Journal of Applied Crystallography

Time resolved X-ray crystallography is experiencing a resurgence, in part, because of serial methods that readily allow scientists to create stop-motion movies of macromolecular function of photoactivation, enzyme catalysed reactions, and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet on demand tape drive sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with electronic states of metal ions within the sample. To our knowledge, such a combined methodology has not been deployed previously at a synchrotron beamline and has been restricted to XFELs. Here we describe prototype experiments along the development pathway to a combined droplet on demand diffraction and XES system at the microfocus beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within hundreds of picolitre-volume droplets deposited on a moving tape. In separate experiments at VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high viscosity extruder. Together, these results demonstrate the feasibility of combined droplet on demand serial crystallography and XES experiments using a third-generation synchrotron beamline; project work currently underway at Diamond Light Source. SynopsisWe describe proof of concept experiments towards correlated serial crystallography (SSX) and X-ray emission spectroscopy (XES) from microcrystals at a microfocus synchrotron beamline. A droplet on demand tape drive system delivers microcrystals to the beam within well-separated, hundreds of picolitre-volume droplets while XES allows validation of redox states of metals within protein crystals.

Diffuse scattering is a promising method to gain additional insight into protein dynamics from macro-molecular crystallography (MX) experiments. Bragg intensities yield the average electron density, while the diffuse scattering can be processed to obtain a three-dimensional reciprocal space map, that is further analyzed to determine correlated motion. To make diffuse scattering techniques more accessible, we have created software for data processing called mdx2 that is both convenient to use and simple to extend and modify. Mdx2 is written in Python, and it interfaces with DIALS to implement self-contained data reduction workflows. Data are stored in NeXusformat for software interchange and convenient visualization. Mdx2 can be run on the command line or imported as a package, for instance to encapsulate a complete workflow in a Jupyter notebook for reproducible computing and education. Here, we describe mdx2 version 1.0, a new release incorporating state-of-the-art techniques for data reduction. We describe the implementation of a complete multi-crystal scaling and merging workflow, and test the methods using a high-redundancy dataset from cubic insulin. We show that redundancy can be leveraged during scaling to correct systematic errors, and obtain accurate and reproducible measurements of weak diffuse signals. SynopsisMdx2 is a Python toolkit for processing diffuse scattering data from macromolecular crystals. We describe multi-crystal scaling and merging procedures implemented in the latest version of mdx2. A high-redundancy dataset from cubic insulin is processed to reveal weak scattering features.

The advent of serial crystallography has rejuvenated and popularised room temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g., large complexes, membrane proteins, and viruses) forming fragile crystals, are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallisation. Many medically relevant samples are expressed in mammalian cell-lines, producing a meagre quantity of protein that is incompatible for batch crystallisation. This can limit the scope of serial crystallography approaches. Direct in-situ data collection from a 96-well crystallisation plate enables not only the identification of the best diffracting crystallisation condition, but also the possibility for structure determination at ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallisation plates, mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility (ESRF). We applied our iSX approach on a challenging project, Autotaxin, a therapeutic target expressed in a stable human cell-line, to determine a structure in the lowest symmetry P1 space group at 3.0 [A] resolution. Our in situ data collection strategy provided a complete dataset for structure determination, while screening various crystallisation conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallisation plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons. SynopsisThe determination of a challenging structure in the P1 space group, the lowest symmetry possible, shows how our in-situ serial crystallography approach expands the application of crystallisation plates as a robust sample delivery method.

Room-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study, demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site.

Monte Carlo neutron ray-tracing simulations of time-of-flight (TOF)-Laue neutron macromolecular crystal diffraction (n-MX) using the McStas software package were done for the upcoming NMX Macromolecular Diffractometer at the European Spallation Source. Splitting neutron rays that arrive at the crystal lead to dramatic improvements in event formation with minimal computational overhead. The simulated event probability data was sampled using a new single-pass weighted reservoir sampling method, and processed like real n-MX data using DIALS. The effects of air and beamstop scatter on simulated data was investigated. SynopsisMonte Carlo simulations of neutron protein diffraction experiments provide useful data that models instrumental components that interact with neutrons, as well as the crystal diffraction itself. These data can be applied to instrument development, such as the commissioning of the NMX Macromolecular Diffractometer at ESS.

