The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.

Corrections are published for the article by Caldararu et al. [(2019), Acta Cryst. D75, 368–380].

Fragment-based molecular-replacement methods can solve a macromolecular structure quasi-ab initio. ARCIMBOLDO, using a common secondary-structure or tertiary-structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high β-sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log-likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary-structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute-force approach through side-chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main-chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Å resolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content.

The three probes of the structure of matter (X-rays, neutrons and electrons) in biology have complementary properties and strengths. The balance between these three probes within their strengths and weaknesses is perceived to change, even dramatically so at times. For the study of combined states of order and disorder, NMR crystallography is also applicable. Of course, to understand biological systems the required perspectives are surely physiologically relevant temperatures and relevant chemical conditions, as well as a minimal perturbation owing to the needs of the probe itself. These remain very tough challenges because, for example, cryoEM by its very nature will never be performed at room temperature, crystallization often requires nonphysiological chemical conditions, and X-rays and electrons cause beam damage. However, integrated structural biology techniques and functional assays provide a package towards physiological relevance of any given study. Reporting of protein crystal structures, and their associated database entries, could usefully indicate how close to the biological situation they are, as discussed in detail in this feature article.

The information gained by making a measurement, termed the Kullback–Leibler divergence, assesses how much more precisely the true quantity is known after the measurement was made (the posterior probability distribution) than before (the prior probability distribution). It provides an upper bound for the contribution that an observation can make to the total likelihood score in likelihood-based crystallographic algorithms. This makes information gain a natural criterion for deciding which data can legitimately be omitted from likelihood calculations. Many existing methods use an approximation for the effects of measurement error that breaks down for very weak and poorly measured data. For such methods a different (higher) information threshold is appropriate compared with methods that account well for even large measurement errors. Concerns are raised about a current trend to deposit data that have been corrected for anisotropy, sharpened and pruned without including the original unaltered measurements. If not checked, this trend will have serious consequences for the reuse of deposited data by those who hope to repeat calculations using improved new methods.

The analysis of large structural databases reveals general features and relationships among proteins, providing useful insight. A different approach is required to characterize ubiquitous secondary-structure elements, where flexibility is essential in order to capture small local differences. The ALEPH software is optimized for the analysis and the extraction of small protein folds by relying on their geometry rather than on their sequence. The annotation of the structural variability of a given fold provides valuable information for fragment-based molecular-replacement methods, in which testing alternative model hypotheses can succeed in solving difficult structures when no homology models are available or are successful. ARCIMBOLDO_BORGES combines the use of composite secondary-structure elements as a search model with density modification and tracing to reveal the rest of the structure when both steps are successful. This phasing method relies on general fold libraries describing variations around a given pattern of β-sheets and helices extracted using ALEPH. The program introduces characteristic vectors defined from the main-chain atoms as a way to describe the geometrical properties of the structure. ALEPH encodes structural properties in a graph network, the exploration of which allows secondary-structure annotation, decomposition of a structure into small compact folds, generation of libraries of models representing a variation of a given fold and finally superposition of these folds onto a target structure. These functions are available through a graphical interface designed to interactively show the results of structure manipulation, annotation, fold decomposition, clustering and library generation. ALEPH can produce pictures of the graphs, structures and folds for publication purposes.

Cryo-electron microscopy (cryo-EM) has rapidly expanded with the introduction of direct electron detectors, improved image-processing software and automated image acquisition. Its recent adoption by industry, particularly in structure-based drug design, creates new requirements in terms of reliability, reproducibility and throughput. In 2016, Thermo Fisher Scientific (then FEI) partnered with the Medical Research Council Laboratory of Molecular Biology, the University of Cambridge Nanoscience Centre and five pharmaceutical companies [Astex Pharmaceuticals, AstraZeneca, GSK, Sosei Heptares and Union Chimique Belge (UCB)] to form the Cambridge Pharmaceutical Cryo-EM Consortium to share the risks of exploring cryo-EM for early-stage drug discovery. The Consortium expanded with a second Themo Scientific Krios Cryo-EM at the University of Cambridge Department of Materials Science and Metallurgy. Several Consortium members have set up in-house facilities, and a full service cryo-EM facility with Krios and Glacios has been created with the Electron Bio-Imaging Centre for Industry (eBIC for Industry) at Diamond Light Source (DLS), UK. This paper will cover the lessons learned during the setting up of these facilities, including two Consortium Krios microscopes and preparation laboratories, several Glacios microscopes at Consortium member sites, and a Krios and Glacios at eBIC for Industry, regarding site evaluation and selection for high-resolution cryo-EM microscopes, the installation process, scheduling, the operation and maintenance of the microscopes and preparation laboratories, and image processing.

