Among different types of polymorphism, disappearing polymorphism deals with the metastable kinetic form which can not be reproduced after its first isolation. In the world of coordination polymers (CPs) and metal–organic frameworks (MOFs), despite the fact that many types of supramolecular isomerism exist, we are unaware of disappearing supramolecular isomerism akin to disappearing polymorphism. This work reports a MOF with dia topology that could not be reproduced, but subsequent synthesis yielded another supramolecular isomer, a double-pillared-layer MOF. When perylene was added in the same reaction, the disappeared dia MOF reappeared with perylene as a guest in the channels. Interestingly, the photoluminescence of the dia MOF with a perylene guest is dominated by the emission of the guest molecule. The influence of guest molecules on the stabilization of the supramolecular isomers of a MOF opens up a strategy to access MOFs with different structures.

Characterizing and controlling the uniformity of nanoparticles is crucial for their application in science and technology because crystalline defects in the nanoparticles strongly affect their unique properties. Recently, ultra-short and ultra-bright X-ray pulses provided by X-ray free-electron lasers (XFELs) opened up the possibility of structure determination of nanometre-scale matter with Å spatial resolution. However, it is often difficult to reconstruct the 3D structural information from single-shot X-ray diffraction patterns owing to the random orientation of the particles. This report proposes an analysis approach for characterizing defects in nanoparticles using wide-angle X-ray scattering (WAXS) data from free-flying single nanoparticles. The analysis method is based on the concept of correlated X-ray scattering, in which correlations of scattered X-ray are used to recover detailed structural information. WAXS experiments of xenon nanoparticles, or clusters, were conducted at an XFEL facility in Japan by using the SPring-8 Ångstrom compact free-electron laser (SACLA). Bragg spots in the recorded single-shot X-ray diffraction patterns showed clear angular correlations, which offered significant structural information on the nanoparticles. The experimental angular correlations were reproduced by numerical simulation in which kinematical theory of diffraction was combined with geometric calculations. We also explain the diffuse scattering intensity as being due to the stacking faults in the xenon clusters.

X-ray diffraction studies of crystals under pressure and quantitative experimental charge density analysis are among the most demanding types of crystallographic research. A successful feasibility study of the electron density in the mineral grossular under 1 GPa pressure conducted at the CRISTAL beamline at the SOLEIL synchrotron is presented in this work. A single crystal was placed in a diamond anvil cell, but owing to its special design (wide opening angle), short synchrotron wavelength and the high symmetry of the crystal, data with high completeness and high resolution were collected. This allowed refinement of a full multipole model of experimental electron distribution. Results are consistent with the benchmark measurement conducted without a diamond-anvil cell and also with the literature describing investigations of similar structures. Results of theoretical calculations of electron density distribution on the basis of dynamic structure factors mimic experimental findings very well. Such studies allow for laboratory simulations of processes which take place in the Earth's mantle.

Herein the nucleation pathway of conformational polymorphs was revealed by studying the relationships and distinctions among a series of α,ω-alkanedicarboxylic acids [HOOC–(CH2)n−2–COOH, named DAn, where n = 5, 7, 9, 11, 13, 15] in the solid state and in solution. Their polymorphic outcomes, with the exception of DA5, show solvent dependence: form I with conformation I crystallizes from solvents with hydrogen-bond donating (HBD) ability, whereas form II with conformation II crystallizes preferentially from solvents with no HBD ability. In contrast, form II of DA5 does not crystallize in any of the solvents used. Quantum mechanical computation showed that there is no direct conformational link between the solvents and the resultant polymorphic outcomes. Surprisingly, solute aggregates were found in no-HBD solvents by Fourier transform infrared spectroscopy, and only monomers could be detected in HBD solvents, suggesting stronger solvation. Furthermore, it was found that all six compounds including DA5 followed the same pattern in solution. Moreover, crystal-packing efficiency calculations and stability tests stated that dimorphs of DA5 bear a greater stability difference than others. These suggest that the rearrangement from conformation II to I could not be limited by hard desolvation in HBD solvents, where form I was also obtained. In other systems, metastable II was produced in the same solvents, probably as a result of the rearrangement being limited by hard desolvation. In this work, a comparative study uncovers the proposed nucleation pathway: difficulty in desolvation has a remarkable effect on the result of rearrangement and nucleation outcome.

Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of ∼3 Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (∼220 kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.

