The structural and chemical evolution of battery electrodes at the nanoscale plays an important role in affecting the cell performance. Nano-resolution X-ray microscopy has been demonstrated as a powerful technique for characterizing the evolution of battery electrodes under operating conditions with sensitivity to their morphology, compositional distribution and redox heterogeneity. In real-world batteries, the electrode could deform upon battery operation, causing challenges for the image registration which is necessary for several experimental modalities, e.g. XANES imaging. To address this challenge, this work develops a deep-learning-based method for automatic particle identification and tracking. This approach was not only able to facilitate image registration with good robustness but also allowed quantification of the degree of sample deformation. The effectiveness of the method was first demonstrated using synthetic datasets with known ground truth. The method was then applied to an experimental dataset collected on an operating lithium battery cell, revealing a high degree of intra- and interparticle chemical complexity in operating batteries.

Superconducting undulators (SCUs) can offer a much higher on-axis undulator field than state-of-the-art cryogenic permanent-magnet undulators with the same period and vacuum gap. The development of shorter-period and high-field SCUs would allow the free-electron laser and synchrotron radiation source community to reduce both the length of undulators and the dimensions of the accelerator. Magnetic measurements are essential for characterizing the magnetic field quality of undulators for operation in a modern light source. Hall probe scanning is so far the most mature technique for local field characterization of undulators. This article focuses on the systematic error caused by thermal contraction that influences Hall probe measurements carried out in a liquid helium cryostat. A novel procedure, based on the redundant measurement of the magnetic field using multiple Hall probes at known relative distance, is introduced for the correction of such systematic error.

Hard X-ray absorption spectroscopy is a valuable in situ probe for non-destructive diagnostics of metal sites. The low-energy interval of a spectrum (XANES) contains information about the metal oxidation state, ligand type, symmetry and distances in the first coordination shell but shows almost no dependency on the bridged metal–metal bond length. The higher-energy interval (EXAFS), on the contrary, is more sensitive to the coordination numbers and can decouple the contribution from distances in different coordination shells. Supervised machine-learning methods can combine information from different intervals of a spectrum; however, computational approaches for the near-edge region of the spectrum and higher energies are different. This work aims to keep all benefits of XANES and extend its sensitivity towards the interatomic distances in the first and second coordination shells. Using a binuclear bridged copper complex as a case study and cross-validation analysis as a quantitative tool it is shown that the first 170 eV above the edge are already sufficient to balance the contributions of Cu–O/N scattering and Cu–Cu scattering. As a more general outcome this work highlights the trivial but often overlooked importance of using `longer' energy intervals of XANES for structural refinement and machine-learning predictions. The first 200 eV above the absorption edge still do not require parametrization of Debye–Waller damping and can be calculated within full multiple scattering or finite difference approximations with only moderately increased computational costs.

Despite the increased brilliance of the new generation synchrotron sources, there is still a challenge with high-resolution scanning of very thick and absorbing samples, such as a whole mouse brain stained with heavy elements, and, extending further, brains of primates. Samples are typically cut into smaller parts, to ensure a sufficient X-ray transmission, and scanned separately. Compared with the standard tomography setup where the sample would be cut into many pillars, the laminographic geometry operates with slab-shaped sections significantly reducing the number of sample parts to be prepared, the cutting damage and data stitching problems. In this work, a laminography pipeline for imaging large samples (>1 cm) at micrometre resolution is presented. The implementation includes a low-cost instrument setup installed at the 2-BM micro-CT beamline of the Advanced Photon Source. Additionally, sample mounting, scanning techniques, data stitching procedures, a fast reconstruction algorithm with low computational complexity, and accelerated reconstruction on multi-GPU systems for processing large-scale datasets are presented. The applicability of the whole laminography pipeline was demonstrated by imaging four sequential slabs throughout an entire mouse brain sample stained with osmium, in total generating approximately 12 TB of raw data for reconstruction.

X-ray ptychography and ptychographic computed tomography have seen a rapid rise since the advent of fourth-generation synchrotrons with a high degree of coherent radiation. In addition to quantitative multiscale structural analysis, ptychography with spectral capabilities has been developed, allowing for spatial-localized multiscale structural and spectral information of samples. The SWING beamline of Synchrotron SOLEIL has recently developed a nanoprobe setup where the endstation's first spectral and resonant ptychographic measurements have been successfully conducted. A metallic nickel wire sample was measured using 2D spectral ptychography in XANES mode and resonant ptychographic tomography. From the 2D spectral ptychography measurements, the spectra of the components of the sample's complex-valued refractive index, δ and β, were extracted, integrated along the sample thickness. By performing resonance ptychographic tomography at two photon energies, 3D maps of the refractive index decrement, δ, were obtained at the Ni K-edge energy and another energy above the edge. These maps allowed the detection of impurities in the Ni wire. The significance of accounting for the atomic scattering factor is demonstrated in the calculation of electron density near a resonance through the use of the δ values. These results indicate that at the SWING beamline it is possible to conduct state-of-the-art spectral and resonant ptychography experiments using the nanoprobe setup.