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air–water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

In processing X-ray diffraction data, the intensities obtained from integration of the diffraction images must be corrected for experimental effects in order to place all intensities on a common scale both within and between data collections. Scaling corrects for effects such as changes in sample illumination, absorption and, to some extent, global radiation damage that cause the measured intensities of symmetry-equivalent observations to differ throughout a data set. This necessarily requires a prior evaluation of the point-group symmetry of the crystal. This paper describes and evaluates the scaling algorithms implemented within the DIALS data-processing package and demonstrates the effectiveness and key features of the implementation on example macromolecular crystallographic rotation data. In particular, the scaling algorithms enable new workflows for the scaling of multi-crystal or multi-sweep data sets, providing the analysis required to support current trends towards collecting data from ever-smaller samples. In addition, the implementation of a free-set validation method is discussed, which allows the quantification of the suitability of scaling-model and algorithm choices.

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.

As one of the most important phenomena in crystallization, the crystal nucleation process has always been the focus of research. In this work, influences of pre-assembly species and the desolvation process on the crystal nucleation process were studied. p-Nitro­benzoic acid (PNBA) was taken as a model compound to investigate the relationship between solution chemistry and nucleation kinetics in seven different solvents. One unsolvated form and four solvates of PNBA were obtained and one of the solvates was newly discovered. The nucleation behaviours and nucleation kinetics of PNBA in the seven solvents were studied and analyzed. Density functional theory (DFT) and solvation energy calculation were adopted to evaluate the strength of solute–solvent interactions. Vibrational spectroscopy combined with molecular simulation was applied to reveal the pre-assembly species in the solution. Based on these results, a comprehensive understanding of the relationship between molecular structure, crystal structure, solution chemistry and nucleation dynamics was proposed and discussed. It was found that the structural similarity between solution chemistry and crystal structure, the interaction between specific sites and the overall strength of solvation will jointly affect the nucleation process.

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.

The surface structure of fluoro­apatite (0001) (FAp0001) under quasi-dry and humid conditions has been probed with surface X-ray diffraction (SXRD). Lateral and perpendicular atomic relaxations corresponding to the FAp0001 termination before and after H2O exposure and the location of the adsorbed water molecules have been determined from experimental analysis of the crystal truncation rod (CTR) intensities. The surface under dry conditions exhibits a bulk termination with relaxations in the outermost atomic layers. The hydrated surface is formed by a disordered partially occupied H2O layer containing one water molecule (33% surface coverage) adsorbed at each of the three surface Ca atoms, and is coupled with one OH group randomly bonded to each of the three topmost P atoms with a 33% surface coverage.

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.

Here, structural parameters of various structure reports on RSi2 and R2TSi3 compounds [where R is an alkaline earth metal, a rare earth metal (i.e. an element of the Sc group or a lathanide), or an actinide and T is a transition metal] are summarized. The parameters comprising composition, lattice parameters a and c, ratio c/a, formula unit per unit cell and structure type are tabulated. The relationships between the underlying structure types are presented within a group–subgroup scheme (Bärnighausen diagram). Additionally, unexpectedly missing compounds within the R2TSi3 compounds were examined with density functional theory and compounds that are promising candidates for synthesis are listed. Furthermore, a correlation was detected between the orthorhombic AlB2-like lattices of, for example, Ca2AgSi3 and the divalence of R and the monovalence of T. Finally, a potential tetragonal structure with ordered Si/T sites is proposed.

Propagation-based phase-contrast X-ray imaging is by now a well established imaging technique, which – as a full-field technique – is particularly useful for tomography applications. Since it can be implemented with synchrotron radiation and at laboratory micro-focus sources, it covers a wide range of applications. A limiting factor in its development has been the phase-retrieval step, which was often performed using methods with a limited regime of applicability, typically based on linearization. In this work, a much larger set of algorithms, which covers a wide range of cases (experimental parameters, objects and constraints), is compiled into a single toolbox – the HoloTomoToolbox – which is made publicly available. Importantly, the unified structure of the implemented phase-retrieval functions facilitates their use and performance test on different experimental data.

