Free-electron lasers (FELs) based on superconducting accelerator technology and storage ring facilities operate with bunch repetition rates in the MHz range, and the need arises for bunch-by-bunch electron and photon diagnostics. For photon-pulse-resolved measurements of spectral distributions, fast one-dimensional profile monitors are required. The linear array detector KALYPSO (KArlsruhe Linear arraY detector for MHz-rePetition rate SpectrOscopy) has been developed for electron bunch or photon pulse synchronous read-out with frame rates of up to 2.7 MHz. At the FLASH facility at DESY, a current version of KALYPSO with 256 pixels has been installed at a grating spectrometer as online diagnostics to monitor the pulse-resolved spectra of the high-repetition-rate FEL pulses. Application-specific front-end electronics based on MicroTCA standard have been developed for data acquisition and processing. Continuous data read-out with low latency in the microsecond range enables the integration into fast feedback applications. In this paper, pulse-resolved FEL spectra recorded at 1.0 MHz repetition rate for various operation conditions at FLASH are presented, and the first application of an adaptive feedback for accelerator control based on photon beam diagnostics is demonstrated.

Being able to visualize biology at the molecular level is essential for our understanding of the world. A structural biology approach reveals the molecular basis of disease processes and can guide the design of new drugs as well as aid in the optimization of existing medicines. However, due to the lack of a synchrotron light source, adequate infrastructure, skilled persons and incentives for scientists in addition to limited financial support, the majority of countries across the African continent do not conduct structural biology research. Nevertheless, with technological advances such as robotic protein crystallization and remote data collection capabilities offered by many synchrotron light sources, X-ray crystallography is now potentially accessible to Africa-based scientists. This leap in technology led to the establishment in 2017 of BioStruct-Africa, a non-profit organization (Swedish corporate ID: 802509-6689) whose core aim is capacity building for African students and researchers in the field of structural biology with a focus on prevalent diseases in the African continent. The team is mainly composed of, but not limited to, a group of structural biologists from the African diaspora. The members of BioStruct-Africa have taken up the mantle to serve as a catalyst in order to facilitate the information and technology transfer to those with the greatest desire and need within Africa. BioStruct-Africa achieves this by organizing workshops onsite at our partner universities and institutions based in Africa, followed by post-hoc online mentoring of participants to ensure sustainable capacity building. The workshops provide a theoretical background on protein crystallography, hands-on practical experience in protein crystallization, crystal harvesting and cryo-cooling, live remote data collection on a synchrotron beamline, but most importantly the links to drive further collaboration through research. Capacity building for Africa-based researchers in structural biology is crucial to win the fight against the neglected tropical diseases, e.g. ascariasis, hookworm, trichuriasis, lymphatic filariasis, active trachoma, loiasis, yellow fever, leprosy, rabies, sleeping sickness, onchocerciasis, schistosomiasis, etc., that constitute significant health, social and economic burdens to the continent. BioStruct-Africa aims to build local and national expertise that will have direct benefits for healthcare within the continent.

X-ray double-crystal monochromators face a shift of the exit beam when the Bragg angle and thus the transmitted photon energy changes. This can be compensated for by moving one or both crystals accordingly. In the case of monolithic channel-cut crystals, which exhibit utmost stability, the shift of the monochromated beam is inevitable. Here we report performance tests of novel, asymmetrically cut, channel-cut crystals which reduce the beam movements by more than a factor of 20 relative to the symmetric case over the typical energy range of an EXAFS spectrum at the Cu K-edge. In addition, the presented formulas for the beam offset including the asymmetry angle directly indicate the importance of this value, which has been commonly neglected so far in the operation of double-crystal monochromators.