The in situ measurement technique for a metal/metal-oxide mixture at extra-high temperature above 2000 K has been desired in the field of nuclear safety engineering. In the present study, we succeeded in simultaneous XAFS–XRD measurements of the Zr oxidation [Zr + O → Zr(O) + ZrO2] up to 1952 K and ZrO2–Y2O3 reaction from 1952 to 2519 K. The chemical shift during Zr oxidation was observed in the absorption spectra around the Zr K-edge, and the interatomic cation–cation and cation–oxygen distances obtained by the fitting analysis of EXAFS during the Y2O3–ZrO2 reaction are explained. Also, the temperature dependency of the anharmonic effect was investigated by comparing the fitted second- and third-order cumulants with the theoretical ones in which the Morse potential was applied as an interatomic potential, giving a good explanation about the local structure dynamics. Finally, the applicability of the developed system to investigation of nuclear fuel materials, such as UO2–Zr, is discussed.

Understanding the correlation between chemical and microstructural properties is critical for unraveling the fundamental relationship between materials chemistry and physical structures that can benefit materials science and engineering. Here, we demonstrate novel in situ correlative imaging of the X-ray Compton scattering computed tomography (XCS-CT) technique for studying this fundamental relationship. XCS-CT can image light elements that do not usually exhibit strong signals using other X-ray characterization techniques. This paper describes the XCS-CT setup and data analysis method for calculating the valence electron momentum density and lithium-ion concentration, and provides two examples of spatially and temporally resolved chemical properties inside batteries in 3D. XCS-CT was applied to study two types of rechargeable lithium batteries in standard coin cell casings: (1) a lithium-ion battery containing a cathode of bespoke microstructure and liquid electrolyte, and (2) a solid-state battery containing a solid-polymer electrolyte. The XCS-CT technique is beneficial to a wide variety of materials and systems to map chemical composition changes in 3D structures.

Understanding and controlling the structure and function of liquid interfaces is a constant challenge in biology, nanoscience and nanotechnology, with applications ranging from molecular electronics to controlled drug release. X-ray reflectivity and grazing incidence diffraction provide invaluable probes for studying the atomic scale structure at liquid–air interfaces. The new time-resolved laser system at the LISA liquid diffractometer situated at beamline P08 at the PETRA III synchrotron radiation source in Hamburg provides a laser pump with X-ray probe. The femtosecond laser combined with the LISA diffractometer allows unique opportunities to investigate photo-induced structural changes at liquid interfaces on the pico- and nanosecond time scales with pump–probe techniques. A time resolution of 38 ps has been achieved and verified with Bi. First experiments include laser-induced effects on salt solutions and liquid mercury surfaces with static and varied time scales measurements showing the proof of concept for investigations at liquid surfaces.

Imaging energy filters in photoelectron microscopes and momentum microscopes use spherical fields with deflection angles of 90°, 180° and even 2 × 180°. These instruments are optimized for high energy resolution, and exhibit image aberrations when operated in high transmission mode at medium energy resolution. Here, a new approach is presented for bandpass-filtered imaging in real or reciprocal space using an electrostatic dodecapole with an asymmetric electrode array. In addition to energy-dispersive beam deflection, this multipole allows aberration correction up to the third order. Here, its use is described as a bandpass prefilter in a time-of-flight momentum microscope at the hard X-ray beamline P22 of PETRA III. The entire instrument is housed in a straight vacuum tube because the deflection angle is only 4° and the beam displacement in the filter is only ∼8 mm. The multipole is framed by transfer lenses in the entrance and exit branches. Two sets of 16 different-sized entrance and exit apertures on piezomotor-driven mounts allow selection of the desired bandpass. For pass energies between 100 and 1400 eV and slit widths between 0.5 and 4 mm, the transmitted kinetic energy intervals are between 10 eV and a few hundred electronvolts (full width at half-maximum). The filter eliminates all higher or lower energy signals outside the selected bandpass, significantly improving the signal-to-background ratio in the time-of-flight analyzer.

