse repetition rates are given.

The high pulse intensity and repetition rate of the European X-ray Free-Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Currently, X-ray crystallography, which typically uses synchrotron sources, remains the dominant method for structural determination of proteins and other biomolecules. However, small protein crystals do not provide sufficiently high-resolution diffraction patterns and suffer radiation damage; therefore, conventional X-ray crystallography needs larger protein crystals. The burgeoning method of serial crystallography using X-ray free-electron lasers (XFELs) avoids these challenges: it affords excellent structural data from weakly diffracting objects, including tiny crystals. An XFEL is implemented by irradiating microjets of suspensions of microcrystals with very intense X-ray beams. However, while the method for creating microcrystalline microjets is well established, little attention is given to the growth of high-quality nano/microcrystals suitable for XFEL experiments. In this study, in order to assist the growth of such crystals, we calculate the mean crystal size and the time needed to grow crystals to the desired size in batch crystallization (the predominant method for preparing the required microcrystalline slurries); this time is reckoned theoretically both for microcrystals and for crystals larger than the upper limit of the Gibbs–Thomson effect. The impact of the omnipresent impurities on the growth of microcrystals is also considered quantitatively. Experiments, performed with the model protein lysozyme, support the theoretical predictions. [ABSTRACT FROM AUTHOR]

The Femtosecond X-ray Experiments (FXE) instrument at the European X-ray Free-Electron Laser (EuXFEL) provides an optimized platform for investigations of ultrafast physical, chemical and biological processes. It operates in the energy range 4.7-20 keVaccommodating flexible and versatile environments for a wide range of samples using diverse ultrafast X-ray spectroscopic, scattering and diffraction techniques. FXE is particularly suitable for experiments taking advantage of the sub-MHz repetition rates provided by the EuXFEL. In this paper a dedicated setup for studies on ultrafast biological and chemical dynamics in solution phase at sub-MHz rates at FXE is presented. Particular emphasis on the different liquid jet sample delivery options and their performance is given. Our portfolio of high-speed jets compatible with sub- MHz experiments includes cylindrical jets, gas dynamic virtual nozzles and flat jets. The capability to perform multi-color X-ray emission spectroscopy (XES) experiments is illustrated by a set of measurements using the dispersive X-ray spectrometer in von Hamos geometry. Static XES data collected using a multicrystal scanning Johann-type spectrometer are also presented. A few examples of experimental results on ultrafast time-resolved X-ray emission spectroscopy and wide-angle X-ray scattering at sub-MHz pulse repetition rates are given. [ABSTRACT FROM AUTHOR]

The high pulse intensity and repetition rate of the European X-ray Free- Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL. [ABSTRACT FROM AUTHOR]

The characterization of X-ray focal spots is of great significance for the diagnosis and performance optimization of focusing systems. X-ray free-electron lasers (XFELs) are the latest generation of X-ray sources with ultrahigh brilliance, ultrashort pulse duration and nearly full transverse coherence. Because each XFEL pulse is unique and has an ultrahigh peak intensity, it is difficult to characterize its focal spot size individually with full power. Herein, a method for characterizing the spot size at the focus position is proposed based on coherent diffraction imaging. A numerical simulation was conducted to verify the feasibility of the proposed method. The focal spot size of the Coherent Scattering and Imaging endstation at the Shanghai Soft X-ray Free Electron Laser Facility was characterized using the method. The full width at half-maxima of the focal spot intensity and spot size in the horizontal and vertical directions were calculated to be 2.10 ± 0.24 µm and 2.00 ± 0.20 µm, respectively. An ablation imprint on the silicon frame was used to validate the results of the proposed method.

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding.

X-ray free-electron lasers (FELs) provide cutting-edge tools for fundamental researches to study nature down to the atomic level at a time-scale that fits this resolution. A precise knowledge of temporal information of FEL pulses is the central issue for its applications. Here we proposed and demonstrated a novel method to determine the FEL temporal profiles online. This robust method, designed for ultrafast FELs, allows researchers to acquire real-time longitudinal profiles of FEL pulses as well as their arrive times with respect to the external optical laser with a resolution better than 6 fs. Based on this method, we can also directly measure various properties of FEL pulses and correlations between them online. This helps us to further understand the FEL lasing processes and realize the generation of stable, nearly fully coherent soft X-ray laser pulses at the Shanghai Soft X-ray FEL facility. This method will enhance the experimental opportunities for ultrafast science in various areas.

