Takaki Hatsui

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 of an X-ray pixel detector with multi-port charge-coupled device for X-ray free-electron laser experiments

This paper presents development of an X-ray pixel detector with a multi-port charge-coupled device (MPCCD) for X-ray Free-Electron laser experiments. The fabrication process of the CCD was selected based on the X-ray radiation hardness against the estimated annual dose of 1.6 × 10(14) photon/mm(2). The sensor device was optimized by maximizing the full well capacity as high as 5 Me- within 50 μm square pixels while keeping the single photon detection capability for X-ray photons higher than 6 keV and a readout speed of 60 frames/s. The system development also included a detector system for the MPCCD sensor. This paper summarizes the performance, calibration methods, and operation status.

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.

A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals 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.

Extreme ultraviolet free electron laser seeded with high-order harmonic of Ti:sapphire laser

The 13th harmonic of a Ti:sapphire (Ti:S) laser in the plateau region was injected as a seeding source to a 250-MeV free-electron-laser (FEL) amplifier. When the amplification conditions were fulfilled, strong enhancement of the radiation intensity by a factor of 650 was observed. The random and uncontrollable spikes, which appeared in the spectra of the Self-Amplified Spontaneous Emission (SASE) based FEL radiation without the seeding source, were found to be suppressed drastically to form to a narrow-band, single peak profile at 61.2 nm. The properties of the seeded FEL radiation were well reproduced by numerical simulations. We discuss the future precept of the seeded FEL scheme to the shorter wavelength region.

Deep Inner-Shell Multiphoton Ionization by Intense X-Ray Free-Electron Laser Pulses

We have investigated multiphoton multiple ionization dynamics of xenon atoms using a new x-ray free-electron laser facility, SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan, and identified that Xe(n+) with n up to 26 is produced at a photon energy of 5.5 keV. The observed high charge states (n≥24) are produced via five-photon absorption, evidencing the occurrence of multiphoton absorption involving deep inner shells. A newly developed theoretical model, which shows good agreement with the experiment, elucidates the complex pathways of sequential electronic decay cascades accessible in heavy atoms. The present study of heavy-atom ionization dynamics in high-intensity hard-x-ray pulses makes a step forward towards molecular structure determination with x-ray free-electron lasers.

Single-shot three-dimensional structure determination of nanocrystals with femtosecond X-ray free-electron laser pulses

Conventional three-dimensional (3D) structure determination methods require either multiple measurements at different sample orientations or a collection of serial sections through a sample. Here we report the experimental demonstration of single-shot 3D structure determination of an object; in this case, individual gold nanocrystals at ~5.5 nm resolution using ~10 fs X-ray free-electron laser pulses. Coherent diffraction patterns are collected from high-index-faceted nanocrystals, each struck by an X-ray free-electron laser pulse. Taking advantage of the symmetry of the nanocrystal and the curvature of the Ewald sphere, we reconstruct the 3D structure of each nanocrystal from a single-shot diffraction pattern. By averaging a sufficient number of identical nanocrystals, this method may be used to determine the 3D structure of nanocrystals at atomic resolution. As symmetry exists in many virus particles, this method may also be applied to 3D structure studies of such particles at nanometer resolution on femtosecond time scales.

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 platform for serial femtosecond crystallography using an X-ray free-electron laser and its applications at SACLA are described.

Macromolecular structures probed by combining single-shot free-electron laser diffraction with synchrotron coherent X-ray imaging

Nanostructures formed from biological macromolecular complexes utilizing the self-assembly properties of smaller building blocks such as DNA and RNA hold promise for many applications, including sensing and drug delivery. New tools are required for their structural characterization. Intense, femtosecond X-ray pulses from X-ray free-electron lasers enable single-shot imaging allowing for instantaneous views of nanostructures at ambient temperatures. When combined judiciously with synchrotron X-rays of a complimentary nature, suitable for observing steady-state features, it is possible to perform ab initio structural investigation. Here we demonstrate a successful combination of femtosecond X-ray single-shot diffraction with an X-ray free-electron laser and coherent diffraction imaging with synchrotron X-rays to provide an insight into the nanostructure formation of a biological macromolecular complex: RNA interference microsponges. This newly introduced multimodal analysis with coherent X-rays can be applied to unveil nano-scale structural motifs from functional nanomaterials or biological nanocomplexes, without requiring a priori knowledge.

Femtosecond x-ray absorption spectroscopy with hard x-ray free electron laser

We have developed a method of dispersive x-ray absorption spectroscopy with a hard x-ray free electron laser (XFEL), generated by a self-amplified spontaneous emission (SASE) mechanism. A transmission grating was utilized for splitting SASE-XFEL light, which has a relatively large bandwidth (ΔE/E ∼ 5 × 10−3), into several branches. Two primary split beams were introduced into a dispersive spectrometer for measuring signal and reference spectra simultaneously. After normalization, we obtained a Zn K-edge absorption spectrum with a photon-energy range of 210 eV, which is in excellent agreement with that measured by a conventional wavelength-scanning method. From the analysis of the difference spectra, the noise ratio was evaluated to be ∼3 × 10−3, which is sufficiently small to trace minute changes in transient spectra induced by an ultrafast optical laser. This scheme enables us to perform single-shot, high-accuracy x-ray absorption spectroscopy with femtosecond time resolution.