Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed in a similar fashion to a synchrotron X-ray beamline. It has been open to the ESRF structural biology user community since November 2017 and will remain open during the 2019 ESRF–EBS shutdown.

Current software tools for the automated building of models for macro­molecular X-ray crystal structures are capable of assembling high-quality models for ordered macromolecule and small-molecule scattering components with minimal or no user supervision. Many of these tools also incorporate robust functionality for modelling the ordered water molecules that are found in nearly all macromolecular crystal structures. However, no current tools focus on differentiating these ubiquitous water molecules from other frequently occurring multi-atom solvent species, such as sulfate, or the automated building of models for such species. PeakProbe has been developed specifically to address the need for such a tool. PeakProbe predicts likely solvent models for a given point (termed a `peak') in a structure based on analysis (`probing') of its local electron density and chemical environment. PeakProbe maps a total of 19 resolution-dependent features associated with electron density and two associated with the local chemical environment to a two-dimensional score space that is independent of resolution. Peaks are classified based on the relative frequencies with which four different classes of solvent (including water) are observed within a given region of this score space as determined by large-scale sampling of solvent models in the Protein Data Bank. Designed to classify peaks generated from difference density maxima, PeakProbe also incorporates functionality for identifying peaks associated with model errors or clusters of peaks likely to correspond to multi-atom solvent, and for the validation of existing solvent models using solvent-omit electron-density maps. When tasked with classifying peaks into one of four distinct solvent classes, PeakProbe achieves greater than 99% accuracy for both peaks derived directly from the atomic coordinates of existing solvent models and those based on difference density maxima. While the program is still under development, a fully functional version is publicly available. PeakProbe makes extensive use of cctbx libraries, and requires a PHENIX licence and an up-to-date phenix.python environment for execution.

The performance of automated model building in crystal structure determination usually decreases with the resolution of the experimental data, and may result in fragmented models and incorrect side-chain assignment. Presented here are new methods for machine-learning-based docking of main-chain fragments to the sequence and for their sequence-independent connection using a dedicated library of protein fragments. The combined use of these new methods noticeably increases sequence coverage and reduces fragmentation of the protein models automatically built with ARP/wARP.

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallo­graphy, were exploited to reach these conclusions and provide additional insights.

Recent developments have resulted in electron cryo-microscopy (cryo-EM) becoming a useful tool for the structure determination of biological macromolecules. For samples containing inherent flexibility, heterogeneity or preferred orientation, the collection of extensive cryo-EM data using several conditions and microscopes is often required. In such a scenario, merging cryo-EM data sets is advantageous because it allows improved three-dimensional reconstructions to be obtained. Since data sets are not always collected with the same pixel size, merging data can be challenging. Here, two methods to combine cryo-EM data are described. Both involve the calculation of a rescaling factor from independent data sets. The effects of errors in the scaling factor on the results of data merging are also estimated. The methods described here provide a guideline for cryo-EM users who wish to combine data sets from the same type of microscope and detector.

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp–Glu–Ser), Nematoda (Asp–Asp–His) and Echinodermata (Asp–Glu–Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe–4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound β-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.

Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data were used to investigate the possibility of solving a real incommensurately modulated protein crystal using a supercell approximation. In this way, the answer was known and the refinement method could be tested. Firstly, an average structure was solved by using the `main' reflections, which represent the subset of the reflections that belong to the subcell and in general are more intense than the `satellite' reflections. The average structure was then expanded to create a supercell and refined using all of the reflections. Surprisingly, the refined solution did not match the expected solution, even though the statistics were excellent. Interestingly, the corresponding superspace group had multiple 3D daughter supercell space groups as possibilities, and it was one of the alternate daughter space groups that the refinement locked in on. The lessons learned here will be applied to a real incommensurately modulated profilin–actin crystal that has the same superspace group.