There is a growing interest in designing and building compact X-ray Free Electron Lasers (FELs) for scientific and industry applications. In this paper, we report an X-ray Regenerative Amplifier FEL (XRAFEL) design based on a proposed Ultra Compact X-ray FEL configuration. Our results show that an XRAFEL can dramatically enhance the temporal coherence and increase the spectral brightness of the radiation in the hard X-ray regime without increasing the footprint of the FEL configuration. The proposed compact, fully coherent, and high-flux hard X-ray source holds promise as a valuable candidate for a wide range of high-impact applications in both academia and industry.

A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization.

Featured Application: Recently upgraded inject-and-transfer system will improve beamtime efficiency for serial femtosecond crystallography. Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX. [ABSTRACT FROM AUTHOR]

Featured Application: Our results provide insight into the material selection and directions to pursue in developing sample delivery devices used in XFEL studies. A crystal delivery system is essential in serial femtosecond crystallography experiments using an X-ray free-electron laser (XFEL). Investigating the XFEL-induced radiation damage to materials potentially applicable to sample delivery devices is vital for developing a sample delivery system. In this study, we investigated the radiation damage caused by an XFEL to polydimethylsiloxane (PDMS) and polyimide (PI), which are widely used as sample delivery materials in synchrotron X-rays. Upon XFEL exposure, the PDMS film produced irregularly shaped and sized holes, whereas the PI film produced relatively regular shaped and sized holes. When XFELs were exposed to the channel of the PDMS-based microfluidic device, holes were generated on the film by the radiation damage and the microfluidic device and the internal channel region were structurally destroyed. The PI-based microfluidic device experienced no structural destruction, except for the holes generated by the XFEL. However, as the XFELs were continuously exposed, bubbles generated from the solution due to radiation damage; the accumulation of these bubbles interfered with the path of the inner channel of the microfluidic device. Our results will not only help understand the phenomenon of radiation damage of PDMS and PI films by XFEL, but also provide insight into the directions to pursue in developing applications of PDMS and PI films in XFEL studies. [ABSTRACT FROM AUTHOR]

Variable gap undulator widely used in X-ray free-electron laser (XFEL) enables the photon energy scan by changing its gap. A phase-shifter should be incorporated to compensate for the phase mismatch between the electron bunches and X-ray pulses arising while those traverse the drift space between undulator segments. The uncertainties in both the undulator parameter and the drift space distance introduce an error in calculating the optimum gap distance of the phase-shifter for the different undulator K. The phase-shifter gap needs to be set where the error is within the tolerable range. The control scheme we propose can maintain full FEL intensity over the scanned photon energies. [ABSTRACT FROM AUTHOR]

Multilayer Laue lenses (MLLs) made from WC and SiC were previously used to focus megahertz X-ray pulse trains of the European XFEL free-electron laser, but suffered damage with trains of 30 pulses or longer at an incident fluence of about 0.13 J/cm2 per pulse. Here, we present numerical simulations of the heating of MLLs of various designs, geometry and material properties, that are exposed to such pulse trains. We find that it should be possible to focus the full beam of about 10 J/cm2 fluence of XFEL using materials of a low atomic number. To achieve high diffraction efficiency, lenses made from such materials should be considerably thicker than those used in the experiments. In addition to the lower absorption, this leads to the deposition of energy over a larger volume of the multilayer structure and hence to a lower dose, a lower temperature increase, and an improved dissipation of heat. [ABSTRACT FROM AUTHOR]

Serial crystallography, using either femtosecond X-ray pulses from free-electron laser sources or short synchrotron-radiation exposures, has the potential to reveal metalloprotein structural details while minimizing damage processes. However, deriving a self-consistent set of Bragg intensities from numerous still-crystal exposures remains a difficult problem, with optimal protocols likely to be quite different from those well established for rotation photography. Here several data processing issues unique to serial crystallography are examined. It is found that the limiting resolution differs for each shot, an effect that is likely to be due to both the sample heterogeneity and pulse-to-pulse variation in experimental conditions. Shots with lower resolution limits produce lower-quality models for predicting Bragg spot positions during the integration step. Also, still shots by their nature record only partial measurements of the Bragg intensity. An approximate model that corrects to the full-spot equivalent (with the simplifying assumption that the X-rays are monochromatic) brings the distribution of intensities closer to that expected from an ideal crystal, and improves the sharpness of anomalous difference Fourier peaks indicating metal positions.

Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX.

A crystal delivery system is essential in serial femtosecond crystallography experiments using an X-ray free-electron laser (XFEL). Investigating the XFEL-induced radiation damage to materials potentially applicable to sample delivery devices is vital for developing a sample delivery system. In this study, we investigated the radiation damage caused by an XFEL to polydimethylsiloxane (PDMS) and polyimide (PI), which are widely used as sample delivery materials in synchrotron X-rays. Upon XFEL exposure, the PDMS film produced irregularly shaped and sized holes, whereas the PI film produced relatively regular shaped and sized holes. When XFELs were exposed to the channel of the PDMS-based microfluidic device, holes were generated on the film by the radiation damage and the microfluidic device and the internal channel region were structurally destroyed. The PI-based microfluidic device experienced no structural destruction, except for the holes generated by the XFEL. However, as the XFELs were continuously exposed, bubbles generated from the solution due to radiation damage; the accumulation of these bubbles interfered with the path of the inner channel of the microfluidic device. Our results will not only help understand the phenomenon of radiation damage of PDMS and PI films by XFEL, but also provide insight into the directions to pursue in developing applications of PDMS and PI films in XFEL studies.

Variable gap undulator widely used in X-ray free-electron laser (XFEL) enables the photon energy scan by changing its gap. A phase-shifter should be incorporated to compensate for the phase mismatch between the electron bunches and X-ray pulses arising while those traverse the drift space between undulator segments. The uncertainties in both the undulator parameter and the drift space distance introduce an error in calculating the optimum gap distance of the phase-shifter for the different undulator K. The phase-shifter gap needs to be set where the error is within the tolerable range. The control scheme we propose can maintain full FEL intensity over the scanned photon energies.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization. [ABSTRACT FROM AUTHOR]

Multilayer Laue lenses (MLLs) made from WC and SiC were previously used to focus megahertz X-ray pulse trains of the European XFEL free-electron laser, but suffered damage with trains of 30 pulses or longer at an incident fluence of about 0.13 J/cm2 per pulse. Here, we present numerical simulations of the heating of MLLs of various designs, geometry and material properties, that are exposed to such pulse trains. We find that it should be possible to focus the full beam of about 10 J/cm2 fluence of XFEL using materials of a low atomic number. To achieve high diffraction efficiency, lenses made from such materials should be considerably thicker than those used in the experiments. In addition to the lower absorption, this leads to the deposition of energy over a larger volume of the multilayer structure and hence to a lower dose, a lower temperature increase, and an improved dissipation of heat.

Understanding the origin of reactive species following ionization in aqueous systems is an important aspect of radiation–matter interactions as the initial reactive species lead to production of radicals and subsequent long-term radiation damage. Tunable ultrafast X-ray free-electron pulses provide a new window to probe events occurring on the sub-picosecond timescale, supplementing other methodologies, such as pulse radiolysis, scavenger studies, and stop flow that capture longer timescale chemical phenomena. We review initial work capturing the fastest chemical processes in liquid water radiolysis using optical pump/X-ray probe spectroscopy in the water window and discuss how ultrafast X-ray pump/X-ray probe spectroscopies can examine ionization-induced processes more generally and with better time resolution. Ultimately, these methods will be applied to understanding radiation effects in complex aqueous solutions present in high-level nuclear waste. [ABSTRACT FROM AUTHOR]

