Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data.

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.

Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

Real-time imaging of acoustic waves in bulk materials with X-ray microscopy.

The dynamics of lattice vibrations govern many material processes, such as acoustic wave propagation, displacive phase transitions, and ballistic thermal transport. The maximum velocity of these processes and their effects is determined by the speed of sound, which therefore defines the temporal resolution (picoseconds) needed to resolve these phenomena on their characteristic length scales (nanometers). Here, we present an X-ray microscope capable of imaging acoustic waves with subpicosecond resolution within mm-sized crystals. We directly visualize the generation, propagation, branching, and energy dissipation of longitudinal and transverse acoustic waves in diamond, demonstrating how mechanical energy thermalizes from picosecond to microsecond timescales. Bulk characterization techniques capable of resolving this level of structural detail have previously been available on millisecond time scales-orders of magnitude too slow to capture these fundamental phenomena in solid-state physics and geoscience. As such, the reported results provide broad insights into the interaction of acoustic waves with the structure of materials, and the availability of ultrafast time-resolved dark-field X-ray microscopy opens a vista of new opportunities for 3D imaging of materials dynamics on their intrinsic submicrosecond time scales.

Protocol using similarity score and improved shrink-wrap algorithm for better convergence of phase-retrieval calculation in X-ray diffraction imaging.

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.

Stimulated X-ray emission spectroscopy.

We describe an emerging hard X-ray spectroscopy technique, stimulated X-ray emission spectroscopy (S-XES). S-XES has the potential to characterize the electronic structure of 3d transition metal complexes with spectral information currently not reachable and might lead to the development of new ultrafast X-ray sources with properties beyond the state of the art. S-XES has become possible with the emergence of X-ray free-electron lasers (XFELs) that provide intense femtosecond X-ray pulses that can be employed to generate a population inversion of core-hole excited states resulting in stimulated X-ray emission. We describe the instrumentation, the various types of S-XES, the potential applications, the experimental challenges, and the feasibility of applying S-XES to characterize dilute systems, including the Mn4Ca cluster in the oxygen evolving complex of photosystem II.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

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.

Ultrafast Energy Transfer Process in Confined Gold Nanospheres Revealed by Femtosecond X-ray Imaging and Diffraction.

Femtosecond laser pulses drive nonequilibrium phase transitions via reaction paths hidden in thermal equilibrium. This stimulates interest to understand photoinduced ultrafast melting processes, which remains incomplete due to challenges in resolving accompanied kinetics at the relevant space-time resolution. Here, by newly establishing a multiplexing femtosecond X-ray probe, we have successfully revealed ultrafast energy transfer processes in confined Au nanospheres. Real-time images of electron density distributions with the corresponding lattice structures elucidate that the energy transfer begins with subpicosecond melting at the specimen boundary earlier than the lattice thermalization, and proceeds by forming voids. Two temperature molecular dynamics simulations uncovered the presence of both heterogeneous melting with the melting front propagation from surface and grain boundaries and homogeneous melting with random melting seeds and nanoscale voids. Supported by experimental and theoretical results, we provide a comprehensive atomic-scale picture that accounts for the ultrafast laser-induced melting and evaporation kinetics.

Single-pulse characterization of the focal spot size of X-ray free-electron lasers using coherent diffraction imaging.

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.

Online dynamic flat-field correction for MHz microscopy data at European XFEL.

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.

Resonant X-ray emission spectroscopy using self-seeded hard X-ray pulses at PAL-XFEL.

Self-seeded hard X-ray pulses at PAL-XFEL were used to commission a resonant X-ray emission spectroscopy experiment with a von Hamos spectrometer. The self-seeded beam, generated through forward Bragg diffraction of the [202] peak in a 100 µm-thick diamond crystal, exhibited an average bandwidth of 0.54 eV at 11.223 keV. A coordinated scanning scheme of electron bunch energy, diamond crystal angle and silicon monochromator allowed us to map the Ir Lß2 X-ray emission lines of IrO2 powder across the Ir L3-absorption edge, from 11.212 to 11.242 keV with an energy step of 0.3 eV. This work provides a reference for hard X-ray emission spectroscopy experiments utilizing self-seeded pulses with a narrow bandwidth, eventually applicable for pump-probe studies in solid-state and diluted systems.

Experimental capabilities for liquid jet samples at sub-MHz rates at the FXE Instrument at European XFEL.

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.

Pair Distribution Function from Liquid Jet Nanoparticle Suspension using Femtosecond X-ray Pulses.

X-ray scattering data measured on femtosecond timescales at the SACLA X-ray Free Electron Laser (XFEL) facility on a suspension of HfO2 nanoparticles in a liquid jet were used for pair distribution function (PDF) analysis. Despite a non-optimal experimental setup resulting in a modest Qmax of ~8 Å-1 , a promising PDF was obtained. The main features were reproduced when comparing the XFEL PDF to a PDF obtained from data measured at the PETRA III synchrotron light source. Refining structural parameters such as unit cell dimension and particle size from the XFEL PDF provided reliable values. Although the reachable Qmax limited the obtainable information, the present results indicate that good quality PDFs can be obtained on femtosecond timescales if the experimental conditions are further optimized. The study therefore encourages a new direction in ultrafast structural science where structural features of amorphous and disordered systems can be studied.

Ferritin-Like Proteins: A Conserved Core for a Myriad of Enzyme Complexes.

Ferritin-like proteins share a common fold, a four α-helix bundle core, often coordinating a pair of metal ions. Although conserved, the ferritin fold permits a diverse set of reactions, and is central in a multitude of macromolecular enzyme complexes. Here, we emphasize this diversity through three members of the ferritin-like superfamily: the soluble methane monooxygenase, the class I ribonucleotide reductase and the aldehyde deformylating oxygenase. They all rely on dinuclear metal cofactors to catalyze different challenging oxygen-dependent reactions through the formation of multi-protein complexes. Recent studies using cryo-electron microscopy, serial femtosecond crystallography at an X-ray free electron laser source, or single-crystal X-ray diffraction, have reported the structures of the active protein complexes, and revealed unprecedented insights into the molecular mechanisms of these three enzymes.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Growing Crystals for X-ray Free-Electron Laser Structural Studies of Biomolecules and Their Complexes.

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.

The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity.

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.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Ultrafast Carrier-Lattice Interactions and Interlayer Modulations of Bi2Se3 by X-ray Free-Electron Laser Diffraction.

As a 3D topological insulator, bismuth selenide (Bi2Se3) has potential applications for electrically and optically controllable magnetic and optoelectronic devices. Understanding the coupling with its topological phase requires studying the interactions of carriers with the lattice on time scales down to the subpicosecond regime. Here, we investigate the ultrafast carrier-induced lattice contractions and interlayer modulations in Bi2Se3 thin films by time-resolved diffraction using an X-ray free-electron laser. The lattice contraction depends on the carrier concentration and is followed by an interlayer expansion accompanied by oscillations. Using density functional theory and the Lifshitz model, the initial contraction can be explained by van der Waals force modulation of the confined free carrier layers. Our theoretical calculations suggest that the band inversion, related to a topological phase transition, is modulated by the expansion of the interlayer distance. These results provide insights into the topological phase control by light-induced structural change on ultrafast time scales.

A Bright Future for Serial Femtosecond Crystallography with XFELs.

X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs.

Determination of X-ray pulse duration via intensity correlation measurement of X-ray fluorescence. Erratum.

Corrections to equations and experimental results in the paper by Inoue et al. [(2019). J. Synchrotron Rad. 26, 2050-2054] are made.