Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Quantifying bunch-mode influence on photon-counting detectors at SPring-8.

Count-loss characteristics of photon-counting 2D detectors are demonstrated for eight bunch-modes at SPring-8 through Monte Carlo simulations. As an indicator, the effective maximum count rate was introduced to signify the X-ray intensity that the detector can count with a linearity of 1% or better after applying a count-loss correction in each bunch-mode. The effective maximum count rate is revealed to vary depending on the bunch-mode and the intrinsic dead time of the detectors, ranging from 0.012 to 0.916 Mcps (megacounts per second) for a 120 ns dead time, 0.009 to 0.807 Mcps for a 0.5 µs dead time and 0.020 to 0.273 Mcps for a 3 µs intrinsic detector dead time. Even with equal-interval bunch-modes at SPring-8, the effective maximum count rate does not exceed 1 Mcps pixel-1. In other words, to obtain data with a linearity better than 1%, the maximum intensity of X-rays entering the detector should be reduced to 1 Mcps pixel-1 or less, and, in some cases, even lower, depending on the bunch-mode. When applying count-loss correction using optimized dead times tailored to each bunch-mode, the effective maximum count rate exceeds the values above. However, differences in the effective maximum count rate due to bunch-modes persist. Users of photon-counting 2D detectors are encouraged to familiarize themselves with the count-loss characteristics dependent on bunch-mode, and to conduct experiments accordingly. In addition, when designing the time structure of bunch-modes at synchrotron radiation facilities, it is essential to take into account the impact on experiments using photon-counting 2D detectors.

High-throughput and high-resolution powder X-ray diffractometer consisting of six sets of 2D CdTe detectors with variable sample-to-detector distance and innovative automation system.

The demand for powder X-ray diffraction analysis continues to increase in a variety of scientific fields, as the excellent beam quality of high-brightness synchrotron light sources enables the acquisition of high-quality measurement data with high intensity and angular resolution. Synchrotron powder diffraction has enabled the rapid measurement of many samples and various in situ/operando experiments in nonambient sample environments. To meet the demands for even higher throughput measurements using high-energy X-rays at SPring-8, a high-throughput and high-resolution powder diffraction system has been developed. This system is combined with six sets of two-dimensional (2D) CdTe detectors for high-energy X-rays, and various automation systems, including a system for automatic switching among large sample environmental equipment, have been developed in the third experimental hutch of the insertion device beamline BL13XU at SPring-8. In this diffractometer system, high-brilliance and high-energy X-rays ranging from 16 to 72 keV are available. The powder diffraction data measured under ambient and various nonambient conditions can be analysed using Rietveld refinement and the pair distribution function. Using the 2D CdTe detectors with variable sample-to-detector distance, three types of scan modes have been established: standard, single-step and high-resolution. A major feature is the ability to measure a whole powder pattern with millisecond resolution. Equally important, this system can measure powder diffraction data with high Q exceeding 30 Å-1 within several tens of seconds. This capability is expected to contribute significantly to new research avenues using machine learning and artificial intelligence by utilizing the large amount of data obtained from high-throughput measurements.

High-resolution and high-sensitivity X-ray ptychographic coherent diffraction imaging using the CITIUS detector.

Ptychographic coherent diffraction imaging (PCDI) is a synchrotron X-ray microscopy technique that provides high spatial resolution and a wide field of view. To improve the performance of PCDI, the performance of the synchrotron radiation source and imaging detector should be improved. In this study, ptychographic diffraction pattern measurements using the CITIUS high-speed X-ray image detector and the corresponding image reconstruction are reported. X-rays with an energy of 6.5 keV were focused by total reflection focusing mirrors, and a flux of ∼2.6 × 1010 photons s-1 was obtained at the sample plane. Diffraction intensity data were collected at up to ∼250 Mcounts s-1 pixel-1 without saturation of the detector. Measurements of tantalum test charts and silica particles and the reconstruction of phase images were performed. A resolution of ∼10 nm and a phase sensitivity of ∼0.01 rad were obtained. The CITIUS detector can be applied to the PCDI observation of various samples using low-emittance synchrotron radiation sources and to the stability evaluation of light sources.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Development and application of a tender X-ray ptychographic coherent diffraction imaging system on BL27SU at SPring-8.