This study made use of a recently developed combination of advanced methods to reveal the atomic structure of a disordered nanocrystalline zeolite using exit wave reconstruction, automated diffraction tomography, disorder modelling and diffraction pattern simulation. By applying these methods, it was possible to determine the so far unknown structures of the hydrous layer silicate RUB-6 and the related zeolite-like material RUB-5. The structures of RUB-5 and RUB-6 contain the same dense layer-like building units (LLBUs). In the case of RUB-5, these building units are interconnected via additional SiO4/2 tetrahedra, giving rise to a framework structure with a 2D pore system consisting of intersecting 8-ring channels. In contrast, RUB-6 contains these LLBUs as separate silicate layers terminated by silanol/sil­oxy groups. Both RUB-6 and RUB-5 show stacking disorder with intergrowths of different polymorphs. The unique structure of RUB-6, together with the possibility for an interlayer expansion reaction to form RUB-5, make it a promising candidate for interlayer expansion with various metal sources to include catalytically active reaction centres.

Nucleation of crystals from solution is fundamental to many natural and industrial processes. In this work, the molecular mechanism of conformational polymorphism nucleation and the links between the molecular conformation in solutions and in crystals were investigated in detail by using 5-nitro­furazone as the model compound. Different polymorphs were prepared, and the conformations in solutions obtained by dissolving different polymorphs were analysed and compared. The solutions of 5-nitro­furazone were proven to contain multiple conformers through quantum chemical computation, Raman spectra analysis, 2D nuclear Overhauser effect spectroscopy spectra analysis and molecular dynamics simulation. The conformational evolution and desolvation path was illustrated according to the 1H NMR spectra of solutions with different concentrations. Finally, based on all the above analysis, the molecular conformational evolution path during nucleation of 5-nitro­furazone was illustrated. The results presented in this work shed a new light on the molecular mechanism of conformational polymorphism nucleation in solution.

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.

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

Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.

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

Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal unit-cell packing reveals different crystallographic and noncrystallographic dimers of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

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

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

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

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

Molecular replacement (MR) is the predominant route to solution of the phase problem in macromolecular crystallography. Where the lack of a suitable homologue precludes conventional MR, one option is to predict the target structure using bioinformatics. Such modelling, in the absence of homologous templates, is called ab initio or de novo modelling. Recently, the accuracy of such models has improved significantly as a result of the availability, in many cases, of residue-contact predictions derived from evolutionary covariance analysis. Covariance-assisted ab initio models representing structurally uncharacterized Pfam families are now available on a large scale in databases, potentially representing a valuable and easily accessible supplement to the PDB as a source of search models. Here, the unconventional MR pipeline AMPLE is employed to explore the value of structure predictions in the GREMLIN and PconsFam databases. It was tested whether these deposited predictions, processed in various ways, could solve the structures of PDB entries that were subsequently deposited. The results were encouraging: nine of 27 GREMLIN cases were solved, covering target lengths of 109–355 residues and a resolution range of 1.4–2.9 Å, and with target–model shared sequence identity as low as 20%. The cluster-and-truncate approach in AMPLE proved to be essential for most successes. For the overall lower quality structure predictions in the PconsFam database, remodelling with Rosetta within the AMPLE pipeline proved to be the best approach, generating ensemble search models from single-structure deposits. Finally, it is shown that the AMPLE-obtained search models deriving from GREMLIN deposits are of sufficiently high quality to be selected by the sequence-independent MR pipeline SIMBAD. Overall, the results help to point the way towards the optimal use of the expanding databases of ab initio structure predictions.

Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.

Although the success of molecular-replacement techniques requires the solution of a six-dimensional problem, this is often subdivided into two three-dimensional problems. REMO09 is one of the programs which have adopted this approach. It has been revisited in the light of a new probabilistic approach which is able to directly derive conditional distribution functions without passing through a previous calculation of the joint probability distributions. The conditional distributions take into account various types of prior information: in the rotation step the prior information may concern a non-oriented model molecule alone or together with one or more located model molecules. The formulae thus obtained are used to derive figures of merit for recognizing the correct orientation in the rotation step and the correct location in the translation step. The phases obtained by this new version of REMO09 are used as a starting point for a pipeline which in its first step extends and refines the molecular-replacement phases, and in its second step creates the final electron-density map which is automatically interpreted by CAB, an automatic model-building program for proteins and DNA/RNA structures.

Good prior estimates of the effective root-mean-square deviation (r.m.s.d.) between the atomic coordinates of the model and the target optimize the signal in molecular replacement, thereby increasing the success rate in difficult cases. Previous studies using protein structures solved by X-ray crystallography as models showed that optimal error estimates (refined after structure solution) were correlated with the sequence identity between the model and target, and with the number of residues in the model. Here, this work has been extended to find additional correlations between parameters of the model and the target and hence improved prior estimates of the coordinate error. Using a graph database, a curated set of 6030 molecular-replacement calculations using models that had been solved by X-ray crystallography was analysed to consider about 120 model and target parameters. Improved estimates were achieved by replacing the sequence identity with the Gonnet score for sequence similarity, as well as by considering the resolution of the target structure and the MolProbity score of the model. This approach was extended by analysing 12 610 additional molecular-replacement calculations where the model was determined by NMR. The median r.m.s.d. between pairs of models in an ensemble was found to be correlated with the estimated r.m.s.d. to the target. For models solved by NMR, the overall coordinate error estimates were larger than for structures determined by X-ray crystallography, and were more highly correlated with the number of residues.

