Crystallization kinetics of atomic crystals revealed by a single-shot and single-particle X-ray diffraction experiment.

Crystallization is a fundamental natural phenomenon and the ubiquitous physical process in materials science for the design of new materials. So far, experimental observations of the structural dynamics in crystallization have been mostly restricted to slow dynamics. We present here an exclusive way to explore the dynamics of crystallization in highly controlled conditions (i.e., in the absence of impurities acting as seeds of the crystallites) as it occurs in vacuum. We have measured the early formation stage of solid Xe nanoparticles nucleated in an expanding supercooled Xe jet by means of an X-ray diffraction experiment with 10-fs X-ray free-electron laser (XFEL) pulses. We found that the structure of Xe nanoparticles is not pure face-centered cubic (fcc), the expected stable phase, but a mixture of fcc and randomly stacked hexagonal close-packed (rhcp) structures. Furthermore, we identified the instantaneous coexistence of the comparably sized fcc and rhcp domains in single Xe nanoparticles. The observations are explained by the scenario of structural aging, in which the nanoparticles initially crystallize in the highly stacking-disordered rhcp phase and the structure later forms the stable fcc phase. The results are reminiscent of analogous observations in hard-sphere systems, indicating the universal role of the stacking-disordered phase in nucleation.

X-Ray Single-Grating Interferometry for Wavefront Measurement and Correction of Hard X-Ray Nanofocusing Mirrors.

X-ray single-grating interferometry was applied to conduct accurate wavefront corrections for hard X-ray nanofocusing mirrors. Systematic errors in the interferometer, originating from a grating, a detector, and alignment errors of the components, were carefully examined. Based on the measured wavefront errors, the mirror shapes were directly corrected using a differential deposition technique. The corrected X-ray focusing mirrors with a numerical aperture of 0.01 attained two-dimensionally diffraction-limited performance. The results of the correction indicate that the uncertainty of the wavefront measurement was less than λ/72 in root-mean-square value.

Characterizing crystalline defects in single nanoparticles from angular correlations of single-shot diffracted X-rays.

Characterizing and controlling the uniformity of nanoparticles is crucial for their application in science and technology because crystalline defects in the nanoparticles strongly affect their unique properties. Recently, ultra-short and ultra-bright X-ray pulses provided by X-ray free-electron lasers (XFELs) opened up the possibility of structure determination of nanometre-scale matter with Å spatial resolution. However, it is often difficult to reconstruct the 3D structural information from single-shot X-ray diffraction patterns owing to the random orientation of the particles. This report proposes an analysis approach for characterizing defects in nanoparticles using wide-angle X-ray scattering (WAXS) data from free-flying single nanoparticles. The analysis method is based on the concept of correlated X-ray scattering, in which correlations of scattered X-ray are used to recover detailed structural information. WAXS experiments of xenon nanoparticles, or clusters, were conducted at an XFEL facility in Japan by using the SPring-8 Ångstrom compact free-electron laser (SACLA). Bragg spots in the recorded single-shot X-ray diffraction patterns showed clear angular correlations, which offered significant structural information on the nanoparticles. The experimental angular correlations were reproduced by numerical simulation in which kinematical theory of diffraction was combined with geometric calculations. We also explain the diffuse scattering intensity as being due to the stacking faults in the xenon clusters.

Refinement for single-nanoparticle structure determination from low-quality single-shot coherent diffraction data.

With the emergence of X-ray free-electron lasers, it is possible to investigate the structure of nanoscale samples by employing coherent diffractive imaging in the X-ray spectral regime. In this work, we developed a refinement method for structure reconstruction applicable to low-quality coherent diffraction data. The method is based on the gradient search method and considers the missing region of a diffraction pattern and the small number of detected photons. We introduced an initial estimate of the structure in the method to improve the convergence. The present method is applied to an experimental diffraction pattern of an Xe cluster obtained in an X-ray scattering experiment at the SPring-8 Angstrom Compact free-electron LAser (SACLA) facility. It is found that the electron density is successfully reconstructed from the diffraction pattern with a large missing region, with a good initial estimate of the structure. The diffraction pattern calculated from the reconstructed electron density reproduced the observed diffraction pattern well, including the characteristic intensity modulation in each ring. Our refinement method enables structure reconstruction from diffraction patterns under difficulties such as missing areas and low diffraction intensity, and it is potentially applicable to the structure determination of samples that have low scattering power.

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.

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.

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity.

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI's quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity.

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.

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA.

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Šwavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routine de novo structural determination of biomacromolecules.

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.

Protein-ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation.

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

Atomic resolution structure of serine protease proteinase K at ambient temperature.

Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

Hydroxyethyl cellulose matrix applied to serial crystallography.

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3,000 diffraction images.

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.

A three-dimensional movie of structural changes in bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

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.

Oil-free hyaluronic acid matrix for serial femtosecond crystallography.

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

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.

Shot-to-shot diagnostic of the longitudinal photon source position at the SPring-8 Angstrom Compact Free Electron Laser by means of x-ray grating interferometry.

We present single-shot measurements of the longitudinal photon source position of the SPring-8 Angstrom Compact Free Electron Laser x-ray free electron laser by means of x-ray grating interferometry. The measurements were performed in order to study the behavior of the source under normal operation conditions and as a dependence on the active undulator length. The retrieved experimental results show that x-ray grating interferometry is a powerful in situ monitoring tool for investigating and tuning an x-ray free electron laser.