MICAL is an oxidoreductase that participates in cytoskeleton reorganization via actin disassembly in the presence of NADPH. Although three MICALs (MICAL1, MICAL2 and MICAL3) have been identified in mammals, only the structure of mouse MICAL1 has been reported. Here, the first crystal structure of human MICAL3, which contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, is reported. MICAL3 has an FAD/NADP-binding Rossmann-fold domain for mono­oxygenase activity like MICAL1. The FMO and CH domains of both MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. Based on kinetic analyses, the catalytic efficiency of MICAL3 dramatically increased on adding F-actin only when the CH domain was available. However, this did not occur when two residues, Glu213 and Arg530, were mutated in the FMO and CH domains, respectively. Overall, MICAL3 is structurally highly similar to MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity.

The preferred orientation growth characteristics and surface roughness of polycrystalline bis­muth (Bi) thin films fabricated on glass substrates using the molecular beam epitaxy method were investigated at temperatures ranging from 18 to 150°C. The crystallization and morphology were analyzed in detail and the polycrystalline metal film structure-zone model (SZM) was modified to fit the polycrystalline Bi thin film. The boundary temperature between Zone T and Zone II in the SZM shifted to higher temperatures with the increase in film thickness or the decrease of growth rate. Furthermore, the effect of the thickness and surface roughness on the transport properties was investigated, especially for Bi thin films in Zone II. A two-transport channels model was adopted to reveal the influence of the film thickness on the competition between the metallic surface states and the semiconducting bulk states, which is consistent with the results of Bi single-crystal films. Therefore, the polycrystalline Bi thin films are expected to replace the single-crystal films in the application of spintronic devices.

Using the typical WC–Co cemented carbide as an example, the interactions of dislocations within the ceramic matrix and the binder metal, as well as the possible cooperation and competition between the matrix and binder during deformation of the nanocrystalline cermets, were studied by molecular dynamics simulations. It was found that at the same level of strain, the dislocations in Co have more complex configurations in the cermet with higher Co content. With loading, the ratio between mobile and sessile dislocations in Co becomes stable earlier in the high-Co cermet. The strain threshold for the nucleation of dislocations in WC increases with Co content. At the later stage of deformation, the growth rate of WC dislocation density increases more rapidly in the cermet with lower Co content, which exhibits an opposite tendency compared with Co dislocation density. The relative contribution of Co and WC to the plasticity of the cermet varies in the deformation process. With a low Co content, the density of WC dislocations becomes higher than that of Co dislocations at larger strains, indicating that WC may contribute more than Co to the plasticity of the nanocrystalline cermet at the final deformation stage. The findings in the present work will be applicable to a large variety of ceramic–metal composite materials.

The first ab initio aspherical structure refinement against experimental X-ray structure factors for polypeptides and proteins using a fragmentation approach to break up the protein into residues and solvent, thereby speeding up quantum-crystallographic Hirshfeld atom refinement (HAR) calculations, is described. It it found that the geometric and atomic displacement parameters from the new fragHAR method are essentially unchanged from a HAR on the complete unfragmented system when tested on dipeptides, tripeptides and hexapeptides. The largest changes are for the parameters describing H atoms involved in hydrogen-bond interactions, but it is shown that these discrepancies can be removed by including the interacting fragments as a single larger fragment in the fragmentation scheme. Significant speed-ups are observed for the larger systems. Using this approach, it is possible to perform a highly parallelized HAR in reasonable times for large systems. The method has been implemented in the TONTO software.

The non-steroidal anti-inflammatory drugs mefenamic acid (MFA) and tolfenamic acid (TFA) have a close resemblance in their molecular scaffold, whereby a methyl group in MFA is substituted by a chloro group in TFA. The present study demonstrates the isomorphous nature of these compounds in a series of their multicomponent solids. Furthermore, the unique nature of MFA and TFA has been demonstrated while excavating their alternate solid forms in that, by varying the drug (MFA or TFA) to coformer [4-di­methyl­amino­pyridine (DMAP)] stoichiometric ratio, both drugs have produced three different types of multicomponent crystals, viz. salt (1:1; API to coformer ratio), salt hydrate (1:1:1) and cocrystal salt (2:1). Interestingly, as anticipated from the close similarity of TFA and MFA structures, these multicomponent solids have shown an isomorphous relation. A thorough characterization and structural investigation of the new multicomponent forms of MFA and TFA revealed their similarity in terms of space group and structural packing with isomorphic nature among the pairs. Herein, the experimental results are generalized in a broader perspective for predictably identifying any possible new forms of comparable compounds by mapping their crystal structure landscapes. The utility of such an approach is evident from the identification of polymorph VI of TFA from hetero-seeding with isomorphous MFA form I from acetone–methanol (1:1) solution. That aside, a pseudopolymorph of TFA with di­methyl­formamide (DMF) was obtained, which also has some structural similarity to that of the solvate MFA:DMF. These new isostructural pairs are discussed in the context of solid form screening using structural landscape similarity.