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

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

Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.

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

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

Fragment-based molecular replacement exploits the use of very accurate yet incomplete search models. In the case of the ARCIMBOLDO programs, consistent phase sets produced from the placement and refinement of fragments with Phaser can be combined in order to increase their signal before proceeding to the step of density modification and autotracing with SHELXE. The program ALIXE compares multiple phase sets, evaluating mean phase differences to determine their common origin, and subsequently produces sets of combined phases that group consistent solutions. In this work, its use on different scenarios of very partial molecular-replacement solutions and its performance after the development of a much-optimized set of algorithms are described. The program is available both standalone and integrated within the ARCIMBOLDO programs. ALIXE has been analysed to identify its rate-limiting steps while exploring the best parameterization to improve its performance and make this software efficient enough to work on modest hardware. The algorithm has been parallelized and redesigned to meet the typical landscape of solutions. Analysis of pairwise correlation between the phase sets has also been explored to test whether this would provide additional insight. ALIXE can be used to exhaustively analyse all partial solutions produced or to complement those already selected for expansion, and also to reduce the number of redundant solutions, which is particularly relevant to the case of coiled coils, or to combine partial solutions from different programs. In each case parallelization and optimization to provide speedup makes its use amenable to typical hardware found in crystallography. ARCIMBOLDO_BORGES and ARCIMBOLDO_SHREDDER now call on ALIXE by default.

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

The 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.

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.

The phase transition of E-vanillyl oxime {1-[(E)-(hydroxyimino)methyl]-4-hydroxy-3-methoxybenzene, C8H9NO3} has been analysed by single-crystal and powder X-ray diffraction. The high-temperature (HT) phase (P21/a, Z' = 1) transforms into the low-temperature (LT) phase (threefold superstructure, Poverline{1}, Z' = 6) at ca 190 K. The point operations lost on cooling, {m[010], 2[010]}, are retained as twin operations and constitute the twin law. The screw rotations and glide reflections are retained in the LT phase as partial operations acting on a subset of Euclidean space {b E}^3. The full symmetry of the LT phase, including partial operations, is described by a disconnected space groupoid which is built of three connected components.

The structure of sodium saccharinate 1.875-hydrate is presented in three- and (3+1)-dimensional space. The present model is more accurate than previously published superstructures, due to an excellent data set collected up to a high resolution of 0.89 Å−1. The present study confirms the unusual complexity of the structure comprising a very large primitive unit cell with Z' = 16. A much smaller degree of correlated disorder of parts of the unit cell is found than is present in the previously published models. As a result of pseudo-symmetry, the structure can be described in a higher-dimensional space. The X-ray diffraction data clearly indicate a (3+1)-dimensional periodic structure with stronger main reflections and weaker superstructure reflections. Furthermore, the structure is established as being commensurate. The structure description in superspace results in a four times smaller unit cell with an additional base centring of the lattice, resulting in an eightfold substructure (Z' = 2) of the 3D superstructure. Therefore, such a superspace approach is desirable to work out this high-Z' structure. The displacement and occupational modulation of the saccharinate anions have been studied, as well as their conformational variation along the fourth dimension.

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.

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

ID30A-3 (or MASSIF-3) is a mini-focus (beam size 18 µm × 14 µm) highly intense (2.0 × 1013 photons s−1), fixed-energy (12.81 keV) beamline for macromolecular crystallography (MX) experiments at the European Synchrotron Radiation Facility (ESRF). MASSIF-3 is one of two fixed-energy beamlines sited on the first branch of the canted undulator setup on the ESRF ID30 port and is equipped with a MD2 micro-diffractometer, a Flex HCD sample changer, and an Eiger X 4M fast hybrid photon-counting detector. MASSIF-3 is recommended for collecting diffraction data from single small crystals (≤15 µm in one dimension) or for experiments using serial methods. The end-station has been in full user operation since December 2014, and here its current characteristics and capabilities are described.

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.

