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The BL09XU beamline of SPring-8 has been reorganized into a beamline dedicated for hard X-ray photoelectron spectroscopy (HAXPES) to provide advanced capabilities with upgraded optical instruments. The beamline has two HAXPES analyzers to cover a wide range of applications. Two sets of double channel-cut crystal monochromators with the Si(220) and (311) reflections were installed to perform resonant HAXPES analyses with a total energy resolution of less than 300 meV over a wide energy range (4.9–12 keV) while achieving a fixed-exit condition. A double-crystal X-ray phase retarder using diamond crystals controls the polarization state with a high degree of polarization over 0.9 in the wide energy range 5.9–9.5 keV. Each HAXPES analyzer is equipped with a focusing mirror to provide a high-flux microbeam. The design and performance of the upgraded instruments are presented.

Serial femtosecond crystallography for small-unit-cell systems has so far seen very limited application despite obvious scientific possibilities. This is because reliable data reduction has not been available for these challenging systems. In particular, important intensity corrections such as the partiality correction critically rely on accurate determination of the crystal orientation, which is complicated by the low number of diffraction spots for small-unit-cell crystals. A data reduction pipeline capable of fully automated handling of all steps of data reduction from spot harvesting to merged structure factors has been developed. The pipeline utilizes sparse indexing based on known unit-cell parameters, seed-skewness integration, intensity corrections including an overlap-based combined Ewald sphere width and partiality correction, and a dynamically adjusted post-refinement routine. Using the pipeline, data measured on the compound K4[Pt2(P2O5H2)4]·2H2O have been successfully reduced and used to solve the structure to an R1 factor of ∼9.1%. It is expected that the pipeline will open up the field of small-unit-cell serial femtosecond crystallography experiments and allow investigations into, for example, excited states and reaction intermediate chemistry.

In this study, double-multilayer monochromators that generate intense, high-energy, pink X-ray beams are designed, installed and evaluated at the SPring-8 medium-length (215 m) bending-magnet beamline BL20B2 for imaging applications. Two pairs of W/B4C multilayer mirrors are designed to utilize photon energies of 110 keV and 40 keV with bandwidths of 0.8% and 4.8%, respectively, which are more than 100 times larger when compared with the Si double-crystal monochromator (DCM) with a bandwidth of less than 0.01%. At an experimental hutch located 210 m away from the source, a large and uniform beam of size 14 mm (V) × 300 mm (H) [21 mm (V) × 300 mm (H)] was generated with a high flux density of 1.6 × 109 photons s−1 mm−2 (6.9 × 1010 photons s−1 mm−2) at 110 keV (40 keV), which marked a 300 (190) times increase in the photon flux when compared with a DCM with Si 511 (111) diffraction. The intense pink beams facilitate advanced X-ray imaging for large-sized objects such as fossils, rocks, organs and electronic devices with high speed and high spatial resolution.

Photosystem II (PSII) catalyzes light-induced water oxidation through an Si-state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Abstract Serial femtosecond crystallography (SFX) has enabled determination of room temperature structures of proteins with minimum radiation damage. A highly viscous grease matrix acting as a crystal carrier for serial sample loading at a low flow rate of ~0.5 μl min−1 was introduced into the beam path of X-ray free-electron laser. This matrix makes it possible to determine the protein structure with a sample consumption of less than 1 mg of the protein. The viscosity of the matrix is an important factor in maintaining a continuous and stable sample column from a nozzle of a high viscosity micro-extrusion injector for serial sample loading. Using conventional commercial grease (an oil-based, viscous agent) with insufficient control of viscosity in a matrix often gives an unexpectedly low viscosity, providing an unstable sample stream, with effects such as curling of the stream. Adjustment of the grease viscosity is extremely difficult since the commercial grease contains unknown compounds, which may act as unexpected inhibitors of proteins. This study introduces two novel grease matrix carriers comprising known compounds with a viscosity higher than that of conventional greases, to determine the proteinase K structure from nano-/microcrystals.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Using photosensitive caged-compound for femtosecond crystallography at X-ray free electron lasers would allow the structure determination of reaction intermediates. Here the authors demonstrate the feasibility of this approach with a caged NO-compound and present the initial NO-bound intermediate structure of cytochrome P450 nitric oxide reductase.

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 Å wavelength 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.

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.

A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K2PtBr6 within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.

Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein.

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many cocrystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å. [ABSTRACT FROM AUTHOR]

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-μs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3. [ABSTRACT FROM AUTHOR]

TheM2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across amembrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5 and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. [ABSTRACT FROM AUTHOR]

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 redoxcoupled 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. [ABSTRACT FROM AUTHOR]

The applicability of the heteroepitaxic nucleant method using molecular sieves (MS) to sparse matrix screening of protein crystallization was examined using xylanase as a test protein. The sparse matrix screening with MS provided three crystal forms I−III under 80% of conditions, which confirmed a much higher yield of crystal formation when compared to the conventional screening without MS. In the form I crystals, an MS-dependent improvement in X-ray diffraction quality was observed. Furthermore, form III of xylanase crystals was obtained only in the presence of MS 5A or 13X, demonstrating an MS-dependent packing-space expansion of protein crystals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]