Co-crystallization is a phenomenon widely employed to enhance the physio-chemical and biological properties of active pharmaceutical ingredients (APIs). Exemestane, or 6-methyl­ideneandrosta-1,4-diene-3,17-dione, is an anabolic steroid used as an irreversible steroidal aromatase inhibitor, which is in clinical use to treat breast cancer. The present study deals with the synthesis of co-crystals of exemestane with thio­urea by liquid-assisted grinding. The purity and homogeneity of the exemestane–thio­urea (1:1) co-crystal were confirmed by single-crystal X-ray diffraction followed by thermal stability analysis on the basis of differential scanning calorimetry and thermogravimetric analysis. Detailed geometric analysis of the co-crystal demonstrated that a 1:1 co-crystal stoichiometry is sustained by N—H⋯O hydrogen bonding between the amine (NH2) groups of thio­urea and the carbonyl group of exemestane. The synthesized co-crystal exhibited potent urease inhibition activity in vitro (IC50 = 3.86 ± 0.31 µg ml−1) compared with the API (exemestane), which was found to be inactive, and the co-former (thio­urea) (IC50 = 21.0 ± 1.25 µg ml−1), which is also an established tested standard for urease inhibition assays in vitro. The promising results of the present study highlight the significance of co-crystallization as a crystal engineering tool to improve the efficacy of pharmaceutical ingredients. Furthermore, the role of various hydrogen bonds in the crystal stability is successfully analysed quantitatively using Hirshfeld surface analysis.

With the increasing trend of using microcrystals and intense microbeams at synchrotron X-ray beamlines, radiation damage becomes a more pressing problem. Theoretical calculations show that the photoelectrons that primarily cause damage can escape microcrystals. This effect would become more pronounced with decreasing crystal size as well as at higher energies. To prove this effect, data from cryocooled lysozyme crystals of dimensions 5 × 3 × 3 and 20 × 8 × 8 µm mounted on cryo-transmission electron microscopy (cryo-TEM) grids were collected at 13.5 and 20.1 keV using a PILATUS CdTe 2M detector, which has a similar quantum efficiency at both energies. Accurate absorbed doses were calculated through the direct measurement of individual crystal sizes using scanning electron microscopy after the experiment and characterization of the X-ray microbeam. The crystal lifetime was then quantified based on the D1/2 metric. In this first systematic study, a longer crystal lifetime for smaller crystals was observed and crystal lifetime increased at higher X-ray energies, supporting the theoretical predictions of photoelectron escape. The use of detector technologies specifically optimized for data collection at energies above 20 keV allows the theoretically predicted photoelectron escape to be quantified and exploited, guiding future beamline-design choices.

Single crystals of the m = 8 member of the low-dimensional monophosphate tungsten bronzes (PO2)4(WO3)2m family were grown by chemical vapour transport technique and the high crystalline quality obtained allowed a reinvestigation of the physical and structural properties. Resistivity measurements revealed three anomalies at TC1 = 258 K, TC2 = 245 K and TC3 = 140 K, never observed until now. Parallel X-ray diffraction investigations showed a specific signature associated with three structural transitions, i.e. the appearance of different sets of satellite reflections below TC1, TC2 and TC3. Several harmonics of intense satellite reflections were observed, reflecting the non-sinusoidal nature of the structural modulations and a strong electron–phonon coupling in the material. These transitions could be associated with the formation of three successive unconventional charge density wave states.