Ptychographic coherent diffraction imaging (CDI) allows the visualization of both the structure and chemical state of materials on the nanoscale, and has been developed for use in the soft and hard X-ray regions. In this study, a ptychographic CDI system with pinhole or Fresnel zone-plate optics for use in the tender X-ray region (2-5 keV) was developed on beamline BL27SU at SPring-8, in which high-precision pinholes optimized for the tender energy range were used to obtain diffraction intensity patterns with a low background, and a temperature stabilization system was developed to reduce the drift of the sample position. A ptychography measurement of a 200 nm thick tantalum test chart was performed at an incident X-ray energy of 2.500 keV, and the phase image of the test chart was successfully reconstructed with approximately 50 nm resolution. As an application to practical materials, a sulfur polymer material was measured in the range of 2.465 to 2.500 keV including the sulfur K absorption edge, and the phase and absorption images were successfully reconstructed and the nanoscale absorption/phase spectra were derived from images at multiple energies. In 3 GeV synchrotron radiation facilities with a low-emittance storage ring, the use of the present system will allow the visualization on the nanoscale of the chemical states of various light elements that play important roles in materials science, biology and environmental science.

Development of a scanning soft X-ray spectromicroscope to investigate local electronic structures on surfaces and interfaces of advanced materials under conditions ranging from low vacuum to helium atmosphere.

A scanning soft X-ray spectromicroscope was recently developed based mainly on the photon-in/photon-out measurement scheme for the investigation of local electronic structures on the surfaces and interfaces of advanced materials under conditions ranging from low vacuum to helium atmosphere. The apparatus was installed at the soft X-ray beamline (BL17SU) at SPring-8. The characteristic features of the apparatus are described in detail. The feasibility of this spectromicroscope was demonstrated using soft X-ray undulator radiation. Here, based on these results, element-specific two-dimensional mapping and micro-XAFS (X-ray absorption fine structure) measurements are reported, as well as the observation of magnetic domain structures from using a reference sample of permalloy micro-dot patterns fabricated on a silicon substrate, with modest spatial resolution (e.g. ∼500 nm). Then, the X-ray radiation dose for Nafion® near the fluorine K-edge is discussed as a typical example of material that is not radiation hardened against a focused X-ray beam, for near future experiments.

A statistical approach to correct X-ray response non-uniformity in microstrip detectors for high-accuracy and high-resolution total-scattering measurements.

An unbiased approach to correct X-ray response non-uniformity in microstrip detectors has been developed based on the statistical estimation that the scattering intensity at a fixed angle from an object is expected to be constant within the Poisson noise. Raw scattering data of SiO2 glass measured by a microstrip detector module was found to show an accuracy of 12σPN at an intensity of 106 photons, where σPN is the standard deviation according to the Poisson noise. The conventional flat-field calibration has failed in correcting the data, whereas the alternative approach used in this article successfully improved the accuracy from 12σPN to 2σPN. This approach was applied to total-scattering data measured by a gapless 15-modular detector system. The quality of the data is evaluated in terms of the Bragg reflections of Si powder, the diffuse scattering of SiO2 glass, and the atomic pair distribution function of TiO2 nanoparticles and Ni powder.

Development of an X-ray imaging detector to resolve 200 nm line-and-space patterns by using transparent ceramics layers bonded by solid-state diffusion.

A high-resolution lens-coupled X-ray imaging detector equipped with a thin-layer transparent ceramics scintillator has been developed. The scintillator consists of a 5 µm thick Ce-doped Lu3Al5O12 layer (LuAG:Ce) bonded onto the support substrate of the non-doped LuAG ceramics by using a solid-state diffusion technique. Secondary electron microscopy of the bonded interface indicated that the crystal grains were densely packed without any pores in the optical wavelength scale, indicating a quasi-uniform refractive index across the interface. This guarantees high transparency and minimum reflection, which are essential properties for X-ray imaging detectors. The LuAG:Ce scintillator was incorporated into an X-ray imaging detector coupled with an objective lens with a numerical aperture of 0.85 and an optical magnification of 100. The scintillation light was imaged onto a complementary metal-oxide-semiconductor image sensor. The effective pixel size on the scintillator plane was 65 nm. X-ray transmission images of 200 nm line-and-space patterns were successfully resolved. The high spatial resolution was demonstrated by X-ray transmission images of large integrated circuits with the wiring patterns clearly visualized.