A tandem-double-slit optical system was constructed to evaluate the practical beam emittance of undulator radiation. The optical system was a combination of an upstream slit (S1) and downstream slit (S2) aligned on the optical axis with an appropriate separation. The intensity distribution after the double slits, I(x1, x2), was measured by scanning S1 and S2 in the horizontal direction. Coordinates having 1/sqrt e intensity were extracted from I(x1, x2), whose contour provided the standard deviation ellipse in the x1–x2 space. I(x1, x2) was converted to the corresponding distribution in the phase space, I(x1, x1'). The horizontal beam emittance was evaluated to be 3.1 nm rad, which was larger than the value of 2.4 nm rad estimated by using ray-tracing. It was found that the increase was mainly due to an increase in beam divergence rather than size.

Different approaches of 2D lens arrays as Shack–Hartmann sensors for hard X-rays are compared. For the first time, a combination of Shack–Hartmann sensors for hard X-rays (SHSX) with a super-resolution imaging approach to perform multi-contrast imaging is demonstrated. A diamond lens is employed as a well known test object. The interleaving approach has great potential to overcome the 2D lens array limitation given by the two-photon polymerization lithography. Finally, the radiation damage induced by continuous exposure of an SHSX prototype with a white beam was studied showing a good performance of several hours. The shape modification and influence in the final image quality are presented.

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.

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 unique diagnostic possibilities of X-ray diffraction, small X-ray scattering and phase-contrast imaging techniques applied with high-intensity coherent X-ray synchrotron and X-ray free-electron laser radiation can only be fully realized if a sufficient dynamic range and/or spatial resolution of the detector is available. In this work, it is demonstrated that the use of lithium fluoride (LiF) as a photoluminescence (PL) imaging detector allows measuring of an X-ray diffraction image with a dynamic range of ∼107 within the sub-micrometre spatial resolution. At the PETRA III facility, the diffraction pattern created behind a circular aperture with a diameter of 5 µm irradiated by a beam with a photon energy of 500 eV was recorded on a LiF crystal. In the diffraction pattern, the accumulated dose was varied from 1.7 × 105 J cm−3 in the central maximum to 2 × 10−2 J cm−3 in the 16th maximum of diffraction fringes. The period of the last fringe was measured with 0.8 µm width. The PL response of the LiF crystal being used as a detector on the irradiation dose of 500 eV photons was evaluated. For the particular model of laser-scanning confocal microscope Carl Zeiss LSM700, used for the readout of the PL signal, the calibration dependencies on the intensity of photopumping (excitation) radiation (λ = 488 nm) and the gain have been obtained.

A gas- and vapour-pressure control system synchronized with the continuous data acquisition of millisecond high-resolution powder diffraction measurements was developed to study structural change processes in gas storage and reaction materials such as metal organic framework compounds, zeolite and layered double hydroxide. The apparatus, which can be set up on beamline BL02B2 at SPring-8, mainly comprises a pressure control system of gases and vapour, a gas cell for a capillary sample, and six one-dimensional solid-state (MYTHEN) detectors. The pressure control system can be remotely controlled via developed software connected to a diffraction measurement system and can be operated in the closed gas and vapour line system. By using the temperature-control system on the sample, high-resolution powder diffraction data can be obtained under gas and vapour pressures ranging from 1 Pa to 130 kPa in temperatures ranging from 30 to 1473 K. This system enables one to perform automatic and high-throughput in situ X-ray powder diffraction experiments even at extremely low pressures. Furthermore, this developed system is useful for studying crystal structures during the adsorption/desorption processes, as acquired by millisecond and continuous powder diffraction measurements. The acquisition of diffraction data can be synchronized with the control of the pressure with a high frame rate of up to 100 Hz. In situ and time-resolved powder diffraction measurements are demonstrated for nanoporous Cu coordination polymer in various gas and vapour atmospheres.

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.

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.