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

Electron microscopy of macromolecular structures is an approach that is in increasing demand in the field of structural biology. The automation of image acquisition has greatly increased the potential throughput of electron microscopy. Here, the focus is on the possibilities in Scipion to implement flexible and robust image-processing workflows that allow the electron-microscope operator and the user to monitor the quality of image acquisition, assessing very simple acquisition measures or obtaining a first estimate of the initial volume, or the data resolution and heterogeneity, without any need for programming skills. These workflows can implement intelligent automatic decisions and they can warn the user of possible acquisition failures. These concepts are illustrated by analysis of the well known 2.2 Å resolution β-galactosidase data set.

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

A nonlinear least-squares method for refining a parametric expression describing the estimated errors of reflection intensities in serial crystallographic (SX) data is presented. This approach, which is similar to that used in the rotation method of crystallographic data collection at synchrotrons, propagates error estimates from photon-counting statistics to the merged data. Here, it is demonstrated that the application of this approach to SX data provides better SAD phasing ability, enabling the autobuilding of a protein structure that had previously failed to be built. Estimating the error in the merged reflection intensities requires the understanding and propagation of all of the sources of error arising from the measurements. One type of error, which is well understood, is the counting error introduced when the detector counts X-ray photons. Thus, if other types of random errors (such as readout noise) as well as uncertainties in systematic corrections (such as from X-ray attenuation) are completely understood, they can be propagated along with the counting error, as appropriate. In practice, most software packages propagate as much error as they know how to model and then include error-adjustment terms that scale the error estimates until they explain the variance among the measurements. If this is performed carefully, then during SAD phasing likelihood-based approaches can make optimal use of these error estimates, increasing the chance of a successful structure solution. In serial crystallography, SAD phasing has remained challenging, with the few examples of de novo protein structure solution each requiring many thousands of diffraction patterns. Here, the effects of different methods of treating the error estimates are estimated and it is shown that using a parametric approach that includes terms proportional to the known experimental uncertainty, the reflection intensity and the squared reflection intensity to improve the error estimates can allow SAD phasing even from weak zinc anomalous signal.

The refinement of biomolecular crystallographic models relies on geometric restraints to help to address the paucity of experimental data typical in these experiments. Limitations in these restraints can degrade the quality of the resulting atomic models. Here, an integration of the full all-atom Amber molecular-dynamics force field into Phenix crystallographic refinement is presented, which enables more complete modeling of biomolecular chemistry. The advantages of the force field include a carefully derived set of torsion-angle potentials, an extensive and flexible set of atom types, Lennard–Jones treatment of nonbonded interactions and a full treatment of crystalline electrostatics. The new combined method was tested against conventional geometry restraints for over 22 000 protein structures. Structures refined with the new method show substantially improved model quality. On average, Ramachandran and rotamer scores are somewhat better, clashscores and MolProbity scores are significantly improved, and the modeling of electrostatics leads to structures that exhibit more, and more correct, hydrogen bonds than those refined using traditional geometry restraints. In general it is found that model improvements are greatest at lower resolutions, prompting plans to add the Amber target function to real-space refinement for use in electron cryo-microscopy. This work opens the door to the future development of more advanced applications such as Amber-based ensemble refinement, quantum-mechanical representation of active sites and improved geometric restraints for simulated annealing.

Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference-free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open-source and distributed within RELION-3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.

Reducing the sample-exchange time is a crucial issue in maximizing the throughput of macromolecular crystallography (MX) beamlines because the diffraction data collection itself is completed within a minute in the era of pixel-array detectors. To this end, an upgraded sample changer, SPACE-II, has been developed on the basis of the previous model, SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the BL41XU beamline at SPring-8. SPACE-II achieves one sample-exchange step within 16 s, of which its action accounts for only 11 s, because of three features: (i) the implementation of twin arms that enable samples to be exchanged in one cycle of mount-arm action, (ii) the implementation of long-stroke mount arms that allow samples to be exchanged without withdrawal of the detector and (iii) the use of a fast-moving translation and rotation stage for the mount arms. By pre-holding the next sample prior to the sample-exchange sequence, the time was further decreased to 11 s in the case of automatic data collection, of which the action of SPACE-II accounted for 8 s. Moreover, the sample capacity was expanded from four to eight Uni-Pucks. The performance of SPACE-II has been demonstrated in over two years of operation at BL41XU; the average number of samples mounted on the diffractometer in one day was increased from 132 to 185, with an error rate of 0.089%, which counted incidents in which users could not continue with an experiment without recovery work by entering the experimental hutch. On the basis of these results, SPACE-II has been installed at three other MX beamlines at SPring-8 as of July 2019. The fast and highly reliable SPACE-II is now one of the most important pieces of infrastructure for the MX beamlines at SPring-8, providing users with the opportunity to fully make use of limited beamtime with brilliant X-rays.

Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2'. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.

Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X-ray absorption edges, which shift to higher energy for progressively more positive valence numbers. Indeed, X-ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X-ray sources at synchrotron facilities with energy resolutions in the range 2–3 × 10−4 (ΔE/E). This paper tests whether X-ray free-electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10−3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength-dependent anomalous scattering factors contributed by two differently charged iron centers in the 110-amino-acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X-ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.

The performance of automated protein model building usually decreases with resolution, mainly owing to the lower information content of the experimental data. This calls for a more elaborate use of the available structural information about macromolecules. Here, a new method is presented that uses structural homologues to improve the quality of protein models automatically constructed using ARP/wARP. The method uses local structural similarity between deposited models and the model being built, and results in longer main-chain fragments that in turn can be more reliably docked to the protein sequence. The application of the homology-based model extension method to the example of a CFA synthase at 2.7 Å resolution resulted in a more complete model with almost all of the residues correctly built and docked to the sequence. The method was also evaluated on 1493 molecular-replacement solutions at a resolution of 4.0 Å and better that were submitted to the ARP/wARP web service for model building. A significant improvement in the completeness and sequence coverage of the built models has been observed.

Model quality assessment programs estimate the quality of protein models and can be used to estimate local error in protein models. ProQ3D is the most recent and most accurate version of our software. Here, it is demonstrated that it is possible to use local error estimates to substantially increase the quality of the models for molecular replacement (MR). Adjusting the B factors using ProQ3D improved the log-likelihood gain (LLG) score by over 50% on average, resulting in significantly more successful models in MR compared with not using error estimates. On a data set of 431 homology models to address difficult MR targets, models with error estimates from ProQ3D received an LLG of >50 for almost half of the models 209/431 (48.5%), compared with 175/431 (40.6%) for the previous version, ProQ2, and only 74/431 (17.2%) for models with no error estimates, clearly demonstrating the added value of using error estimates to enable MR for more targets. ProQ3D is available from http://proq3.bioinfo.se/ both as a server and as a standalone download.

The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallo­graphy has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid-state nuclear magnetic resonance and two-dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular-dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.

Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.

Electron diffraction tomography (EDT) has gained increasing interest, starting with the development of automated electron diffraction tomography (ADT) which enables the collection of three-dimensional electron diffraction data from nano-sized crystals suitable for ab initio structure analysis. A basic description of the ADT method, nowadays recognized as a reliable and established method, as well as its special features and general applicability to different transmission electron microscopes is provided. In addition, the usability of ADT for crystal structure analysis of single nano-sized crystals with and without special crystallographic features, such as twinning, modulations and disorder is demonstrated.

Multi-slice simulations of electron diffraction by three-dimensional protein crystals have indicated that structure solution would be severely impeded by dynamical diffraction, especially when crystals are more than a few unit cells thick. In practice, however, dynamical diffraction turned out to be less of a problem than anticipated on the basis of these simulations. Here it is shown that two scattering phenomena, which are usually omitted from multi-slice simulations, reduce the dynamical effect: solvent scattering reduces the phase differences within the exit beam and inelastic scattering followed by elastic scattering results in diffusion of dynamical scattering out of Bragg peaks. Thus, these independent phenomena provide potential reasons for the apparent discrepancy between theory and practice in protein electron crystallography.