This paper recounts the first successful cryo-cooling of protein crystals that demonstrated the reduction in X-ray damage to macromolecular crystals. The project was suggested by David C. Phillips in 1965 at the Royal Institution of Great Britain and continued in 1967 at the Weizmann Institute of Science, where the first cryo-cooling experiments were performed on lysozyme crystals, and was completed in 1969 at Purdue University on lactate dehydrogenase crystals. A 1970 publication in Acta Crystallographica described the cryo-procedures, the use of cryo-protectants to prevent ice formation, the importance of fast, isotropic cryo-cooling and the collection of analytical data showing more than a tenfold decrease in radiation damage in cryo-cooled lactate dehydrogenase crystals. This was the first demonstration of any method that reduced radiation damage in protein crystals, which provided crystallographers with suitable means to employ synchrotron X-ray sources for protein-crystal analysis. Today, fifty years later, more than 90% of the crystal structures deposited in the Protein Data Bank have been cryo-cooled.

Transforming growth factor β-1 (TGFβ-1) is a secreted signalling protein that directs many cellular processes and is an attractive target for the treatment of several diseases. The primary endogenous activity regulatory mechanism for TGFβ-1 is sequestration by its pro-peptide, latency-associated peptide (LAP), which sterically prohibits receptor binding by caging TGFβ-1. As such, recombinant LAP is promising as a protein-based therapeutic for modulating TGFβ-1 activity; however, the mechanism of binding is incompletely understood. Comparison of the crystal structure of unbound LAP (solved here to 3.5 Å resolution) with that of the bound complex shows that LAP is in a more open and extended conformation when unbound to TGFβ-1. Analysis suggests a mechanism of binding TGFβ-1 through a large-scale conformational change that includes contraction of the inter-monomer interface and caging by the `straight-jacket' domain that may occur in partnership through a loop-to-helix transition in the core jelly-roll fold. This conformational change does not appear to include a repositioning of the integrin-binding motif as previously proposed. X-ray scattering-based modelling supports this mechanism and reveals possible orientations and ensembles in solution. Although native LAP is heavily glycosylated, solution scattering experiments show that the overall folding and flexibility of unbound LAP are not influenced by glycan modification. The combination of crystallography, solution scattering and biochemical experiments reported here provide insight into the mechanism of LAP sequestration of TGFβ-1 that is of fundamental importance for therapeutic development.

Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.

Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer's disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson's disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination.

Efficient infiltration of a mesoporous titania matrix with conducting organic polymers or small molecules is one key challenge to overcome for hybrid photovoltaic devices. A quantitative analysis of the backfilling efficiency with time-of-flight grazing incidence small-angle neutron scattering (ToF-GISANS) and scanning electron microscopy (SEM) measurements is presented. Differences in the morphology due to the backfilling of mesoporous titania thin films are compared for the macromolecule poly[4,8-bis­(5-(2-ethyl­hexyl)­thio­phen-2-yl)benzo[1,2-b;4,5-b']di­thio­phene-2,6-diyl-alt-(4-(2-ethyl­hexyl)-3-fluoro­thieno[3,4-b]thio­phene-)-2-carboxyl­ate-2-6-diyl)] (PTB7-Th) and the heavy-element containing small molecule 2-pinacol­boronate-3-phenyl­phen­anthro[9,10-b]telluro­phene (PhenTe-BPinPh). Hence, a 1.7 times higher backfilling efficiency of almost 70% is achieved for the small molecule PhenTe-BPinPh compared with the polymer PTB7-Th despite sharing the same volumetric mass density. The precise characterization of structural changes due to backfilling reveals that the volumetric density of backfilled materials plays a minor role in obtaining good backfilling efficiencies and interfaces with large surface contact.

Mitochondrial calcium uptake proteins 1 and 2 (MICU1 and MICU2) mediate mitochondrial Ca2+ influx via the mitochondrial calcium uniporter (MCU). Its molecular action for Ca2+ uptake is tightly controlled by the MICU1–MICU2 heterodimer, which comprises Ca2+ sensing proteins which act as gatekeepers at low [Ca2+] or facilitators at high [Ca2+]. However, the mechanism underlying the regulation of the Ca2+ gatekeeping threshold for mitochondrial Ca2+ uptake through the MCU by the MICU1–MICU2 heterodimer remains unclear. In this study, we determined the crystal structure of the apo form of the human MICU1–MICU2 heterodimer that functions as the MCU gatekeeper. MICU1 and MICU2 assemble in the face-to-face heterodimer with salt bridges and me­thio­nine knobs stabilizing the heterodimer in an apo state. Structural analysis suggests how the heterodimer sets a higher Ca2+ threshold than the MICU1 homodimer. The structure of the heterodimer in the apo state provides a framework for understanding the gatekeeping role of the MICU1–MICU2 heterodimer.