GIDVis is a software package based on MATLAB specialized for, but not limited to, the visualization and analysis of grazing-incidence thin-film X-ray diffraction data obtained during sample rotation around the surface normal. GIDVis allows the user to perform detector calibration, data stitching, intensity corrections, standard data evaluation (e.g. cuts and integrations along specific reciprocal-space directions), crystal phase analysis etc. To take full advantage of the measured data in the case of sample rotation, pole figures can easily be calculated from the experimental data for any value of the scattering angle covered. As an example, GIDVis is applied to phase analysis and the evaluation of the epitaxial alignment of pentacene­quinone crystallites on a single-crystalline Au(111) surface.

The open-source Python program PDB2INS is designed to prepare a .ins file for refinement with SHELXL [Sheldrick (2015). Acta Cryst. C71, 3–8], taking atom coordinates and other information from a Protein Data Bank (PDB)-format file. If PDB2INS is provided with a four-character PDB code, both the PDB file and the accompanying mmCIF-format reflection data file (if available) are accessed via the internet from the PDB public archive [Read et al. (2011). Structure, 19, 1395–1412] or optionally from the PDB_REDO server [Joosten, Long, Murshudov & Perrakis (2014). IUCrJ, 1, 213–220]. The SHELX-format .ins (refinement instructions and atomic coordinates) and .hkl (reflection data) files can then be generated without further user intervention, appropriate restraints etc. being added automatically. PDB2INS was tested on the 23 974 X-ray structures deposited in the PDB between 2008 and 2018 that included reflection data to 1.7 Å or better resolution in a recognizable format. After creating the two input files for SHELXL without user intervention, ten cycles of conjugate-gradient least-squares refinement were performed. For 96% of these structures PDB2INS and SHELXL completed successfully without error messages.

The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures.

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome by the crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating and phasing properties. This article presents examples of new crystallization conditions induced by the presence of Tb-Xo4. These new crystalline forms bypass crystal defects often encountered by crystallographers, such as low-resolution diffracting samples or crystals with twinning. Thanks to Tb-Xo4's high phasing power, the structure determination process is greatly facilitated and can be extended to serial crystallography approaches.

Rotation is a core crystallographic operation. Two sets of Cartesian coordinates of each point of a rotated object, those before and after rotation, are linearly related, and the coefficients of these linear combinations can be represented in matrix form. This 3 × 3 matrix is unique for all points and thus describes unambiguously a particular rotation. However, its nine elements are mutually dependent and are not interpretable in a straightforward way. To describe rotations by independent and comprehensible parameters, crystallographic software usually refers to Euler or to polar angles. In crystallography and cryo-electron microscopy, there exists a large choice of conventions, making direct comparison of rotation parameters difficult and sometimes confusing. The program py_convrot, written in Python, is a converter of parameters describing rotations. In particular, it deals with all possible choices of polar angles and with all kinds of Euler angles, including all choices of rotation axes and rotation directions. Using a menu, a user can build their own rotation parameterization; its action can be viewed with an interactive graphical tool, Demo. The tables in this article and the extended help pages of the program describe details of these parameterizations and the decomposition of rotation matrices into all types of parameters. The program allows orthogonalization conventions and symmetry operations to be taken into account. This makes the program and its supporting materials both an illustrative teaching material, especially for non-specialists in mathematics and computing, and a tool for practical use.

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.

Cryocooling for macromolecular crystallography is usually performed via plunging the crystal into a liquid cryogen or placing the crystal in a cold gas stream. These two approaches are compared here for the case of nitro­gen cooling. The results show that gas stream cooling, which typically cools the crystal more slowly, yields lower mosaicity and, in some cases, a stronger anomalous signal relative to rapid plunge cooling. During plunging, moving the crystal slowly through the cold gas layer above the liquid surface can produce mosaicity similar to gas stream cooling. Annealing plunge cooled crystals by warming and recooling in the gas stream allows the mosaicity and anomalous signal to recover. For tetragonal thermolysin, the observed effects are less pronounced when the cryosolvent has smaller thermal contraction, under which conditions the protein structures from plunge cooled and gas stream cooled crystals are very similar. Finally, this work also demonstrates that the resolution dependence of the reflecting range is correlated with the cooling method, suggesting it may be a useful tool for discerning whether crystals are cooled too rapidly. The results support previous studies suggesting that slower cooling methods are less deleterious to crystal order, as long as ice formation is prevented and dehydration is limited.