A part of the system CaO-SiO2–Al2O3–Fe2O3–MgO which is of relevance to iron-ore sintering has been studied in detail. For a bulk composition corresponding to 10.45 wt% CaO, 5.49 wt% MgO, 69.15 wt% Fe2O3, 13.37 wt% Al2O3 and 1.55 wt% SiO2 synthesis runs have been performed in air in the range between 1100 and 1300°C. Products have been characterized using reflected-light microscopy, electron microprobe analysis and diffraction techniques. At 1250°C, an almost phase-pure material with composition Ca2.99Mg2.67Fe3+14.58Fe2+0.77Al4.56Si0.43O36 has been obtained. The compound corresponds to the first Si-containing representative of the M14+6nO20+8n polysomatic series of so-called SFCA phases (Silico-Ferrites of Calcium and Aluminum) with n = 2 and is denoted as SFCA-III. Single-crystal diffraction investigations using synchrotron radiation at the X06DA beamline of the Swiss Light Source revealed that the chemically homogenous sample contained both a triclinic and monoclinic polytype. Basic crystallographic data are as follows: triclinic form: a = 10.3279 (2) Å, b = 10.4340 (2) Å, c = 14.3794 (2) Å, α = 93.4888 (12)°, β = 107.3209 (14)° and γ = 109.6626 (14)°, V = 1370.49 (5) Å3, Z = 2, space group P{overline 1}; monoclinic form: a = 10.3277 (2) Å, b = 27.0134 (4) Å, c = 10.4344 (2) Å, β = 109.668 (2)°, V = 2741.22 (9) Å3, Z = 4, space group P21/n. Structure determination of both modifications was successful using diffraction data from the same allotwinned crystal. A description of the observed polytypism within the framework of OD-theory is presented. Triclinic and monoclinic SFCA-III actually correspond to the two possible maximum degree of order structures based on OD-layers containing three spinel (S) and one pyroxene (P) modules (〈S3P〉). The existence of SFCA-III in industrial iron-ore sinters has yet to be confirmed. Polytypism is likely to occur in other SFCA-members (SFCA, SFCA-I) relevant to sintering as well, but has so far been neglected in the characterization of industrial samples. Our results shed light on this phenomenon and may therefore be also helpful for better interpretation of the powder diffraction patterns that are used for phase analysis of iron-ore sinters.

In this paper, the design and functionalities of the high-throughput TELL sample exchange system for macromolecular crystallography is presented. TELL was developed at the Paul Scherrer Institute with a focus on speed, storage capacity and reliability to serve the three macromolecular crystallography beamlines of the Swiss Light Source, as well as the SwissMX instrument at SwissFEL.

In recent years, there have been numerous efforts worldwide to develop the synchrotron X-ray scanning tunneling microscopy (SX-STM) technique. Here, the inauguration of XTIP, the world's first beamline fully dedicated to SX-STM, is reported. The XTIP beamline is located at Sector 4 of the Advanced Photon Source at Argonne National Laboratory. It features an insertion device that can provide left- or right-circular as well as horizontal- and vertical-linear polarization. XTIP delivers monochromatic soft X-rays of between 400 and 1900 eV focused into an environmental enclosure that houses the endstation instrument. This article discusses the beamline system design and its performance.

Inelastic X-ray scattering is a powerful and versatile technique for studying lattice dynamics in materials of scientific and technological importance. In this article, the design and capabilities of the momentum-resolved high-energy-resolution inelastic X-ray spectrometer (HERIX) at beamline 30-ID of the Advanced Photon Source are reported. The instrument operates at 23.724 keV and has an energy resolution of 1.3–1.7 meV. It can accommodate momentum transfers of up to 72  nm−1, at a typical X-ray flux of 4.5 × 109 photons s−1 meV−1 at the sample. A suite of in situ sample environments are provided, including high pressure, static magnetic fields and uniaxial strains, all at high or cryogenic temperatures.