Calibration of area detectors from powder diffraction standards is widely used at synchrotron beamlines. From a single diffraction image, it is not possible to determine both the sample-to-detector distance and the wavelength, but, with images taken from multiple positions along the beam direction and where the relative displacement is known, the sample-to-detector distance and wavelength can both be determined with good precision. An example calibration using the GSAS-II software package is presented.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Multilayer monochromator devices are commonly used at (imaging) beamlines of synchrotron facilities to shape the X-ray beam to relatively small bandwidth and high intensity. However, stripe artefacts are often observed and can deteriorate the image quality. Although the intensity distribution of these artefacts has been described in the literature, their spectral distribution is currently unknown. To assess the spatio-spectral properties of the monochromated X-ray beam, the direct beam has been measured for the first time using a hyperspectral X-ray detector. The results show a large number of spectral features with different spatial distributions for a [Ru, B4C] strip monochromator, associated primarily with the higher-order harmonics of the undulator and monochromator. It is found that their relative contributions are sufficiently low to avoid an influence on the imaging data. The [V, B4C] strip suppresses these high-order harmonics even more than the former, yet at the cost of reduced efficiency.

Detection of heavy elements, such as metals, in macromolecular crystallography (MX) samples by X-ray fluorescence is a function traditionally covered at synchrotron MX beamlines by silicon drift detectors, which cannot be used at X-ray free-electron lasers because of the very short duration of the X-ray pulses. Here it is shown that the hybrid pixel charge-integrating detector JUNGFRAU can fulfill this function when operating in a low-flux regime. The feasibility of precise position determination of micrometre-sized metal marks is also demonstrated, to be used as fiducials for offline prelocation in serial crystallography experiments, based on the specific fluorescence signal measured with JUNGFRAU, both at the synchrotron and at SwissFEL. Finally, the measurement of elemental absorption edges at a synchrotron beamline using JUNGFRAU is also demonstrated.

Beamline 8A (BL 8A) is an undulator-based soft X-ray beamline at Pohang Accelerator Laboratory. This beamline is aimed at high-resolution ambient-pressure X-ray photoelectron spectroscopy (AP-XPS), soft X-ray absorption spectroscopy (soft-XAS) and scanning photoemission microscopy (SPEM) experiments. BL 8A has two branches, 8A1 SPEM and 8A2 AP-XPS, that share a plane undulator, the first mirror (M1) and the monochromator. The photon beam is switched between the two branches by changing the refocusing mirrors after the monochromator. The acceptance angle of M1 is kept glancing at 1.2°, and Pt is coated onto the mirrors to achieve high reflectance, which ensures a wide photon energy range (100–2000 eV) with high resolution at a photon flux of ∼1013 photons s−1. In this article, the main properties and performance of the beamline are reported, together with selected experiments performed on the new beamline and experimental system.

MAX IV is a fourth-generation, or diffraction-limited, synchrotron light source with a number of state-of-the-art beamlines. The performance of a beamline is, to a high degree, set by the energy resolution it can achieve, which in turn is governed to a large extent by the monochromator. During the design phase of a monochromator, the mechanical requirements must be fully understood and met with margin. During commissioning, the performance must be verified and optimized. In this paper, six soft X-ray monochromators at MAX IV beamlines (Bloch, Veritas, HIPPIE, SPECIES, FinEstBeAMS and SoftiMAX) are examined with a focus on their resolving power, energy range and the time required to change measurement range, as those parameters are dependent on each other. The monochromators have a modern commercial design, planned and developed in close collaboration with the vendors. This paper aims to present the current status of the commissioning at MAX IV with emphasis on elucidating the mechanical limitations on the performance of the monochromators. It contains analysis of the outcome and our approach to achieve fast and high-resolution monochromators.

Mammalian Munc18 proteins are essential for membrane fusion and human health. Here, we review the literature describing structural and in vitro data, and identify a possible explanation for the conflicting functional roles that have been reported.

Leading scientists set out resource challenge of meeting net zero emissions in the UK by 2050  The Natural History Museum

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The bond-valence method was performed on 51 crystal data sets from nitrogenase proteins, indicating the presence of molybdenum(III) in FeMo cofactors and vanadium(III) with more reduced iron complements in FeV cofactors.