Recent developments in neutron scattering instrumentation and sample handling have enabled studies of more complex biological samples and measurements at shorter exposure times. The experiments are typically conducted in D2O-based buffers to emphasize or diminish scattering from a particular components or to minimize background noise in the experiment. To extract most information from such experiments it is thus desirable to determine accurate estimates of how and when closely bound hydrogen atoms from the biomolecule exchange with the deuterium in the solvent. We introduce and document software, PSX, for exploring the effect of hydrogen-deuterium exchange for proteins solubilized in D2O as well as the underlying bioinformatical models. The software aims to be generally applicable for any atomistic structure of a protein and its surrounding environment, and thus captures effects of both heterogenous exchange rates throughout the protein structure and by varying the experimental conditions such as pH and temperature. The paper concludes with examples of applications and estimates of the effect in typical scenarios emerging in small-angle neutron scattering on biological macromolecules in solution. Our analysis suggests that the common assumption of 90% exchange is in many cases an overestimate with the rapid sample handling systems currently available, which leads to fitting and calibration issues when analysing the data. Source code for the presented software is available from an online repository in which it is published under version 3 of the GNU publishing license.

We describe the design and implementation of a drop on fixed target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume (40 - 90 pL) aqueous droplets, containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals immobilised on a solid support. The system was tested with various enzyme systems, including lysozyme and two -lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free electron laser facilities was evaluated. This drop on fixed target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

We report on recent methodological advances at the Life Science X-ray Scattering (LiX) beamline of the National Synchrotron Light Source II (NSLS-II) to support small-angle X-ray scattering experiments on skeletal and cardiac muscle tissues. These experiments have been routinely performed at the BioCAT beamline of the Advanced Photon Source (APS) over the past two decades to measure sarcomeric protein organization within healthy and diseased muscle tissues and provide direct molecular evidence for their functional roles and dynamics. Many recent advances in our understanding of sarcomeric proteins relied on diffraction data and include, as examples, MyBP-C, crossbridge SRX/DRX states, and titin. With LiX now available for muscle experimentation, more muscle users can be supported which will speed up research of sarcomeric proteins, muscle biomechanics, and skeletal and cardiac myopathies. LiX explicitly focuses on high-throughput muscle diffraction with rapid sample turnover and semi-automated data processing. These operations have been tested and validated on skeletal and cardiac tissues sourced from both humans and multiple animal models including pig, rat, mouse, and zebrafish.

Diffuse scattering is a powerful technique to study disorder and dynamics of macromolecules at atomic resolution. Although diffuse scattering is always present in diffraction images from macromolecular crystals, the signal is weak compared with Bragg peaks and background, making it a challenge to visualize and measure accurately. Recently, this challenge has been addressed using the reciprocal space mapping technique, which leverages ideal properties of modern X-ray detectors to reconstruct the complete three-dimensional volume of continuous diffraction from diffraction images of a crystal (or crystals) in many different orientations. This chapter will review recent progress in reciprocal space mapping with a particular focus on the strategy implemented in the mdx-lib and mdx2 software packages. The chapter concludes with an introductory data processing tutorial using Python packages DIALS, NeXpy, and mdx2.

Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

The cctbx.xfel suite of processing programs and tools allows fast, visual analysis of serial diffraction images from synchrotrons and XFELs. Built on DIALS and cctbx, cctbx.xfel is designed for real-time and post-experiment processing with a fully featured graphical user interface. Users can quickly identify hitrates, view diffraction patterns, analyze unit-cell isomorphism using clustering, and merge data using a metadata tagging approach that allows on-the-fly organization and visualization of processing results. This paper describes the fundamental algorithms and command-line programs used by cctbx.xfel, including the two main program dials.stills process, which performs spot-finding, indexing, geometric refinement, and integration, and cctbx.xfel.merge, which performs scaling, post-refinement, and merging. A discussion of merging statis-tics is presented and newer features are described, including random sub-sampling for indexing multi-lattice hits and {Delta}CC1/2 filtering to remove outliers. Finally we show a complex, heterogeneous sample containing hexagonal and monoclinic isoforms in P 63 and P 21. The isoforms are separated by unit cell clustering, and for each isoform we resolve a (pseudo-)merohedral indexing ambiguity.

Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED), is an emerging method for determining structures of submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single MicroED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of MicroED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based graphical user interface for automated real-time and offline batch 3D ED/MicroED data processing (AutoLEI). We illustrate the functionality of the GUI through four examples, demonstrating both offline and real-time data processing capabilities. These examples include small organic molecules, metal-organic frameworks (MOFs), and proteins, showcasing the versatility and efficiency of the GUI in various applications. SynopsisA graphical user interface for real-time and offline 3D ED/MicroED data processing by XDS was developed. The GUI aims to improve efficiency, minimize redundant data processing work, and provide users with real-time feedback during data collection.

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 [A] and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

Due to their high penetration depth, X-rays enable us to obtain information from the interior of whole, unsliced cells. Scanning small angle X-ray scattering (SAXS), in particular, reveals real-space images in dark field representation as well as structural information in reciprocal space. However, obtaining information on anisotropy and orientation from cells in an aqueous, close-to-physiological environment remains challenging. Here, we extend the recently introduced fast scanning SAXS mode with short exposure times of few milliseconds to such hydrated samples by combining a newly developed, X-ray compatible microfluidic sample chamber and innovative data analysis that includes an effective noise-filtering method. This strategy enables the systematic analysis of radiation damage by quantifying the SAXS signal. Our results demonstrate that scanning SAXS can be used to obtain intracellular information of fixed-hydrated cells and the approach may in the future be applicable to living cells as well. SynopsisFast scanning SAXS on biological cells in aqueous environment reveals intracellular anisotropy and orientation and allows for systematic assessment of radiation damage caused by the measurements.

The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands1. Significantly less is even known about multiple binding orientations for fragments (< 300 Da) although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tuneable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to advance the rational optimisation of SARS-CoV-2 nsp1 targeting fragment hits.

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no simple correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9[A] resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. SynopsisMembrane protein diffraction anisotropy originates from a peak at 4.9 [A] resolution in intensity profiles, due to secondary structure collinearity.

In standard rotational data collection for macromolecular crystallography data are normally collected from a single crystal, and the resulting data processing delivers metrics for data completeness and signal to noise that are well established. However, in serial crystallography it can be difficult to assess quickly whether enough data have been recorded to deliver a well scaled and complete dataset with sufficient signal to noise to address the scientific question being asked. Completeness alone is not an appropriate metric, as a nominally complete dataset can be obtained with a much smaller number of images, and thus multiplicity, than is needed to produce a final dataset with well estimated merged intensity values. Insufficient data result in alarmingly reasonable processing statistics and plausible electron density maps that contain almost no experimental signal, instead being dominated by the phases from the phasing model. We have therefore established a simple electron density-based test to determine whether enough data have been collected, and implemented this in the autoprocessing pipeline at the T-REXX endstation on beamline P14 at PETRA III. Importantly, the results of this test help guide decisions as to whether more data should be collected, or whether the experimenter can move onto a new time-point or sample. SynopsisWe describe a simple test to determine whether sufficient data have been collected during a serial crystallographic experiment, and its incorporation into the autoprocessing pipeline at the T- REXX endstation on beamline P14 at the PETRA III synchrotron.

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method -with SAD and MAD being the most popular ones- as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well-known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, poly-alanine tracing. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, in all model building cycles sequence is docked and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests. SynopsisSide chain tracing now completes model building in SHELXE to enhance density modification. All alternative SHELXE modes, using single or combined sources of starting phase information, are described. O_FD O_INLINEFIG[Formula 1]C_INLINEFIGM_FD(1)C_FD