The electron linear accelerators driving modern X-ray free-electron lasers can emit intense, tunable, quasi-monochromatic terahertz (THz) transients with peak electric fields of V Å−1 and peak magnetic fields in excess of 10 T when a purpose-built, compact, superconducting THz undulator is implemented. New research avenues such as X-ray movies of THz-driven mode-selective chemistry come into reach by making dual use of the ultra-short GeV electron bunches, possible by a rather minor extension of the infrastructure.

A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.

The optical design of a Hettrick–Underwood-style soft X-ray spectrometer with Wolter type 1 mirrors is presented. The spectrometer with a nominal length of 3.1 m can achieve a high resolving power (resolving power higher than 10000) in the soft X-ray regime when a small source beam (<3 µm in the grating dispersion direction) and small pixel detector (5 µm effective pixel size) are used. Adding Wolter mirrors to the spectrometer before its dispersive elements can realize the spatial imaging capability, which finds applications in the spectroscopic studies of spatially dependent electronic structures in tandem catalysts, heterostructures, etc. In the pump–probe experiments where the pump beam perturbs the materials followed by the time-delayed probe beam to reveal the transient evolution of electronic structures, the imaging capability of the Wolter mirrors can offer the pixel-equivalent femtosecond time delay between the pump and probe beams when their wavefronts are not collinear. In combination with some special sample handing systems, such as liquid jets and droplets, the imaging capability can also be used to study the time-dependent electronic structure of chemical transformation spanning multiple time domains from microseconds to nanoseconds. The proposed Wolter mirrors can also be adopted to the existing soft X-ray spectrometers that use the Hettrick–Underwood optical scheme, expanding their capabilities in materials research.

The composition of occupied and unoccupied electronic states in the vicinity of Fermi energies is vital for all materials and relates to their physical, chemical and mechanical properties. This work demonstrates how the combination of resonant and non-resonant X-ray emission spectroscopies supplemented with theoretical modelling allows for quantitative analysis of electronic states in 5d transition metal and metal-oxide materials. Application of X-rays provides element selectivity that, in combination with the penetrating properties of hard X-rays, allows determination of the composition of electronic states under working conditions, i.e. non-vacuum environment. Tungsten metal and tungsten oxide are evaluated to show the capability to simultaneously assess composition of around-band-gap electronic states as well as the character and magnitude of the crystal field splitting.

Quantum beats in fluorescence decay from Zeeman-split magnetic sublevels have been measured for helium Rydberg states excited by synchrotron radiation. The Zeeman quantum beats observed in this prototypical case were fitted with an equation from a theoretical formulation. It is proposed that Zeeman quantum beat measurement can be a useful way to simply evaluate the polarization characteristics of extreme ultraviolet light.

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.

Strain tensor measurements are important for understanding elastic and plastic deformation, but full bulk strain tensor measurement techniques are still lacking, in particular for dynamic loading. Here, such a methodology is reported, combining imaging-based strain field mapping and simultaneous X-ray diffraction for four typical loading modes: one-dimensional strain/stress compression/tension. Strain field mapping resolves two in-plane principal strains, and X-ray diffraction analysis yields volumetric strain, and thus the out-of-plane principal strain. This methodology is validated against direct molecular dynamics simulations on nanocrystalline tantalum. This methodology can be implemented with simultaneous X-ray diffraction and digital image correlation in synchrotron radiation or free-electron laser experiments.

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X-ray free-electron laser (XFEL) to provide intense and short X-ray pulses. Considering the limited opportunities to use these facilities, it is necessary to verify the plausibility of a target experiment prior to the actual experiment. For this purpose, a program has been developed, referred to as S-cube, which is short for a Solution Scattering Simulator. This code allows the routine estimation of the shape and signal-to-noise ratio (SNR) of TRXL data from known experimental parameters. Specifically, S-cube calculates the difference scattering curve and the associated quantum noise on the basis of the molecular structure of the target reactant and product, the target solvent, the energy of the pump laser pulse and the specifications of the beamline to be used. Employing a simplified form for the pair-distribution function required to calculate the solute–solvent cross term greatly increases the calculation speed as compared with a typical TRXL data analysis. Demonstrative applications of S-cube are presented, including the estimation of the expected TRXL data and SNR level for the future LCLS-II HE beamlines.

