Data reduction in protein serial crystallography.

Serial crystallography (SX) has become an established technique for protein structure determination, especially when dealing with small or radiation-sensitive crystals and investigating fast or irreversible protein dynamics. The advent of newly developed multi-megapixel X-ray area detectors, capable of capturing over 1000 images per second, has brought about substantial benefits. However, this advancement also entails a notable increase in the volume of collected data. Today, up to 2 PB of data per experiment could be easily obtained under efficient operating conditions. The combined costs associated with storing data from multiple experiments provide a compelling incentive to develop strategies that effectively reduce the amount of data stored on disk while maintaining the quality of scientific outcomes. Lossless data-compression methods are designed to preserve the information content of the data but often struggle to achieve a high compression ratio when applied to experimental data that contain noise. Conversely, lossy compression methods offer the potential to greatly reduce the data volume. Nonetheless, it is vital to thoroughly assess the impact of data quality and scientific outcomes when employing lossy compression, as it inherently involves discarding information. The evaluation of lossy compression effects on data requires proper data quality metrics. In our research, we assess various approaches for both lossless and lossy compression techniques applied to SX data, and equally importantly, we describe metrics suitable for evaluating SX data quality.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

XDSGUI: a graphical user interface for XDS, SHELX and ARCIMBOLDO.

XDSGUI is a lightweight graphical user interface (GUI) for the XDS, SHELX and ARCIMBOLDO program packages that serves both novice and experienced users in obtaining optimal processing and phasing results for X-ray, neutron and electron diffraction data. The design of the program enables data processing and phasing without command line usage, and supports advanced command flows in a simple user-modifiable and user-extensible way. The GUI supplies graphical information based on the tabular log output of the programs, which is more intuitive, comprehensible and efficient than text output can be.

Crystal diffraction prediction and partiality estimation using Gaussian basis functions.

The recent diversification of macromolecular crystallographic experiments including the use of pink beams, convergent electron diffraction and serial snapshot crystallography has shown the limitations of using the Laue equations for diffraction prediction. This article gives a computationally efficient way of calculating approximate crystal diffraction patterns given varying distributions of the incoming beam, crystal shapes and other potentially hidden parameters. This approach models each pixel of a diffraction pattern and improves data processing of integrated peak intensities by enabling the correction of partially recorded reflections. The fundamental idea is to express the distributions as weighted sums of Gaussian functions. The approach is demonstrated on serial femtosecond crystallography data sets, showing a significant decrease in the required number of patterns to refine a structure to a given error.

Automatic bad-pixel mask maker for X-ray pixel detectors with application to serial crystallography.

X-ray crystallography has witnessed a massive development over the past decade, driven by large increases in the intensity and brightness of X-ray sources and enabled by employing high-frame-rate X-ray detectors. The analysis of large data sets is done via automatic algorithms that are vulnerable to imperfections in the detector and noise inherent with the detection process. By improving the model of the behaviour of the detector, data can be analysed more reliably and data storage costs can be significantly reduced. One major requirement is a software mask that identifies defective pixels in diffraction frames. This paper introduces a methodology and program based upon concepts of machine learning, called robust mask maker (RMM), for the generation of bad-pixel masks for large-area X-ray pixel detectors based on modern robust statistics. It is proposed to discriminate normally behaving pixels from abnormal pixels by analysing routine measurements made with and without X-ray illumination. Analysis software typically uses a Bragg peak finder to detect Bragg peaks and an indexing method to detect crystal lattices among those peaks. Without proper masking of the bad pixels, peak finding methods often confuse the abnormal values of bad pixels in a pattern with true Bragg peaks and flag such patterns as useful regardless, leading to storage of enormous uninformative data sets. Also, it is computationally very expensive for indexing methods to search for crystal lattices among false peaks and the solution may be biased. This paper shows how RMM vastly improves peak finders and prevents them from labelling bad pixels as Bragg peaks, by demonstrating its effectiveness on several serial crystallography data sets.

A Lithium-Sulfur Battery Using Binder-Free Graphene-Coated Aluminum Current Collector.