The demand for powder X-ray diffraction analysis continues to increase in a variety of scientific fields, as the excellent beam quality of high-brightness synchrotron light sources enables the acquisition of high-quality measurement data with high intensity and angular resolution. Synchrotron powder diffraction has enabled the rapid measurement of many samples and various in situ/operando experiments in nonambient sample environments. To meet the demands for even higher throughput measurements using high-energy X-rays at SPring-8, a high-throughput and high-resolution powder diffraction system has been developed. This system is combined with six sets of two-dimensional (2D) CdTe detectors for high-energy X-rays, and various automation systems, including a system for automatic switching among large sample environmental equipment, have been developed in the third experimental hutch of the insertion device beamline BL13XU at SPring-8. In this diffractometer system, high-brilliance and high-energy X-rays ranging from 16 to 72 keV are available. The powder diffraction data measured under ambient and various nonambient conditions can be analysed using Rietveld refinement and the pair distribution function. Using the 2D CdTe detectors with variable sample-to-detector distance, three types of scan modes have been established: standard, single-step and high-resolution. A major feature is the ability to measure a whole powder pattern with millisecond resolution. Equally important, this system can measure powder diffraction data with high Q exceeding 30 Å−1 within several tens of seconds. This capability is expected to contribute significantly to new research avenues using machine learning and artificial intelligence by utilizing the large amount of data obtained from high-throughput measurements.

Structural biology experiments benefit significantly from state-of-the-art synchrotron data collection. One can acquire macromolecular crystallography (MX) diffraction data on large-area photon-counting pixel-array detectors at framing rates exceeding 1000 frames per second, using 200 Gbps network connectivity, or higher when available. In extreme cases this represents a raw data throughput of about 25 GB s−1, which is nearly impossible to deliver at reasonable cost without compression. Our field has used lossless compression for decades to make such data collection manageable. Many MX beamlines are now fitted with DECTRIS Eiger detectors, all of which are delivered with optimized compression algorithms by default, and they perform well with current framing rates and typical diffraction data. However, better lossless compression algorithms have been developed and are now available to the research community. Here one of the latest and most promising lossless compression algorithms is investigated on a variety of diffraction data like those routinely acquired at state-of-the-art MX beamlines.

Laser-induced projectile impact testing (LIPIT) based on synchrotron imaging is proposed and validated. This emerging high-velocity, high-strain microscale dynamic loading technique offers a unique perspective on the strain and energy dissipation behavior of materials subjected to high-speed microscale single-particle impacts. When combined with synchrotron radiation imaging techniques, LIPIT allows for in situ observation of particle infiltration. Two validation experiments were carried out, demonstrating the potential of LIPIT in the roentgenoscopy of the dynamic properties of various materials. With a spatial resolution of 10 µm and a temporal resolution of 33.4 µs, the system was successfully realized at the Beijing Synchrotron Radiation Facility 3W1 beamline. This innovative approach opens up new avenues for studying the dynamic properties of materials in situ.

In a photoinjector electron source, the initial transverse electron bunch properties are determined by the spatial properties of the laser beam on the photocathode. Spatial shaping of the laser is commonly achieved by relay imaging an illuminated circular mask onto the photocathode. However, the Gibbs phenomenon shows that recreating the sharp edge and discontinuity of the cut profile at the mask on the cathode is not possible with an optical relay of finite aperture. Furthermore, the practical injection of the laser into the photoinjector results in the beam passing through small or asymmetrically positioned apertures. This work uses wavefront propagation to show how the transport apertures cause ripple structures to appear in the transverse laser profile even when effectively the full laser power is transmitted. The impact of these structures on the propagated electron bunch has also been studied with electron bunches of high and low charge density. With high charge density, the ripples in the initial charge distribution rapidly wash-out through space charge effects. However, for bunches with low charge density, the ripples can persist through the bunch transport. Although statistical properties of the electron bunch in the cases studied are not greatly affected, there is the potential for the distorted electron bunch to negatively impact machine performance. Therefore, these effects should be considered in the design phase of accelerators using photoinjectors.

L3-edge high-energy-resolution fluorescence-detection X-ray absorption near-edge structure (XANES) spectra for palladium and rhodium compounds are presented, with focus on their electronic structures. The data are compared with transmission XANES spectra recorded at the K-edge. A correlation between the absorption edge energy and the metal ion oxidation state is not observed. Despite the different filling of the 4d orbitals and different local coordination, the Rh and Pd compounds show remarkably similar spectral shapes. Calculation of the density of states and of the L3-XANES data reproduce the experimental results.