In chemistry, stereochemically active lone pairs are typically described as an important non-bonding effect, and recent interest has centred on understanding the derived effect of lone pair expression on physical properties such as thermal conductivity. To manipulate such properties, it is essential to understand the conditions that lead to lone pair expression and provide a quantitative chemical description of their identity to allow comparison between systems. Here, density functional theory calculations are used first to establish the presence of stereochemically active lone pairs on antimony in the archetypical chalcogenide MnSb2O4. The lone pairs are formed through a similar mechanism to those in binary post-transition metal compounds in an oxidation state of two less than their main group number [e.g. Pb(II) and Sb(III)], where the degree of orbital interaction (covalency) determines the expression of the lone pair. In MnSb2O4 the Sb lone pairs interact through a void space in the crystal structure, and their their mutual repulsion is minimized by introducing a deflection angle. This angle increases significantly with decreasing Sb—Sb distance introduced by simulating high pressure, thus showing the highly destabilizing nature of the lone pair interactions. Analysis of the chemical bonding in MnSb2O4 shows that it is dominated by polar covalent interactions with significant contributions both from charge accumulation in the bonding regions and from charge transfer. A database search of related ternary chalcogenide structures shows that, for structures with a lone pair (SbX3 units), the degree of lone pair expression is largely determined by whether the antimony–chalcogen units are connected or not, suggesting a cooperative effect. Isolated SbX3 units have larger X—Sb—X bond angles and therefore weaker lone pair expression than connected units. Since increased lone pair expression is equivalent to an increased orbital interaction (covalent bonding), which typically leads to increased heat conduction, this can explain the previously established correlation between larger bond angles and lower thermal conductivity. Thus, it appears that for these chalcogenides, lone pair expression and thermal conductivity may be related through the degree of covalency of the system.

Nanocrystallography has transformed our ability to interrogate the atomic structures of proteins, peptides, organic molecules and materials. By probing atomic level details in ordered sub-10 nm regions of nanocrystals, scanning nanobeam electron diffraction extends the reach of nanocrystallography and in principle obviates the need for diffraction from large portions of one or more crystals. Scanning nanobeam electron diffraction is now applied to determine atomic structures from digitally defined regions of beam-sensitive peptide nanocrystals. Using a direct electron detector, thousands of sparse diffraction patterns over multiple orientations of a given crystal are recorded. Each pattern is assigned to a specific location on a single nanocrystal with axial, lateral and angular coordinates. This approach yields a collection of patterns that represent a tilt series across an angular wedge of reciprocal space: a scanning nanobeam diffraction tomogram. Using this diffraction tomogram, intensities can be digitally extracted from any desired region of a scan in real or diffraction space, exclusive of all other scanned points. Intensities from multiple regions of a crystal or from multiple crystals can be merged to increase data completeness and mitigate missing wedges. It is demonstrated that merged intensities from digitally defined regions of two crystals of a segment from the OsPYL/RCAR5 protein produce fragment-based ab initio solutions that can be refined to atomic resolution, analogous to structures determined by selected-area electron diffraction. In allowing atomic structures to now be determined from digitally outlined regions of a nanocrystal, scanning nanobeam diffraction tomography breaks new ground in nanocrystallography.

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.

In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.

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

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

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

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

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

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.

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.

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.

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.

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

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.

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

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.

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

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

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

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

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

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

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

Small-angle scattering tensor tomography (SASTT) is a recently developed technique able to tomographically reconstruct the 3D reciprocal space from voxels within a bulk volume. SASTT extends the concept of X-ray computed tomography, which typically reconstructs scalar values, by reconstructing a tensor per voxel, which represents the local nanostructure 3D organization. In this study, the nanostructure orientation in a human trabecular-bone sample obtained by SASTT was validated by sectioning the sample and using 3D scanning small-angle X-ray scattering (3D sSAXS) to measure and analyze the orientation from single voxels within each thin section. Besides the presence of cutting artefacts from the slicing process, the nanostructure orientations obtained with the two independent methods were in good agreement, as quantified with the absolute value of the dot product calculated between the nanostructure main orientations obtained in each voxel. The average dot product per voxel over the full sample containing over 10 000 voxels was 0.84, and in six slices, in which fewer cutting artefacts were observed, the dot product increased to 0.91. In addition, SAXS tensor tomography not only yields orientation information but can also reconstruct the full 3D reciprocal-space map. It is shown that the measured anisotropic scattering for individual voxels was reproduced from the SASTT reconstruction in each voxel of the 3D sample. The scattering curves along different 3D directions are validated with data from single voxels, demonstrating SASTT's potential for a separate analysis of nanostructure orientation and structural information from the angle-dependent intensity distribution.