The paper reports the structure of a Δ1-pyrroline-2-carboxylate reductase from the archaeon Thermococcus litoralis, a key enzyme involved in the second step of trans-4-Hydroxy-L-proline metabolism, conserved in archaea, bacteria and humans.

Reductive cyclization of 1,3,5-triphenyl- and 3-(2-meth­oxy­phen­yl)-1,5-di­phenyl­pentane-1,5-diones by zinc in acetic acid medium leads to the formation of 1,2,4-tri­phenyl­cyclo­pentane-1,2-diol [1,2,4-Ph3C5H5-1,2-(OH)2, C23H22O2, (I)] and 4-(2-meth­oxy­phen­yl)-1,2-di­phenyl­cyclo­pentane-1,2-diol [4-(2-MeOC6H4)-1,2-Ph2C5H5-1,2-(OH)2, C24H24O3, (II)]. Their single crystals have been obtained by crystallization from a THF/hexane solvent mixture. Diols (I) and (II) crystallize in ortho­rhom­bic (Pbca) and triclinic (Poverline{1}) space groups, respectively, at 150 K. Their asymmetric units comprise one [in the case of (I)] and three [in the case of (II)] crystallographically independent mol­ecules of the achiral (1R,2S,4r)-diol isomer. Each hydroxyl group is involved in one intra­molecular and one inter­molecular O—H⋯O hydrogen bond, forming one-dimensional chains. Compounds (I) and (II) have been used successfully as precatalyst activators for the ring-opening polymerization of ∊-caprolactone.

The title compound, C15H22N4O5S, was prepared via alkyl­ation of 3-(chloro­meth­yl)-5-(pentan-3-yl)-1,2,4-oxa­diazole in anhydrous dioxane in the presence of tri­ethyl­amine. The thia­diazine ring has an envelope conformation with the S atom displaced by 0.4883 (6) Å from the mean plane through the other five atoms. The planar 1,2,4-oxa­diazole ring is inclined to the mean plane of the thia­diazine ring by 77.45 (11)°. In the crystal, mol­ecules are linked by C—H⋯N hydrogen bonds, forming chains propagating along the b-axis direction. Hirshfeld surface analysis and two-dimensional fingerprint plots have been used to analyse the inter­molecular contacts present in the crystal. Mol­ecular docking studies were use to evaluate the title compound as a potential system that inter­acts effectively with the capsid of the Hepatitis B virus (HBV), supported by an experimental in vitro HBV replication model.

The title compound, C24H18FNO3, crystallizes in the monoclinic centrosymmetric space group P21/n and its mol­ecular conformation is stabilized via C—H⋯O intra­molecular inter­actions. The supra­molecular network mainly comprises C—H⋯O, C—H⋯F and C—H⋯π inter­actions, which contribute towards the formation of the crystal structure. The different inter­molecular inter­actions have been further analysed via Hirshfeld surface analysis and fingerprint plots.

Exact and approximate mathematical formulas of equatorial aberration for powder diffraction data collected with an Si strip X-ray detector in continuous-scan integration mode are presented. An approximate formula is applied to treat the experimental data measured with a commercial powder diffractometer.