TEMPUS is a new detector system being developed for photon science. It is based on the Timepix4 chip and, thus, it can be operated in two distinct modes: a photon-counting mode, which allows for conventional full-frame readout at rates up to 40 kfps; and an event-driven time-stamping mode, which allows excellent time resolution in the nanosecond regime in measurements with moderate X-ray flux. In this paper, the initial prototype, a single-chip device, is introduced, and the readout system described. Moreover, and in order to evaluate its capabilities, some tests were performed at PETRA III and ESRF for which results are also presented.

The differentially pumped rare-gas filter at the end of the VUV beamline of the Swiss Light Source has been adapted to house a windowless absorption cell for gases. Absorption spectra can be recorded from 7 eV to up to 21 eV photon energies routinely, as shown by a new water and nitrous oxide absorption spectrum. By and large, the spectra agree with previously published ones both in terms of resonance energies and absorption cross sections, but that of N2O exhibits a small shift in the { ilde{f D}} band and tentative fine structures that have not yet been fully described. This setup will facilitate the measurement of absorption spectra in the VUV above the absorption edge of LiF and MgF2 windows. It will also allow us to carry out condensed-phase measurements on thin liquid sheets and solid films. Further development options are discussed, including the recording of temperature-dependent absorption spectra, a stationary gas cell for calibration measurements, and the improvement of the photon energy resolution.

Aerosol science is of utmost importance for both climate and public health research, and in recent years X-ray techniques have proven effective tools for aerosol-particle characterization. To date, such methods have often involved the study of particles collected onto a substrate, but a high photon flux may cause radiation damage to such deposited particles and volatile components can potentially react with the surrounding environment after sampling. These and many other factors make studies on collected aerosol particles challenging. Therefore, a new aerosol sample-delivery system dedicated to X-ray photoelectron spectroscopy studies of aerosol particles and gas molecules in-flight has been developed at the MAX IV Laboratory. The aerosol particles are brought from atmospheric pressure to vacuum in a continuous flow, ensuring that the sample is constantly renewed, thus avoiding radiation damage, and allowing measurements on the true unsupported aerosol. At the same time, available gas molecules can be used for energy calibration and to study gas-particle partitioning. The design features of the aerosol sample-delivery system and important information on the operation procedures are described in detail here. Furthermore, to demonstrate the experimental range of the aerosol sample-delivery system, results from aerosol particles of different shape, size and composition are presented, including inorganic atmospheric aerosols, secondary organic aerosols and engineered nanoparticles.

The paper considers the possibility of using the diamond-silicon carbide composite Skeleton® with a technological coating of polycrystalline silicon as a substrate for X-ray mirrors used with powerful synchrotron radiation sources (third+ and fourth generation). Samples were studied after polishing to provide the following surface parameters: root-mean-square flatness ≃ 50 nm, micro-roughness on the frame 2 µm × 2 µm σ ≃ 0.15 nm. The heat capacity, thermal conductivity and coefficient of linear thermal expansion were investigated. For comparison, a monocrystalline silicon sample was studied under the same conditions using the same methods. The value of the coefficient of linear thermal expansion turned out to be higher than that of monocrystalline silicon and amounted to 4.3 × 10−6 K−1, and the values of thermal conductivity (5.0 W cm−1 K−1) and heat capacity (1.2 J K−1 g−1) also exceeded the values for Si. Thermally induced deformations of both Skeleton® and monocrystalline silicon samples under irradiation with a CO2 laser beam have also been experimentally studied. Taking into account the obtained thermophysical constants, the calculation of thermally induced deformation under irradiation with hard (20 keV) X-rays showed almost three times less deformation of the Skeleton® sample than of the monocrystalline silicon sample.

The split-and-delay unit (SDU) at FLASH2 will be upgraded to enable the simultaneous operation of two temporally, spatially and spectrally separated probe beams when the free-electron laser undulators are operated in a two-color scheme. By means of suitable thin filters and an optical grating beam path a wide range of combinations of photon energies in the spectral range from 150 eV to 780 eV can be chosen. In this paper, simulations of the spectral transmission and performance parameters of the filter technique are discussed, along with a monochromator with dispersion compensation presently under construction.

Addressing the demand for high stability of beamline instruments at the SHINE facility, a high stability mirror regulating mechanism has been developed for mirror adjustments. Active mass damping was adopted to attenuate pitch angle vibrations of mirrors caused by structural vibrations. An internal absolute velocity feedback was used to reduce the negative impact of spillover effects and to improve performance. The experiment was conducted on a prototype structure of a mirror regulating mechanism, and results showed that the vibration RMS of the pitch angle was effectively attenuated from 47 nrad to 27 nrad above 1 Hz.