This study relates to the INFN project SYRMA-3D for in vivo phase-contrast breast computed tomography using the SYRMEP synchrotron radiation beamline at the ELETTRA facility in Trieste, Italy. This peculiar imaging technique uses a novel dosimetric approach with respect to the standard clinical procedure. In this study, optimization of the acquisition procedure was evaluated in terms of dose delivered to the breast. An offline dose monitoring method was also investigated using radiochromic film dosimetry. Various irradiation geometries have been investigated for scanning the prone patient's pendant breast, simulated by a 14 cm-diameter polymethylmethacrylate cylindrical phantom containing pieces of calibrated radiochromic film type XR-QA2. Films were inserted mid-plane in the phantom, as well as wrapped around its external surface, and irradiated at 38 keV, with an air kerma value that would produce an estimated mean glandular dose of 5 mGy for a 14 cm-diameter 50% glandular breast. Axial scans were performed over a full rotation or over 180°. The results point out that a scheme adopting a stepped rotation irradiation represents the best geometry to optimize the dose distribution to the breast. The feasibility of using a piece of calibrated radiochromic film wrapped around a suitable holder around the breast to monitor the scan dose offline is demonstrated.

A versatile, compact heater designed at National Synchrotron Light Source-II for in situ X-ray nano-imaging in a full-field transmission X-ray microscope is presented. Heater design for nano-imaging is challenging, combining tight spatial constraints with stringent design requirements for the temperature range and stability. Finite-element modeling and analytical calculations were used to determine the heater design parameters. Performance tests demonstrated reliable and stable performance, including maintaining the exterior casing close to room temperature while the heater is operating at above 1100°C, a homogenous heating zone and small temperature fluctuations. Two scientific experiments are presented to demonstrate the heater capabilities: (i) in situ 3D nano-tomography including a study of metal dealloying in a liquid molten salt extreme environment, and (ii) a study of pore formation in icosahedral quasicrystals. The progression of structural changes in both studies were clearly resolved in 3D, showing that the new heater enables powerful capabilities to directly visualize and quantify 3D morphological evolution of materials under real conditions by X-ray nano-imaging at elevated temperature during synthesis, fabrication and operation processes. This heater design concept can be applied to other applications where a precise, compact heater design is required.

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.

A scanning soft X-ray spectromicroscope was recently developed based mainly on the photon-in/photon-out measurement scheme for the investigation of local electronic structures on the surfaces and interfaces of advanced materials under conditions ranging from low vacuum to helium atmosphere. The apparatus was installed at the soft X-ray beamline (BL17SU) at SPring-8. The characteristic features of the apparatus are described in detail. The feasibility of this spectromicroscope was demonstrated using soft X-ray undulator radiation. Here, based on these results, element-specific two-dimensional mapping and micro-XAFS (X-ray absorption fine structure) measurements are reported, as well as the observation of magnetic domain structures from using a reference sample of permalloy micro-dot patterns fabricated on a silicon substrate, with modest spatial resolution (e.g. ∼500 nm). Then, the X-ray radiation dose for Nafion® near the fluorine K-edge is discussed as a typical example of material that is not radiation hardened against a focused X-ray beam, for near future experiments.

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

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

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

The Advanced Photon Source 1-ID beamline, operating in the 40–140 keV X-ray energy range, has successfully employed continuously tunable saw-tooth refractive lenses to routinely deliver beams focused in both one and two dimensions to experiments for over 15 years. The practical experience of implementing such lenses, made of silicon and aluminium, is presented, including their properties, control, alignment, and diagnostic methods, achieving ∼1 µm focusing (vertically). Ongoing development and prospects towards submicrometre focusing at these high energies are also mentioned.

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.