Lithium-sulfur battery of practical interest requires thin-layer support to achieve acceptable volumetric energy density. However, the typical aluminum current collector of Li-ion battery cannot be efficiently used in the Li/S system due to the insulating nature of sulfur and a reaction mechanism involving electrodeposition of dissolved polysulfides. We study the electrochemical behavior of a Li/S battery using a carbon-coated Al current collector in which the low thickness, the high electronic conductivity, and, at the same time, the host ability for the reaction products are allowed by a binder-free few-layer graphene (FLG) substrate. The FLG enables a sulfur electrode having a thickness below 100 µm, fast kinetics, low impedance, and an initial capacity of 1000 mAh gS -1 with over 70% retention after 300 cycles. The Li/S cell using FLG shows volumetric and gravimetric energy densities of 300 Wh L-1 and 500 Wh kg-1, respectively, which are values well competing with commercially available Li-ion batteries.

Data reduction for serial crystallography using a robust peak finder.

A peak-finding algorithm for serial crystallography (SX) data analysis based on the principle of 'robust statistics' has been developed. Methods which are statistically robust are generally more insensitive to any departures from model assumptions and are particularly effective when analysing mixtures of probability distributions. For example, these methods enable the discretization of data into a group comprising inliers (i.e. the background noise) and another group comprising outliers (i.e. Bragg peaks). Our robust statistics algorithm has two key advantages, which are demonstrated through testing using multiple SX data sets. First, it is relatively insensitive to the exact value of the input parameters and hence requires minimal optimization. This is critical for the algorithm to be able to run unsupervised, allowing for automated selection or 'vetoing' of SX diffraction data. Secondly, the processing of individual diffraction patterns can be easily parallelized. This means that it can analyse data from multiple detector modules simultaneously, making it ideally suited to real-time data processing. These characteristics mean that the robust peak finder (RPF) algorithm will be particularly beneficial for the new class of MHz X-ray free-electron laser sources, which generate large amounts of data in a short period of time.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Synchronous RNA conformational changes trigger ordered phase transitions in crystals.

Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.

Macromolecular phasing using diffraction from multiple crystal forms.

A phasing algorithm for macromolecular crystallography is proposed that utilizes diffraction data from multiple crystal forms - crystals of the same molecule with different unit-cell packings (different unit-cell parameters or space-group symmetries). The approach is based on the method of iterated projections, starting with no initial phase information. The practicality of the method is demonstrated by simulation using known structures that exist in multiple crystal forms, assuming some information on the molecular envelope and positional relationships between the molecules in the different unit cells. With incorporation of new or existing methods for determination of these parameters, the approach has potential as a method for ab initio phasing.

pinkIndexer - a universal indexer for pink-beam X-ray and electron diffraction snapshots.

A crystallographic indexing algorithm, pinkIndexer, is presented for the analysis of snapshot diffraction patterns. It can be used in a variety of contexts including measurements made with a monochromatic radiation source, a polychromatic source or with radiation of very short wavelength. As such, the algorithm is particularly suited to automated data processing for two emerging measurement techniques for macromolecular structure determination: serial pink-beam X-ray crystallography and serial electron crystallography, which until now lacked reliable programs for analyzing many individual diffraction patterns from crystals of uncorrelated orientation. The algorithm requires approximate knowledge of the unit-cell parameters of the crystal, but not the wavelengths associated with each Bragg spot. The use of pinkIndexer is demonstrated by obtaining 1005 lattices from a published pink-beam serial crystallography data set that had previously yielded 140 indexed lattices. Additionally, in tests on experimental serial crystallography diffraction data recorded with quasi-monochromatic X-rays and with electrons the algorithm indexed more patterns than other programs tested.

Serial protein crystallography in an electron microscope.

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

XGANDALF - extended gradient descent algorithm for lattice finding.