To date, computed tomography experiments, carried-out at synchrotron radiation facilities worldwide, pose a tremendous challenge in terms of the breadth and complexity of the experimental datasets produced. Furthermore, near real-time three-dimensional reconstruction capabilities are becoming a crucial requirement in order to perform high-quality and result-informed synchrotron imaging experiments, where a large amount of data is collected and processed within a short time window. To address these challenges, we have developed and deployed a synchrotron computed tomography framework designed to automatically process online the experimental data from the synchrotron imaging beamlines, while leveraging the high-performance computing cluster capabilities to accelerate the real-time feedback to the users on their experimental results. We have, further, integrated it within a modern unified national authentication and data management framework, which we have developed and deployed, spanning the entire data lifecycle of a large-scale scientific facility. In this study, the overall architecture, functional modules and workflow design of our synchrotron computed tomography framework are presented in detail. Moreover, the successful integration of the imaging beamlines at the Shanghai Synchrotron Radiation Facility into our scientific computing framework is also detailed, which, ultimately, resulted in accelerating and fully automating their entire data processing pipelines. In fact, when compared with the original three-dimensional tomography reconstruction approaches, the implementation of our synchrotron computed tomography framework led to an acceleration in the experimental data processing capabilities, while maintaining a high level of integration with all the beamline processing software and systems.

Xray free-electron lasers (XFELs) enable experiments that would have been impractical or impossible at conventional X-ray laser facilities. Indeed, more XFEL facilities are being built and planned, with their aim to deliver larger pulse energies and higher peak brilliance. While seeking to increase the pulse power, it is quintessential to consider the maximum pulse fluence that a grazing-incidence FEL mirror can withstand. To address this issue, several studies were conducted on grazing-incidence damage by soft X-ray FEL pulses at the European XFEL facility. Boron carbide (B4C) coatings on polished silicon substrate were investigated using 1 keV photon energy, similar to the X-ray mirrors currently installed at the soft X-ray beamlines (SASE3). The purpose of this study is to compare the damage threshold of B4C and Si to determine the advantages, tolerance and limits of using B4C coatings.

By studying the structures of (μ-1,4,10,13-tetra­thia-7,16-di­aza­cyclo­octa­deca­ne)bis[iodidopalladium(II)] diiodide penta­(diiodine), [Pd2I2(C12H26N2S4)](I)2·5I2 or [Pd2I2([18]aneN2S4)](I)2·(I2)5, and 4,7,13,16,21,24-hexa­oxa-1,10-diazo­niabi­cyclo­[8.8.8]hexa­cosane triiodide iodide hemi­penta­(diiodine) di­chloro­methane mono­solvate, C18H38N2O62+·I3−·I−·2.5I2·CH2Cl2 or [H2([2.2.2]cryptand)](I3)(I)(I2)2.5·CH2Cl2, we confirm the structural variety of extended polyiodides achievable upon changing the shape, charge and dimensions of the cation template, by altering the synthetic strategy adopted and/or the experimental conditions. Although it is still often difficult to characterize discrete [I2m+n]n− polyiodides higher than I3− on the basis of structural parameters, such as I—I bond distances, FT–Raman spectroscopy appears to identify them as aggregates of I2, I− and (symmetric or slightly asymmetric) I3− building blocks linked by I⋯I inter­actions of varying strengths. However, because FT–Raman spectroscopy carries no information about the topological features of extended polyiodides, the two techniques should therefore be applied in combination to enhance the analysis of this kind of compounds.

Historically, knowledge of the mol­ecular packing within the crystal structures of organic semi­con­duc­tors has been instrumental in understanding their solid-state electronic properties. Nowadays, crystal structures are thus becoming increasingly important for enabling engineering properties, understanding poly­mor­phism in bulk and in thin films, exploring dynamics and elucidating phase-transition mech­a­nisms. This review article introduces the most salient and recent results of the field.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system.