The ePix10ka2M (ePix10k) is a new large area detector specifically developed for X-ray free-electron laser (XFEL) applications. The hybrid pixel detector was developed at SLAC to provide a hard X-ray area detector with a high dynamic range, running at the 120 Hz repetition rate of the Linac Coherent Light Source (LCLS). The ePix10k consists of 16 modules, each with 352 × 384 pixels of 100 µm × 100 µm distributed on four ASICs, resulting in a 2.16 megapixel detector, with a 16.5 cm × 16.5 cm active area and ∼80% coverage. The high dynamic range is achieved with three distinct gain settings (low, medium, high) as well as two auto-ranging modes (high-to-low and medium-to-low). Here the three fixed gain modes are evaluated. The resulting dynamic range (from single photon counting to 10000 photons pixel−1 pulse−1 at 8 keV) makes it suitable for a large number of different XFEL experiments. The ePix10k replaces the large CSPAD in operation since 2011. The dimensions of the two detectors are similar, making the upgrade from CSPAD to ePix10k straightforward for most setups, with the ePix10k improving on experimental performance. The SLAC-developed ePix cameras all utilize a similar platform, are tailored to target different experimental conditions and are designed to provide an upgrade path for future high-repetition-rate XFELs. Here the first measurements on this new ePix10k detector are presented and the performance under typical XFEL conditions evaluated during an LCLS X-ray diffuse scattering experiment measuring the 9.5 keV X-ray photons scattered from a thin liquid jet.

The first experimental results from a new transmissive diagnostic instrument for synchrotron X-ray beamlines are presented. The instrument utilizes a single-crystal chemical-vapour-deposition diamond plate as the detector material, with graphitic wires embedded within the bulk diamond acting as electrodes. The resulting instrument is an all-carbon transmissive X-ray imaging detector. Within the instrument's transmissive aperture there is no surface metallization that could absorb X-rays, and no surface structures that could be damaged by exposure to synchrotron X-ray beams. The graphitic electrodes are fabricated in situ within the bulk diamond using a laser-writing technique. Two separate arrays of parallel graphitic wires are fabricated, running parallel to the diamond surface and perpendicular to each other, at two different depths within the diamond. One array of wires has a modulated bias voltage applied; the perpendicular array is a series of readout electrodes. X-rays passing through the detector generate charge carriers within the bulk diamond through photoionization, and these charge carriers travel to the nearest readout electrode under the influence of the modulated electrical bias. Each of the crossing points between perpendicular wires acts as an individual pixel. The simultaneous read-out of all pixels is achieved using a lock-in technique. The parallel wires within each array are separated by 50 µm, determining the pixel pitch. Readout is obtained at 100 Hz, and the resolution of the X-ray beam position measurement is 600 nm for a 180 µm size beam.

Double-sided Fresnel zone plates are diffractive lenses used for high-resolution hard X-ray microscopy. The double-sided structures have significantly higher aspect ratios compared with single-sided components and hence enable more efficient imaging. The zone plates discussed in this paper are fabricated on each side of a thin support membrane, and the alignment of the zone plates with respect to each other is critical. Here, a simple and reliable way of quantifying misalignments by recording efficiency maps and measuring the absolute diffraction efficiency of the zone plates as a function of tilting angle in two directions is presented. The measurements are performed in a setup based on a tungsten-anode microfocus X-ray tube, providing an X-ray energy of 8.4 keV through differential measurements with a Cu and an Ni filter. This study investigates the sources of the misalignments and concludes that they can be avoided by decreasing the structure heights on both sides of the membrane and by pre-programming size differences between the front- and back-side zone plates.