Ultrafast Structural Dynamics of Nanoparticles in Intense Laser Fields.

Femtosecond laser pulses have opened new frontiers for the study of ultrafast phase transitions and nonequilibrium states of matter. In this Letter, we report on structural dynamics in atomic clusters pumped with intense near-infrared (NIR) pulses into a nanoplasma state. Employing wide-angle scattering with intense femtosecond x-ray pulses from a free-electron laser source, we find that highly excited xenon nanoparticles retain their crystalline bulk structure and density in the inner core long after the driving NIR pulse. The observed emergence of structural disorder in the nanoplasma is consistent with a propagation from the surface to the inner core of the clusters.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Grease matrix as a versatile carrier of proteins for serial crystallography.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

Software for the data analysis of the arrival-timing monitor at SACLA.

X-ray free-electron laser (XFEL) pulses from SPring-8 Ångstrom Compact free-electron LAser (SACLA) with a temporal duration of <10 fs have provided a variety of benefits in scientific research. In a previous study, an arrival-timing monitor was developed to improve the temporal resolution in pump-probe experiments at beamline 3 by rearranging data in the order of the arrival-timing jitter between the XFEL and the synchronized optical laser pulses. This paper presents Timing Monitor Analyzer (TMA), a software package by which users can conveniently obtain arrival-timing data in the analysis environment at SACLA. The package is composed of offline tools that pull stored data from cache storage, and online tools that pull data from a data-handling server in semi-real time during beam time. Users can select the most suitable tool for their purpose, and share the results through a network connection between the offline and online analysis environments.

Advancement of X-ray radiography using microfocus X-ray source in conjunction with amplitude grating and SOI pixel detector, SOPHIAS.

We show how to improve microfocus X-ray radiography by using the SOPHIAS silicon-on-insulator pixel detector in conjunction with an amplitude grating. Single-exposure multi-energy absorption and differential phase contrast imaging was performed using the single amplitude grating method. The sensitivity in differential phase contrast imaging in a two-pixel-pitch setup was enhanced by 39% in comparison with the previously reported method [Rev. Sci. Instrum. 81, 113702 (2010).] by analyzing charge-sharing effects. Small-angle-scattering imaging was also possible in the two-pixel-pitch setup by counting the number of X-ray photons passing the pixel boundaries.

Nanosecond pump-probe device for time-resolved serial femtosecond crystallography developed at SACLA.

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Šis successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

Data acquisition system for X-ray free-electron laser experiments at SACLA.

A data acquisition system for X-ray free-electron laser experiments at SACLA has been developed. The system has been designed for reliable shot-to-shot data storage with a high data stream greater than 4 Gbps and massive data analysis. Configuration of the system and examples of prompt data analysis during experiments are presented. Upgrade plans for the system to extend flexibility are described.

Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser.

An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Šwas obtained.

Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser.

X-ray free-electron lasers (FELs) enable crystallographic data collection using extremely bright femtosecond pulses from microscopic crystals beyond the limitations of conventional radiation damage. This diffraction-before-destruction approach requires a new crystal for each FEL shot and, since the crystals cannot be rotated during the X-ray pulse, data collection requires averaging over many different crystals and a Monte Carlo integration of the diffraction intensities, making the accurate determination of structure factors challenging. To investigate whether sufficient accuracy can be attained for the measurement of anomalous signal, a large data set was collected from lysozyme microcrystals at the newly established `multi-purpose spectroscopy/imaging instrument' of the SPring-8 Ångstrom Compact Free-Electron Laser (SACLA) at RIKEN Harima. Anomalous difference density maps calculated from these data demonstrate that serial femtosecond crystallography using a free-electron laser is sufficiently accurate to measure even the very weak anomalous signal of naturally occurring S atoms in a protein at a photon energy of 7.3 keV.