Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.

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.

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

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

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

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

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.

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

Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference-free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open-source and distributed within RELION-3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.

Noncrystallographic symmetry (NCS) averaging following molecular-replacement phasing is generally the major technique used to solve a structure with several molecules in one asymmetric unit, such as a spherical icosahedral viral particle. As an alternative method to NCS averaging, a new approach to optimize or to refine the electron density directly under NCS constraints is proposed. This method has the same effect as the conventional NCS-averaging method but does not include the process of Fourier synthesis to generate the electron density from amplitudes and the corresponding phases. It has great merit for the solution of structures with limited data that are either twinned or incomplete at low resolution. This method was applied to the case of the T = 1 shell-domain subviral particle of Penaeus vannamei nodavirus with data affected by twinning using the REFMAC5 refinement software.

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

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

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

The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.

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

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

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

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

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

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

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

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

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

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

Confidence maps provide complementary information for interpreting cryo-EM densities as they indicate statistical significance with respect to background noise. They can be thresholded by specifying the expected false-discovery rate (FDR), and the displayed volume shows the parts of the map that have the corresponding level of significance. Here, the basic statistical concepts of confidence maps are reviewed and practical guidance is provided for their interpretation and usage inside the CCP-EM suite. Limitations of the approach are discussed and extensions towards other error criteria such as the family-wise error rate are presented. The observed map features can be rendered at a common isosurface threshold, which is particularly beneficial for the interpretation of weak and noisy densities. In the current article, a practical guide is provided to the recommended usage of confidence maps.

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.

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

The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallo­graphy has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid-state nuclear magnetic resonance and two-dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular-dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.

Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.

The photovoltaic perovskite, methyl­ammonium lead triiodide [CH3NH3PbI3 (MAPbI3)], is one of the most efficient materials for solar energy conversion. Various kinds of chemical and physical modifications have been applied to MAPbI3 towards better understanding of the relation between composition, structure, electronic properties and energy conversion efficiency of this material. Pressure is a particularly useful tool, as it can substantially reduce the interatomic spacing in this relatively soft material and cause significant modifications to the electronic structure. Application of high pressure induces changes in the crystal symmetry up to a threshold level above which it leads to amorphization. Here, a detailed structural study of MAPbI3 at high hydro­static pressures using Ne and Ar as pressure transmitting media is reported. Single-crystal X-ray diffraction experiments with synchrotron radiation at room temperature in the 0–20 GPa pressure range show that atoms of both gaseous media, Ne and Ar, are gradually incorporated into MAPbI3, thus leading to marked structural changes of the material. Specifically, Ne stabilizes the high-pressure phase of NexMAPbI3 and prevents amorphization up to 20 GPa. After releasing the pressure, the crystal has the composition of Ne0.97MAPbI3, which remains stable under ambient conditions. In contrast, above 2.4 GPa, Ar accelerates an irreversible amorphization. The distinct impacts of Ne and Ar are attributed to differences in their chemical reactivity under pressure inside the restricted space between the PbI6 octahedra.

In this paper, the conformational polymorphism of a chlorinated diketo­pyrrolo­pyrrole (DPP) dye having flexible substituents in a non-hydrogen-bonding system is reported. The propyl-substituted DPP derivative (PR3C) has three polymorphic forms, each showing a different colour (red, orange and yellow). All polymorphs could be obtained concomitantly under various crystallization conditions. The results of the crystal structure analysis indicate that PR3C adopts different conformations in each polymorph. The packing effect caused by the difference in the arrangement of neighbouring molecules was found to play an important role in the occurrence of the observed polymorphism. The thermodynamic stability relationship between the three polymorphs was identified by thermal analysis and indicates that the yellow polymorph is the thermally stable form. The results indicate that the yellow form and orange form are enantiotropically related, and the other polymorph is monotropically related to the others.

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.

Multi-slice simulations of electron diffraction by three-dimensional protein crystals have indicated that structure solution would be severely impeded by dynamical diffraction, especially when crystals are more than a few unit cells thick. In practice, however, dynamical diffraction turned out to be less of a problem than anticipated on the basis of these simulations. Here it is shown that two scattering phenomena, which are usually omitted from multi-slice simulations, reduce the dynamical effect: solvent scattering reduces the phase differences within the exit beam and inelastic scattering followed by elastic scattering results in diffusion of dynamical scattering out of Bragg peaks. Thus, these independent phenomena provide potential reasons for the apparent discrepancy between theory and practice in protein electron crystallography.

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

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

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

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

This paper discusses the full structural solution of the hybrid perovskite formamidinium lead tribromide (FAPbBr3) and its temperature-dependent phase transitions in the range from 3 K to 300 K using neutron powder diffraction and synchrotron X-ray diffraction. Special emphasis is put on the influence of deuteration on formamidinium, its position in the unit cell and disordering in comparison to fully hydrogenated FAPbBr3. The temperature-dependent measurements show that deuteration critically influences the crystal structures, i.e. results in partially-ordered temperature-dependent structural modifications in which two symmetry-independent molecule positions with additional dislocation of the molecular centre atom and molecular angle inclinations are present.

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.

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

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.

The design and first results of a large-solid-angle X-ray emission spectrometer that is optimized for energies between 1.5 keV and 5.5 keV are presented. The spectrometer is based on an array of 11 cylindrically bent Johansson crystal analyzers arranged in a non-dispersive Rowland circle geometry. The smallest achievable energy bandwidth is smaller than the core hole lifetime broadening of the absorption edges in this energy range. Energy scanning is achieved using an innovative design, maintaining the Rowland circle conditions for all crystals with only four motor motions. The entire spectrometer is encased in a high-vacuum chamber that allocates a liquid helium cryostat and provides sufficient space for in situ cells and operando catalysis reactors.