Performance of Self Amplifed Spontaneous Emission (SASE) operation for an X-ray Free-Electron Laser (FEL), is optimized by Multiple Objective Evolutionary Algorithms (MOEA). Different types of hybrid planar undulators are considered to achieve 0.5 Å = FEL = 1.5 Å wavelength range. This demand is regarded as a continuous optimization problem, and hence, an evolutionary algorithm is designed to find optimal FEL performance parameters. An encoding scheme comprising of undulator period (u) and undulator gap (g) is adopted here to find optimal values for saturation power (Psat) and FEL subject to several physical constraints on u and g such as the ratio of g/u and Lsat. It is shown that MOEA gives optimal solutions for estimation of Lsat with plausible Psat values. [ABSTRACT FROM AUTHOR]

Ultimate focusing of an X‐ray free‐electron laser (XFEL) enables the generation of ultrahigh‐intensity X‐ray pulses. Although sub‐10 nm focusing has already been achieved using synchrotron light sources, the sub‐10 nm focusing of XFEL beams remains difficult mainly because the insufficient stability of the light source hinders the evaluation of a focused beam profile. This problem is specifically disadvantageous for the Kirkpatrick–Baez (KB) mirror focusing system, in which a slight misalignment of ∼300 nrad can degrade the focused beam. In this work, an X‐ray nanobeam of a free‐electron laser was generated using reflective KB focusing optics combined with speckle interferometry. The speckle profiles generated by 2 nm platinum particles were systematically investigated on a single‐shot basis by changing the alignment of the multilayer KB mirror system installed at the SPring‐8 Angstrom Compact Free‐Electron Laser, in combination with computer simulations. It was verified that the KB mirror alignments were optimized with the required accuracy, and a focused vertical beam of 5.8 nm (±1.2 nm) was achieved after optimization. The speckle interferometry reported in this study is expected to be an effective tool for optimizing the alignment of nano‐focusing systems and for generating an unprecedented intensity of up to 1022 W cm−2 using XFEL sources. [ABSTRACT FROM AUTHOR]

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies. [ABSTRACT FROM AUTHOR]

The long‐anticipated high‐resolution structures of the human melatonin G protein‐coupled receptors MT1 and MT2, involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti‐insomnia and antidepression drugs using X‐ray free‐electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane‐buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT2 structure allowed accurate mapping of type 2 diabetes‐related single‐nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein–membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT1 and MT2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next‐generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects. [ABSTRACT FROM AUTHOR]

Structural information on protein dynamics is a critical factor in fully understanding the protein functions. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) is a recently established technique for visualizing the structural changes or reactions in proteins that are at work with high spatial and temporal resolution. In the pump-probe method, protein microcrystals are continuously delivered from an injector and exposed to an X-ray free-electron laser (XFEL) pulse after a trigger to initiate a reaction, such as light, chemicals, temperature, and electric field, which affords the structural snapshots of intermediates that occur in the protein. We are in the process of developing the device and techniques for pump-probe TR-SFX while using XFEL produced at SPring-8 Angstrom Compact Free-Electron Laser (SACLA). In this paper, we described our current development details and data collection strategies for the optical pump X-ray probe TR-SFX experiment at SACLA and then reported the techniques of in crystallo TR spectroscopy, which is useful in clarifying the nature of reaction that takes place in crystals in advance. [ABSTRACT FROM AUTHOR]

Accelerator-based fourth-generation light sources are utilized in a wide range of interdisciplinary applications such as nanotechnology, materials science, biosciences, and medicine. A hard X-ray free-electron laser (FEL), as a state-of-the-art light source, was optimized using evolutionary algorithms for dedicated user applications such as X-ray Raman scattering (XRS), resonant inelastic X-ray scattering (RIXS), and X-ray emission spectroscopies (XES). Optimal parameter sets were obtained for an in-vacuum planar undulator driven by an 8 GeV electron beam. Performance parameters of self-amplified spontaneous emission (SASE) operation (i.e. optimized SASE performance parameters through evolutionary algorithms) were found to be consistent with operating X-ray FEL facilities around the world. It is shown that FEL characteristics for specific user experiments can be optimized by finding several evolutionary algorithm solutions within the range of 5 keV to 10 keV. [ABSTRACT FROM AUTHOR]