A dedicated X-ray imaging detector for 200 keV high-energy X-ray microtomography was developed to realize high-efficiency high-resolution imaging while keeping the field of view wide.

It is shown that the 2p3d resonant inelastic X-ray scattering intensity is distorted by saturation and self-absorption effects, i.e. by incident-energy-dependent saturation and by emission-energy-dependent self-absorption.

Nanoporous anodic aluminium oxide (AAO) membranes are promising host systems for confinement of condensed matter. Characterizing their structure and composition is thus of primary importance for studying the behavior of confined objects. Here a novel methodology to extract quantitative information on the structure and composition of well defined AAO membranes by combining small-angle neutron scattering (SANS) measurements and scanning electron microscopy (SEM) imaging is reported. In particular, (i) information about the pore hexagonal arrangement is extracted from SEM analysis, (ii) the best SANS experimental conditions to perform reliable measurements are determined and (iii) a detailed fitting method is proposed, in which the probed length in the fitting model is a critical parameter related to the longitudinal pore ordering. Finally, to validate this strategy, it is applied to characterize AAOs prepared under different conditions and it is shown that the experimental SANS data can be fully reproduced by a core/shell model, indicating the existence of a contaminated shell. This original approach, based on a detailed and complete description of the SANS data, can be applied to a variety of confining media and will allow the further investigation of condensed matter under confinement.

Bone crystallite chemistry and structure change during bone maturation. However, these properties of bone can also be affected by limited uptake of the chemical constituents of the mineral by the animal. This makes probing the effect of bone-mineralization-related diseases a complicated task. Here it is shown that the combination of vibrational spectroscopy with two-dimensional X-ray diffraction can provide unparalleled information on the changes in bone chemistry and structure associated with different bone pathologies (phosphate deficiency) and/or health conditions (pregnancy, lactation). Using a synergistic analytical approach, it was possible to trace the effect that changes in the remodelling regime have on the bone mineral chemistry and structure in normal and mineral-deficient (hypophosphatemic) mice. The results indicate that hypophosphatemic mice have increased bone remodelling, increased carbonate content and decreased crystallinity of the bone mineral, as well as increased misalignment of crystallites within the bone tissue. Pregnant and lactating mice that are normal and hypophosphatemic showed changes in the chemistry and misalignment of the apatite crystals that can be related to changes in remodelling rates associated with different calcium demand during pregnancy and lactation.

The Inorganic Crystal Structure Database (ICSD) is the world's largest database of fully evaluated and published crystal structure data, mostly obtained from experimental results. However, the purely experimental approach is no longer the only route to discover new compounds and structures. In the past few decades, numerous computational methods for simulating and predicting structures of inorganic solids have emerged, creating large numbers of theoretical crystal data. In order to take account of these new developments the scope of the ICSD was extended in 2017 to include theoretical structures which are published in peer-reviewed journals. Each theoretical structure has been carefully evaluated, and the resulting CIF has been extended and standardized. Furthermore, a first classification of theoretical data in the ICSD is presented, including additional categories used for comparison of experimental and theoretical information.

Single-crystal elastic constants have been derived by lattice strain measurements using neutron diffraction on polycrystalline Ti-6Al-4V, Ti-6Al-2Sn-4Zr-6Mo and Ti-3Al-8V-6Cr-4Zr-4Mo alloy samples. A variety of model approximations for the grain-to-grain interactions, namely approaches by Voigt, Reuss, Hill, Kroener, de Wit and Matthies, including texture weightings, have been applied and compared. A load-transfer approach for multiphase alloys was also implemented and the results are compared with single-phase data. For the materials under investigation, the results for multiphase alloys agree well with the results for single-phase materials in the corresponding phases. In this respect, all eight elastic constants in the dual-phase Ti-6Al-2Sn-4Zr-6Mo alloy have been derived for the first time.

Errors in the article by Opara, Martiel, Arnold, Braun, Stahlberg, Makita, David & Padeste [J. Appl. Cryst. (2017), 50, 909–918] are corrected.