Serial crystallography records still diffraction patterns from single, randomly oriented crystals, then merges data from hundreds or thousands of them to form a complete data set. To process the data, the diffraction patterns must first be indexed, equivalent to determining the orientation of each crystal. A novel automatic indexing algorithm is presented, which in tests usually gives significantly higher indexing rates than alternative programs currently available for this task. The algorithm does not require prior knowledge of the lattice parameters but can make use of that information if provided, and also allows indexing of diffraction patterns generated by several crystals in the beam. Cases with a small number of Bragg spots per pattern appear to particularly benefit from the new approach. The algorithm has been implemented and optimized for fast execution, making it suitable for real-time feedback during serial crystallography experiments. It is implemented in an open-source C++ library and distributed under the LGPLv3 licence. An interface to it has been added to the CrystFEL software suite.

Publisher Correction: Structural basis of ligand recognition at the human MT1 melatonin receptor.

Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Megahertz serial crystallography.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a ß-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

From Lithium-Ion to Sodium-Ion Batteries: Advantages, Challenges, and Surprises.

Mobile and stationary energy storage by rechargeable batteries is a topic of broad societal and economical relevance. Lithium-ion battery (LIB) technology is at the forefront of the development, but a massively growing market will likely put severe pressure on resources and supply chains. Recently, sodium-ion batteries (SIBs) have been reconsidered with the aim of providing a lower-cost alternative that is less susceptible to resource and supply risks. On paper, the replacement of lithium by sodium in a battery seems straightforward at first, but unpredictable surprises are often found in practice. What happens when replacing lithium by sodium in electrode reactions? This review provides a state-of-the art overview on the redox behavior of materials when used as electrodes in lithium-ion and sodium-ion batteries, respectively. Advantages and challenges related to the use of sodium instead of lithium are discussed.

Identification of rogue datasets in serial crystallography.

Advances in beamline optics, detectors and X-ray sources allow new techniques of crystallographic data collection. In serial crystallography, a large number of partial datasets from crystals of small volume are measured. Merging of datasets from different crystals in order to enhance data completeness and accuracy is only valid if the crystals are isomorphous, i.e. sufficiently similar in cell parameters, unit-cell contents and molecular structure. Identification and exclusion of non-isomorphous datasets is therefore indispensable and must be done by means of suitable indicators. To identify rogue datasets, the influence of each dataset on CC1/2 [Karplus & Diederichs (2012 ▸). Science, 336, 1030-1033], the correlation coefficient between pairs of intensities averaged in two randomly assigned subsets of observations, is evaluated. The presented method employs a precise calculation of CC1/2 that avoids the random assignment, and instead of using an overall CC1/2, an average over resolution shells is employed to obtain sensible results. The selection procedure was verified by measuring the correlation of observed (merged) intensities and intensities calculated from a model. It is found that inclusion and merging of non-isomorphous datasets may bias the refined model towards those datasets, and measures to reduce this effect are suggested.

Recent developments in CrystFEL.

CrystFEL is a suite of programs for processing data from 'serial crystallography' experiments, which are usually performed using X-ray free-electron lasers (FELs) but also increasingly with other X-ray sources. The CrystFEL software suite has been under development since 2009, just before the first hard FEL experiments were performed, and has been significantly updated and improved since then. This article describes the most important improvements which have been made to CrystFEL since the first release version. These changes include the addition of new programs to the suite, the ability to resolve 'indexing ambiguities' and several ways to improve the quality of the integrated data by more accurately modelling the underlying diffraction physics.

Breaking the indexing ambiguity in serial crystallography.

In serial crystallography, a very incomplete partial data set is obtained from each diffraction experiment (a `snapshot'). In some space groups, an indexing ambiguity exists which requires that the indexing mode of each snapshot needs to be established with respect to a reference data set. In the absence of such re-indexing information, crystallographers have thus far resorted to a straight merging of all snapshots, yielding a perfectly twinned data set of higher symmetry which is poorly suited for structure solution and refinement. Here, two algorithms have been designed for assembling complete data sets by clustering those snapshots that are indexed in the same way, and they have been tested using 15,445 snapshots from photosystem I [Chapman et al. (2011), Nature (London), 470, 73-77] and with noisy model data. The results of the clustering are unambiguous and enabled the construction of complete data sets in the correct space group P63 instead of (twinned) P6322 that researchers have been forced to use previously in such cases of indexing ambiguity. The algorithms thus extend the applicability and reach of serial crystallography.