A Thomson scattering X-ray source can provide quasi-monochromatic, continuously energy-tunable, polarization-controllable and high-brightness X-rays, which makes it an excellent tool for X-ray fluorescence computed tomography (XFCT). In this paper, we examined the suppression of Compton scattering background in XFCT using the linearly polarized X-rays and the implementation feasibility of linearly polarized XFCT based on this type of light source, concerning the influence of phantom attenuation and the sampling strategy, its advantage over K-edge subtraction computed tomography (CT), the imaging time, and the potential pulse pile-up effect by Monte Carlo simulations. A fan beam and pinhole collimator geometry were adopted in the simulation and the phantom was a polymethyl methacrylate cylinder inside which were gadolinium (Gd)-loaded water solutions with Gd concentrations ranging from 0.2 to 4.0 wt%. Compared with the case of vertical polarization, Compton scattering was suppressed by about 1.6 times using horizontal polarization. An accurate image of the Gd-containing phantom was successfully reconstructed with both spatial and quantitative identification, and good linearity between the reconstructed value and the Gd concentration was verified. When the attenuation effect cannot be neglected, one full cycle (360°) sampling and the attenuation correction became necessary. Compared with the results of K-edge subtraction CT, the contrast-to-noise ratio values of XFCT were improved by 2.03 and 1.04 times at low Gd concentrations of 0.2 and 0.5 wt%, respectively. When the flux of a Thomson scattering light source reaches 1013 photons s−1, it is possible to finish the data acquisition of XFCT at the minute or second level without introducing pulse pile-up effects.

The laser annealing process for AuNi nanoparticles has been visualized using coherent X-ray diffraction imaging (CXDI). AuNi bimetallic alloy nanoparticles, originally phase separated due to the miscibility gap, transform to metastable mixed alloy particles with rounded surface as they are irradiated by laser pulses. A three-dimensional CXDI shows that the internal part of the AuNi particles is in the mixed phase with preferred compositions at ∼29 at% of Au and ∼90 at% of Au.

Active cathode particles are fundamental architectural units for the composite electrode of Li-ion batteries. The microstructure of the particles has a profound impact on their behavior and, consequently, on the cell-level electrochemical performance. LiCoO2 (LCO, a dominant cathode material) is often in the form of well-shaped particles, a few micrometres in size, with good crystallinity. In contrast to secondary particles (an agglomeration of many fine primary grains), which are the other common form of battery particles populated with structural and chemical defects, it is often anticipated that good particle crystallinity leads to superior mechanical robustness and suppressed charge heterogeneity. Yet, sub-particle level charge inhomogeneity in LCO particles has been widely reported in the literature, posing a frontier challenge in this field. Herein, this topic is revisited and it is demonstrated that X-ray absorption spectra on single-crystalline particles with highly anisotropic lattice structures are sensitive to the polarization configuration of the incident X-rays, causing some degree of ambiguity in analyzing the local spectroscopic fingerprint. To tackle this issue, a methodology is developed that extracts the white-line peak energy in the X-ray absorption near-edge structure spectra as a key data attribute for representing the local state of charge in the LCO crystal. This method demonstrates significantly improved accuracy and reveals the mesoscale chemical complexity in LCO particles with better fidelity. In addition to the implications on the importance of particle engineering for LCO cathodes, the method developed herein also has significant impact on spectro-microscopic studies of single-crystalline materials at synchrotron facilities, which is broadly applicable to a wide range of scientific disciplines well beyond battery 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.

Synchrotron X-ray diffraction (XRD) measured on the XMaS beamline at the ESRF was used to characterize the alloy composition and crystalline surface corrosion of three copper alloy Tudor artefacts recovered from the undersea wreck of King Henry VIII's warship the Mary Rose. The XRD method adopted has a dynamic range ∼1:105 and allows reflections <0.002% of the height of major reflections in the pattern to be discerned above the background without smoothing. Laboratory XRD, scanning electron microscopy–energy dispersive spectroscopy, synchrotron X-ray fluorescence and X-ray excited optical luminescence–X-ray near-edge absorption structure were used as supporting techniques, and the combination revealed structural and compositional features of importance to both archaeology and conservation. The artefacts were brass links believed to be fragments of chainmail and were excavated from the seabed during 1981 and 1982. Their condition reflects very different treatment just after recovery, viz. complete cleaning and conservation, chemical corrosion inhibition and chloride removal only, and distilled water soaking only (to remove the chlorides). The brass composition has been determined for all three at least in the top 7 µm or so as Cu(73%)Zn(27%) from the lattice constant. Measurement of the peak widths showed significant differences in the crystallite size and microstrain between the three samples. All of the links are found to be almost chloride-free with the main corrosion products being spertiniite, sphalerite, zincite, covellite and chalcocite. The balance of corrosion products between the links reflects the conservation treatment applied to one and points to different corrosion environments for the other two.

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

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.

This article describes a high-efficiency experimental configuration for a self-referenced lattice comparator with a `brush beam' of synchrotron radiation from a bending magnet and two linear position-sensitive photon-counting-type X-ray detectors. The efficiency is more than ten times greater compared with the `pencil-beam' configuration and a pair of zero-dimensional detectors. A solution for correcting the systematic deviation of d-spacing measurements caused by the horizontal non-uniformity of the brush beam is provided. Also, the use of photon-counting-type one-dimensional detectors not only improves the spatial resolution of the measurements remarkably but can also adjust the sample's attitude angles easily.