Cryo-electron tomography (cryo-ET) enables molecular-resolution 3D imaging of complex biological specimens such as viral particles, cellular sections and, in some cases, whole cells. This enables the structural characterization of molecules in their near-native environments, without the need for purification or separation, thereby preserving biological information such as conformational states and spatial relationships between different molecular species. Subtomogram averaging is an image-processing workflow that allows users to leverage cryo-ET data to identify and localize target molecules, determine high-resolution structures of repeating molecular species and classify different conformational states. Here, STOPGAP, an open-source package for subtomogram averaging that is designed to provide users with fine control over each of these steps, is described. In providing detailed descriptions of the image-processing algorithms that STOPGAP uses, this manuscript is also intended to serve as a technical resource to users as well as for further community-driven software development.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.

For cryo-electron tomography (cryo-ET) of beam-sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal-to-noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample-environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo-ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low-symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron-beam damage to the specimen is an issue and the first images acquired will transfer more high-resolution information than those acquired last. There is also an inherent trade-off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n-helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose-symmetric tilt schemes for cryo-ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose-symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced. Tested on endothiapepsin crystals with four soaked fragments, thaumatin and SARS-CoV-2 3CLpro, Cryo2RT reveals unique ligand-binding poses, offers a comparable throughput to cryo-crystallography and eases the exploration of structural dynamics at various temperatures.

Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered ortho­rhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the β-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.

A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.

The availability of highly accurate protein structure predictions from AlphaFold2 (AF2) and similar tools has hugely expanded the applicability of molecular replacement (MR) for crystal structure solution. Many structures can be solved routinely using raw models, structures processed to remove unreliable parts or models split into distinct structural units. There is therefore an open question around how many and which cases still require experimental phasing methods such as single-wavelength anomalous diffraction (SAD). Here, this question is addressed using a large set of PDB depositions that were solved by SAD. A large majority (87%) could be solved using unedited or minimally edited AF2 predictions. A further 18 (4%) yield straightforwardly to MR after splitting of the AF2 prediction using Slice'N'Dice, although different splitting methods succeeded on slightly different sets of cases. It is also found that further unique targets can be solved by alternative modelling approaches such as ESMFold (four cases), alternative MR approaches such as ARCIMBOLDO and AMPLE (two cases each), and multimeric model building with AlphaFold-Multimer or UniFold (three cases). Ultimately, only 12 cases, or 3% of the SAD-phased set, did not yield to any form of MR tested here, offering valuable hints as to the number and the characteristics of cases where experimental phasing remains essential for macromolecular structure solution.

The structures of the simplest symmetric primary ethers [(CnH2n+1)2O, n = 1–3] determined under high pressure revealed their conformational preferences and intermolecular interactions. In three new polymorphs of di­ethyl ether (C2H5)2O, high pressure promotes intermolecular CH⋯O contacts and enforces a conversion from the trans–trans conformer present in the α, β and γ phases to the trans–gauche conformer, which is higher in energy by 6.4 kJ mol−1, in the δ phase. Two new polymorphs of di­methyl ether (CH3)2O display analogous transformations of the CH⋯O bonds. The crystal structure of di-n-propyl ether (C3H7)2O, determined for the first time, is remarkably stable over the whole pressure range investigated from 1.70 up to 5.30 GPa.

Highly accurate protein structure prediction can generate accurate models of protein and protein–protein complexes in X-ray crystallography. However, the question of how to make more effective use of predicted models for completing structure analysis, and which strategies should be employed for the more challenging cases such as multi-helical structures, multimeric structures and extremely large structures, both in the model preparation and in the completion steps, remains open for discussion. In this paper, a new strategy is proposed based on the framework of direct methods and dual-space iteration, which can greatly simplify the pre-processing steps of predicted models both in normal and in challenging cases. Following this strategy, full-length models or the conservative structural domains could be used directly as the starting model, and the phase error and the model bias between the starting model and the real structure would be modified in the direct-methods-based dual-space iteration. Many challenging cases (from CASP14) have been tested for the general applicability of this constructive strategy, and almost complete models have been generated with reasonable statistics. The hybrid strategy therefore provides a meaningful scheme for X-ray structure determination using a predicted model as the starting point.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB–TcC subcomplex that makes a hollow shell. This TcB–TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB–TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