Hybrid photon-counting detectors are widely established at third-generation synchrotron facilities and the specifications of the Pilatus3 X CdTe were quickly recognized as highly promising in charge-density investigations. This is mainly attributable to the detection efficiency in the high-energy X-ray regime, in combination with a dynamic range and noise level that should overcome the perpetual problem of detecting strong and weak data simultaneously. These benefits, however, come at the expense of a persistent problem for high diffracted beam flux, which is particularly problematic in single-crystal diffraction of materials with strong scattering power and sharp diffraction peaks. Here, an in-depth examination of data collected on an inorganic material, FeSb2, and an organic semiconductor, rubrene, revealed systematic differences in strong intensities for different incoming beam fluxes, and the implemented detector intensity corrections were found to be inadequate. Only significant beam attenuation for the collection of strong reflections was able to circumvent this systematic error. All data were collected on a bending-magnet beamline at a third-generation synchrotron radiation facility, so undulator and wiggler beamlines and fourth-generation synchrotrons will be even more prone to this error. On the other hand, the low background now allows for an accurate measurement of very weak intensities, and it is shown that it is possible to extract structure factors of exceptional quality using standard crystallographic software for data processing (SAINT-Plus, SADABS and SORTAV), although special attention has to be paid to the estimation of the background. This study resulted in electron-density models of substantially higher accuracy and precision compared with a previous investigation, thus for the first time fulfilling the promise of photon-counting detectors for very accurate structure factor measurements.

In fungi, the mitochondrial respiratory chain complexes (complexes I–IV) are responsible for oxidative phosphorylation, as in higher eukaryotes. Cryo-EM was used to identify a 200 kDa membrane protein from Neurospora crassa in lipid nanodiscs as cytochrome c oxidase (complex IV) and its structure was determined at 5.5 Å resolution. The map closely resembles the cryo-EM structure of complex IV from Saccharomyces cerevisiae. Its ten subunits are conserved in S. cerevisiae and Bos taurus, but other transmembrane subunits are missing. The different structure of the Cox5a subunit is typical for fungal complex IV and may affect the interaction with complex III in a respiratory supercomplex. Additional density was found between the matrix domains of the Cox4 and Cox5a subunits that appears to be specific to N. crassa.

Model building into experimental maps is a key element of structural biology, but can be both time consuming and error prone for low-resolution maps. Here we present Namdinator, an easy-to-use tool that enables the user to run a molecular dynamics flexible fitting simulation followed by real-space refinement in an automated manner through a pipeline system. Namdinator will modify an atomic model to fit within cryo-EM or crystallography density maps, and can be used advantageously for both the initial fitting of models, and for a geometrical optimization step to correct outliers, clashes and other model problems. We have benchmarked Namdinator against 39 deposited cryo-EM models and maps, and observe model improvements in 34 of these cases (87%). Clashes between atoms were reduced, and the model-to-map fit and overall model geometry were improved, in several cases substantially. We show that Namdinator is able to model large-scale conformational changes compared to the starting model. Namdinator is a fast and easy tool for structural model builders at all skill levels. Namdinator is available as a web service (https://namdinator.au.dk), or it can be run locally as a command-line tool.

This study explores the possibility of measuring the dynamics of proteins in solution using X-ray photon correlation spectroscopy (XPCS) at nearly diffraction-limited storage rings (DLSRs). We calculate the signal-to-noise ratio (SNR) of XPCS experiments from a concentrated lysozyme solution at the length scale of the hydrodynamic radius of the protein molecule. We take into account limitations given by the critical X-ray dose and find expressions for the SNR as a function of beam size, sample-to-detector distance and photon energy. Specifically, we show that the combined increase in coherent flux and coherence lengths at the DLSR PETRA IV yields an increase in SNR of more than one order of magnitude. The resulting SNR values indicate that XPCS experiments of biological macromolecules on nanometre length scales will become feasible with the advent of a new generation of synchrotron sources. Our findings provide valuable input for the design and construction of future XPCS beamlines at DLSRs.

Reliable sample delivery and efficient use of limited beam time have remained bottlenecks for serial crystallography (SX). Using a high-intensity polychromatic X-ray beam in combination with a newly developed charge-integrating JUNGFRAU detector, we have applied the method of fixed-target SX to collect data at a rate of 1 kHz at a synchrotron-radiation facility. According to our data analysis for the given experimental conditions, only about 3 000 diffraction patterns are required for a high-quality diffraction dataset. With indexing rates of up to 25%, recording of such a dataset takes less than 30 s.

Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) is a multidomain protein whose main function is to block apoptosis by caspase inhibition. XIAP is also involved in other signalling pathways, including NF-κB activation and copper homeostasis. XIAP is overexpressed in tumours, potentiating cell survival and resistance to chemotherapeutics, and has therefore become an important target for the treatment of malignancy. Despite the fact that the structure of each single domain is known, the conformation of the full-length protein has never been determined. Here, the first structural model of the full-length XIAP dimer, determined by an integrated approach using nuclear magnetic resonance, small-angle X-ray scattering and electron paramagnetic resonance data, is presented. It is shown that XIAP adopts a compact and relatively rigid conformation, implying that the spatial arrangement of its domains must be taken into account when studying the interactions with its physiological partners and in developing effective inhibitors.

Protein-engineering methods have been exploited to produce a surrogate system for the extracellular neurotransmitter-binding site of a heteromeric human ligand-gated ion channel, the glycine receptor. This approach circumvents two major issues: the inherent experimental difficulties in working with a membrane-bound ion channel and the complication that a heteromeric assembly is necessary to create a key, physiologically relevant binding site. Residues that form the orthosteric site in a highly stable ortholog, acetylcholine-binding protein, were selected for substitution. Recombinant proteins were prepared and characterized in stepwise fashion exploiting a range of biophysical techniques, including X-ray crystallography, married to the use of selected chemical probes. The decision making and development of the surrogate, which is termed a glycine-binding protein, are described, and comparisons are provided with wild-type and homomeric systems that establish features of molecular recognition in the binding site and the confidence that the system is suited for use in early-stage drug discovery targeting a heteromeric α/β glycine receptor.

Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy (cryo-EM) by facilitating the correction of beam-induced motion and radiation damage, and also by providing high-resolution image capture. A new-generation DED, the DE64, has been developed by Direct Electron that has good performance in both integrating and counting modes. The camera has been characterized in both modes in terms of image quality, throughput and resolution of cryo-EM reconstructions. The modulation transfer function, noise power spectrum and detective quantum efficiency (DQE) were determined for both modes, as well as the number of images per unit time. Although the DQE for counting mode was superior to that for integrating mode, the data-collection throughput for this mode was more than ten times slower. Since throughput and resolution are related in single-particle cryo-EM, data for apoferritin were collected and reconstructed using integrating mode, integrating mode in conjunction with a Volta phase plate (VPP) and counting mode. Only the counting-mode data resulted in a better than 3 Å resolution reconstruction with similar numbers of particles, and this increased performance could not be compensated for by the increased throughput of integrating mode or by the increased low-frequency contrast of integrating mode with the VPP. These data show that the superior image quality provided by counting mode is more important for high-resolution cryo-EM reconstructions than the superior throughput of integrating mode.

Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

High-resolution single-crystal X-ray measurements of the monoclinic polymorph of bicalutamide and the aspherical atom databank approach have served as a basis for a reconstruction of the charge density distribution of the drug and its androgen receptor (AR) and albumin complexes. The contributions of various types of intermolecular interactions to the total crystal energy or ligand:AR energy were estimated. The cyan and amide groups secured the ligand placement in the albumin (Lys-137) and the AR binding pocket (Leu-704, Asn-705, Arg-752), and also determined the packing of the small-molecule crystals. The total electrostatic interaction energy on average was −230 kJ mol−1, comparable with the electrostatic lattice energy of the monoclinic bicalutamide polymorph. This is the result of similar distributions of electropositive and electronegative regions on the experimental and theoretical molecular electrostatic potential maps despite differences in molecular conformations. In general, bicalutamide interacted with the studied proteins with similar electrostatic interaction energies and adjusted its conformation and electrostatic potential to fit the binding pocket in such a way as to enhance the interactions, e.g. hydrogen bonds and π⋯π stacking.

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.

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.

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.

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.

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.

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.

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.

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.

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.