DatView is a new graphical user interface (GUI) for plotting parameters to explore correlations, identify outliers and export subsets of data. It was designed to simplify and expedite analysis of very large unmerged serial femtosecond crystallography (SFX) data sets composed of indexing results from hundreds of thousands of microcrystal diffraction patterns. However, DatView works with any tabulated data, offering its functionality to many applications outside serial crystallography. In DatView's user-friendly GUI, selections are drawn onto plots and synchronized across all other plots, so correlations between multiple parameters in large multi-parameter data sets can be rapidly identified. It also includes an item viewer for displaying images in the current selection alongside the associated metadata. For serial crystallography data processed by indexamajig from CrystFEL [White, Kirian, Martin, Aquila, Nass, Barty & Chapman (2012). J. Appl. Cryst. 45, 335–341], DatView generates a table of parameters and metadata from stream files and, optionally, the associated HDF5 files. By combining the functionality of several commonly needed tools for SFX in a single GUI that operates on tabulated data, the time needed to load and calculate statistics from large data sets is reduced. This paper describes how DatView facilitates (i) efficient feedback during data collection by examining trends in time, sample position or any parameter, (ii) determination of optimal indexing and integration parameters via the comparison mode, (iii) identification of systematic errors in unmerged SFX data sets, and (iv) sorting and highly flexible data filtering (plot selections, Boolean filters and more), including direct export of subset CrystFEL stream files for further processing.

The program Mercury, developed at the Cambridge Crystallographic Data Centre, was originally designed primarily as a crystal structure visualization tool. Over the years the fields and scientific communities of chemical crystallography and crystal engineering have developed to require more advanced structural analysis software. Mercury has evolved alongside these scientific communities and is now a powerful analysis, design and prediction platform which goes a lot further than simple structure visualization.

It is shown that it is possible to perform combined X-ray and neutron single-crystal studies in the same diamond anvil cell (DAC). A modified Merrill–Bassett DAC equipped with an inflatable membrane filled with He gas has been developed. It can be used on laboratory X-ray and synchrotron diffractometers as well as on neutron instruments. The data processing procedures and a joint structural refinement of the high-pressure synchrotron and neutron single-crystal data are presented and discussed for the first time.

Silicon nanowire-based sensors find many applications in micro- and nano-electromechanical systems, thanks to their unique characteristics of flexibility and strength that emerge at the nanoscale. This work is the first study of this class of micro- and nano-fabricated silicon-based structures adopting the scanning X-ray diffraction microscopy technique for mapping the in-plane crystalline strain (∊044) and tilt of a device which includes pillars with suspended nanowires on a substrate. It is shown how the micro- and nanostructures of this new type of nanowire system are influenced by critical steps of the fabrication process, such as electron-beam lithography and deep reactive ion etching. X-ray analysis performed on the 044 reflection shows a very low level of lattice strain (<0.00025 Δd/d) but a significant degree of lattice tilt (up to 0.214°). This work imparts new insights into the crystal structure of micro- and nanomaterial-based sensors, and their relationship with critical steps of the fabrication process.

BornAgain is a free and open-source multi-platform software framework for simulating and fitting X-ray and neutron reflectometry, off-specular scattering, and grazing-incidence small-angle scattering (GISAS). This paper concentrates on GISAS. Support for reflectometry and off-specular scattering has been added more recently, is still under intense development and will be described in a later publication. BornAgain supports neutron polarization and magnetic scattering. Users can define sample and instrument models through Python scripting. A large subset of the functionality is also available through a graphical user interface. This paper describes the software in terms of the realized non-functional and functional requirements. The web site https://www.bornagainproject.org/ provides further documentation.

Molybdenum oxides and sulfides on various low-cost high-surface-area supports are excellent catalysts for several industrially relevant reactions. The surface layer structure of these materials is, however, difficult to characterize due to small and disordered MoOx domains. Here, it is shown how X-ray total scattering can be applied to gain insights into the structure through differential pair distribution function (d-PDF) analysis, where the scattering signal from the support material is subtracted to obtain structural information on the supported structure. MoOx catalysts supported on alumina nanoparticles and on zeolites are investigated, and it is shown that the structure of the hydrated molybdenum oxide layer is closely related to that of disordered and polydisperse polyoxometalates. By analysing the PDFs with a large number of automatically generated cluster structures, which are constructed in an iterative manner from known polyoxometalate clusters, information is derived on the structural motifs in supported MoOx.