The Protein Data Bank (PDB) was established as the first open-access digital data resource in biology and medicine in 1971 with seven X-ray crystal structures of proteins. Today, the PDB houses >210 000 experimentally determined, atomic level, 3D structures of proteins and nucleic acids as well as their complexes with one another and small molecules (e.g. approved drugs, enzyme cofactors). These data provide insights into fundamental biology, biomedicine, bioenergy and biotechnology. They proved particularly important for understanding the SARS-CoV-2 global pandemic. The US-funded Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and other members of the Worldwide Protein Data Bank (wwPDB) partnership jointly manage the PDB archive and support >60 000 `data depositors' (structural biologists) around the world. wwPDB ensures the quality and integrity of the data in the ever-expanding PDB archive and supports global open access without limitations on data usage. The RCSB PDB research-focused web portal at https://www.rcsb.org/ (RCSB.org) supports millions of users worldwide, representing a broad range of expertise and interests. In addition to retrieving 3D structure data, PDB `data consumers' access comparative data and external annotations, such as information about disease-causing point mutations and genetic variations. RCSB.org also provides access to >1 000 000 computed structure models (CSMs) generated using artificial intelligence/machine-learning methods. To avoid doubt, the provenance and reliability of experimentally determined PDB structures and CSMs are identified. Related training materials are available to support users in their RCSB.org explorations.

A new polymorph of 1,3-diacetylpyrene has been obtained from its melt and thoroughly characterized using single-crystal X-ray diffraction, steady-state UV–Vis spectroscopy and periodic density functional theory calculations. Experimental studies covered the temperature range from 90 to 390 K and the pressure range from atmospheric to 4.08 GPa. Optimal sample placement in a diamond anvil cell according to our previously presented methodology ensured over 80% data coverage up to 0.8 Å for a monoclinic sample. Unrestrained Hirshfeld atom refinement of the high-pressure crystal structures was successful and anharmonic behavior of carbonyl oxygen atoms was observed. Unlike the previously characterized polymorph, the structure of 2°AP-β is based on infinite π-stacks of antiparallel 2°AP molecules. 2°AP-β displays piezochromism and piezofluorochromism which are directly related to the variation in interplanar distances within the π-stacking. The importance of weak intermolecular interactions is reflected in the substantial negative thermal expansion coefficient of −55.8 (57) MK−1 in the direction of C—H⋯O interactions.

Platinum(II) complexes of square-planar geometry are interesting from a crystal engineering viewpoint because they exhibit strong luminescence based on the self-assembly of molecular units. The luminescence color changes in response to gentle stimuli, such as vapor exposure or weak mechanical forces. Both the molecular and the crystal designs for soft crystals are critical to effectively generate the chromic luminescence phenomenon of Pt(II) complexes. In this topical review, strategies for fabricating chromic luminescent Pt(II) complexes are described from a crystal design perspective, focusing on the structural regulation of Pt(II) complexes that exhibit assembly-induced luminescence via metal–metal interactions and structural control of anionic Pt(II) complexes using cations. The research progress on the evolution of various chromic luminescence properties of Pt(II) complexes, including the studies conducted by our group, are presented here along with the latest research outcomes, and an overview of the frontiers and future potential of this research field is provided.

This paper was motivated by the articles `Same or different – that is the question' in CrystEngComm (July 2020) and `Change to the definition of a crystal' in the IUCr Newsletter (June 2021). Experimental approaches to crystal comparisons require rigorously defined classifications in crystallography and beyond. Since crystal structures are determined in a rigid form, their strongest equivalence in practice is rigid motion, which is a composition of translations and rotations in 3D space. Conventional representations based on reduced cells and standardizations theoretically distinguish all periodic crystals. However, all cell-based representations are inherently discontinuous under almost any atomic displacement that can arbitrarily scale up a reduced cell. Hence, comparison of millions of known structures in materials databases requires continuous distance metrics.

The functionality and efficiency of proteins within a biological membrane are highly dependent on both the membrane lipid composition and the physiochemical properties of the solution. Lipid mesophases are directly influenced by changes in temperature, pH, water content or due to individual properties of single lipids such as photoswitchability. In this work, we were able to induce light- and temperature-driven mesophase transitions in a model membrane system containing a mixture of 1,2-dipalmitoyl-phosphatidylcholine phospho­lipids and azo­benzene amphiphiles. We observed reversible and reproducible transitions between the lamellar and Pn3m cubic phase after illuminating the sample for 5 min with light of 365 and 455 nm wavelengths, respectively, to switch between the cis and trans states of the azo­benzene N=N double bond. These light-controlled mesophase transitions were found for mixed complexes with up to 20% content of the photosensitive molecule and at temperatures below the gel-to-liquid crystalline phase transition temperature of 33°C. Our results demonstrate the potential to design bespoke model systems to study the response of membrane lipids and proteins upon changes in mesophase without altering the environment and thus provide a possible basis for drug delivery systems.

The hardware for data archiving has expanded capacities for digital storage enormously in the past decade or more. The IUCr evaluated the costs and benefits of this within an official working group which advised that raw data archiving would allow ground truth reproducibility in published studies. Consultations of the IUCr's Commissions ensued via a newly constituted standing advisory committee, the Committee on Data. At all stages, the IUCr financed workshops to facilitate community discussions and possible methods of raw data archiving implementation. The recent launch of the IUCrData journal's Raw Data Letters is a milestone in the implementation of raw data archiving beyond the currently published studies: it includes diffraction patterns that have not been fully interpreted, if at all. The IUCr 75th Congress in Melbourne included a workshop on raw data reuse, discussing the successes and ongoing challenges of raw data reuse. This article charts the efforts of the IUCr to facilitate discussions and plans relating to raw data archiving and reuse within the various communities of crystallography, diffraction and scattering.

In the folded state, biomolecules exchange between multiple conformational states crucial for their function. However, most structural models derived from experiments and computational predictions only encode a single state. To represent biomolecules accurately, we must move towards modeling and predicting structural ensembles. Information about structural ensembles exists within experimental data from X-ray crystallography and cryo-electron microscopy. Although new tools are available to detect conformational and compositional heterogeneity within these ensembles, the legacy PDB data structure does not robustly encapsulate this complexity. We propose modifications to the macromolecular crystallographic information file (mmCIF) to improve the representation and interrelation of conformational and compositional heterogeneity. These modifications will enable the capture of macromolecular ensembles in a human and machine-interpretable way, potentially catalyzing breakthroughs for ensemble–function predictions, analogous to the achievements of AlphaFold with single-structure prediction.

A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.

X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump–probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light–oxygen–voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump–probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thio­ether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.

This study examines various methods for modelling the electron density and, thus, the electrostatic potential of an organometallic complex for use in crystal structure refinement against 3D electron diffraction (ED) data. It focuses on modelling the scattering factors of iron(III), considering the electron density distribution specific for coordination with organic linkers. We refined the structural model of the metal–organic complex, iron(III) acetyl­acetonate (FeAcAc), using both the independent atom model (IAM) and the transferable aspherical atom model (TAAM). TAAM refinement initially employed multipolar parameters from the MATTS databank for acetyl­acetonate, while iron was modelled with a spherical and neutral approach (TAAM ligand). Later, custom-made TAAM scattering factors for Fe—O coordination were derived from DFT calculations [TAAM-ligand-Fe(III)]. Our findings show that, in this compound, the TAAM scattering factor corresponding to Fe3+ has a lower scattering amplitude than the Fe3+ charged scattering factor described by IAM. When using scattering factors corresponding to the oxidation state of iron, IAM inaccurately represents electrostatic potential maps and overestimates the scattering potential of the iron. In addition, TAAM significantly improved the fitting of the model to the data, shown by improved R1 values, goodness-of-fit (GooF) and reduced noise in the Fourier difference map (based on the residual distribution analysis). For 3D ED, R1 values improved from 19.36% (IAM) to 17.44% (TAAM-ligand) and 17.49% (TAAM-ligand-Fe3+), and for single-crystal X-ray diffraction (SCXRD) from 3.82 to 2.03% and 1.98%, respectively. For 3D ED, the most significant R1 reductions occurred in the low-resolution region (8.65–2.00 Å), dropping from 20.19% (IAM) to 14.67% and 14.89% for TAAM-ligand and TAAM-ligand-Fe(III), respectively, with less improvement in high-resolution ranges (2.00–0.85 Å). This indicates that the major enhancements are due to better scattering modelling in low-resolution zones. Furthermore, when using TAAM instead of IAM, there was a noticeable improvement in the shape of the thermal ellipsoids, which more closely resembled those of an SCXRD-refined model. This study demonstrates the applicability of more sophisticated scattering factors to improve the refinement of metal–organic complexes against 3D ED data, suggesting the need for more accurate modelling methods and highlighting the potential of TAAM in examining the charge distribution of large molecular structures using 3D ED.

A pressure-induced triclinic-to-monoclinic phase transition has been caught `in the act' over a wider series of high-pressure synchrotron diffraction experiments conducted on a large, photoluminescent organo-gold(I) compound. Here, we describe the mechanism of this single-crystal-to-single-crystal phase transition, the onset of which occurs at ∼0.6 GPa, and we report a high-quality structure of the new monoclinic phase, refined using aspherical atomic scattering factors. Our case illustrates how conducting a fast series of diffraction experiments, enabled by modern equipment at synchrotron facilities, can lead to overestimation of the actual pressure of a phase transition due to slow transformation kinetics.

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL. Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydro­phobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.