Small-angle neutron scattering (SANS) was recently applied to the in situ and operando study of the charge/discharge process in Li-ion battery full-cells based on a pouch cell design. Here, this work is continued in a half-cell with a graphite electrode cycled versus a metallic lithium counter electrode, in a study conducted on the SANS-1 instrument of the neutron source FRM II at the Heinz Maier-Leibnitz Zentrum in Garching, Germany. It is confirmed that the SANS integrated intensity signal varies as a function of graphite lithiation, and this variation can be explained by changes in the squared difference in scattering length density between graphite and the electrolyte. The scattering contrast change upon graphite lithiation/delithiation calculated from a multi-phase neutron scattering model is in good agreement with the experimentally measured values. Due to the finite coherence length, the observed SANS contrast, which mostly stems from scattering between the (lithiated) graphite and the electrolyte phase, contains local information on the mesoscopic scale, which allows the development of lithiated phases in the graphite to be followed. The shape of the SANS signal curve can be explained by a core–shell model with step-wise (de)lithiation from the surface. Here, for the first time, X-ray diffraction, SANS and theory are combined to give a full picture of graphite lithiation in a half-cell. The goal of this contribution is to confirm the correlation between the integrated SANS data obtained during operando measurements of an Li-ion half-cell and the electrochemical processes of lithiation/delithiation in micro-scaled graphite particles. For a deeper understanding of this correlation, modelling and experimental data for SANS and results from X-ray diffraction were taken into account.

Clays and soils produce strong small-angle X-ray scattering (SAXS) because they contain large numbers of nanoparticles, namely allophane and ferrihydrite. These nanoparticles are amorphous and have approximately spherical shape with a size of around 3–10 nm. The weight ratios of these nanoparticles will affect the properties of the clays and soils. However, the nanoparticles in clays and soils are not generally quantified and are sometimes ignored because there is no standard method to quantify them. This paper describes a method to quantify nanoparticles in clays and soils with SAXS. This is achieved by deriving normalized SAXS intensities from unit weight of the sample, which are not affected by absorption. By integrating the normalized SAXS intensities over the reciprocal space, one obtains a value that is proportional to the weight ratio of the nanoparticles, proportional to the square of the difference of density between the nanoparticles and the liquid surrounding the nanoparticles, and inversely proportional to the density of the nanoparticles. If the density of the nanoparticles is known, the weight ratio of the nanoparticles can be calculated from the SAXS intensities. The density of nanoparticles was estimated from the chemical composition of the sample. Nanoparticles in colloidal silica, silica gels, mixtures of silica gel and α-aluminium oxide, and synthetic clays have been quantified with the integral SAXS method. The results show that the errors of the weight ratios of nanoparticles are around 25% of the weight ratio. It is also shown that some natural clays contain large fractions of nanoparticles; montmorillonite clay from the Mikawa deposit, pyrophillite clay from the Shokozan deposit and kaolinite clay from the Kanpaku deposit contain 25 (7), 10 (2) and 19 (5) wt% nanoparticles, respectively, where errors are shown in parentheses.

A historical tool for crystallographic analysis is provided by the Hilton net, which can be used for manually surveying the crystal lattice as it is manifested by the Kikuchi bands in a gnomonic projection. For a quantitative analysis using the Hilton net, the projection centre as the relative position of the signal source with respect to the detector plane needs to be known. Interplanar angles are accessible with a precision and accuracy which is estimated to be ≤0.3°. Angles between any directions, e.g. zone axes, are directly readable. Finally, for the rare case of an unknown projection-centre position, its determination is demonstrated by adapting an old approach developed for photogrammetric applications. It requires the indexing of four zone axes [uvw]i in a backscattered Kikuchi diffraction pattern of a known phase collected under comparable geometric conditions.

An online knowledge base on the alternate conformations adopted by main-chain and side-chain atoms in protein structures solved by X-ray crystallography is described.

The unique nucleating and phasing capabilities of the crystallophore, Tb-Xo4, are illustrated through challenging cases.

A new temperature-controllable humidifier for X-ray diffraction has been developed. It is shown that the humidifier can successfully maintain protein crystal growth at a temperature lower than room temperature.