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.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Heterogeneity in M. tuberculosis ß-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

Imaging via Correlation of X-Ray Fluorescence Photons.

We demonstrate that x-ray fluorescence emission, which cannot maintain a stationary interference pattern, can be used to obtain images of structures by recording photon-photon correlations in the manner of the stellar intensity interferometry of Hanbury Brown and Twiss. This is achieved utilizing femtosecond-duration pulses of a hard x-ray free-electron laser to generate the emission in exposures comparable to the coherence time of the fluorescence. Iterative phasing of the photon correlation map generated a model-free real-space image of the structure of the emitters. Since fluorescence can dominate coherent scattering, this may enable imaging uncrystallised macromolecules.

Dose-efficient scanning Compton X-ray microscopy.

The highest resolution of images of soft matter and biological materials is ultimately limited by modification of the structure, induced by the necessarily high energy of short-wavelength radiation. Imaging the inelastically scattered X-rays at a photon energy of 60 keV (0.02 nm wavelength) offers greater signal per energy transferred to the sample than coherent-scattering techniques such as phase-contrast microscopy and projection holography. We present images of dried, unstained, and unfixed biological objects obtained by scanning Compton X-ray microscopy, at a resolution of about 70 nm. This microscope was realised using novel wedged multilayer Laue lenses that were fabricated to sub-ångström precision, a new wavefront measurement scheme for hard X rays, and efficient pixel-array detectors. The doses required to form these images were as little as 0.02% of the tolerable dose and 0.05% of that needed for phase-contrast imaging at similar resolution using 17 keV photon energy. The images obtained provide a quantitative map of the projected mass density in the sample, as confirmed by imaging a silicon wedge. Based on these results, we find that it should be possible to obtain radiation damage-free images of biological samples at a resolution below 10 nm.

JINXED: just in time crystallization for easy structure determination of biological macromolecules.

Macromolecular crystallography is a well established method in the field of structural biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now under development towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand-soaking and cryo-protection. These handling steps can cause significant crystal damage, and hence reduce data quality. Furthermore, in time-resolved experiments based on serial crystallography, which use micrometre-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method that combines protein crystallization and data collection in a novel one-step process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg-white lysozyme and crystallization times of only a few seconds. This method, called JINXED (Just IN time Crystallization for Easy structure Determination), promises high-quality data due to the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

Speckle contrast of interfering fluorescence X-rays.

With the development of X-ray free-electron lasers (XFELs), producing pulses of femtosecond durations comparable with the coherence times of X-ray fluorescence, it has become possible to observe intensity-intensity correlations due to the interference of emission from independent atoms. This has been used to compare durations of X-ray pulses and to measure the size of a focusedX-ray beam, for example. Here it is shown that it is also possible to observe the interference of fluorescence photons through the measurement of the speckle contrast of angle-resolved fluorescence patterns. Speckle contrast is often used as a measure of the degree of coherence of the incident beam or the fluctuations of the illuminated sample as determined from X-ray diffraction patterns formed by elastic scattering, rather than from fluorescence patterns as addressed here. Commonly used approaches to estimate speckle contrast were found to suffer when applied to XFEL-generated fluorescence patterns due to low photon counts and a significant variation of the excitation pulse energy from shot to shot. A new method to reliably estimate speckle contrast under such conditions, using a weighting scheme, is introduced. The method is demonstrated by comparing the speckle contrast of fluorescence observed with pulses of 3 fs to 15 fs duration.

Heterogeneity in the M. tuberculosis ß-Lactamase Inhibition by Sulbactam.

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) 1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals 6 . Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating 7-9 , cooperativity, induced fit 10,11 and conformational selection 11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans- enamine. This was made possible in part by the application of the singular value decomposition 14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

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.

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio-logical temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system - the CFEL TapeDrive - with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 ß-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

Robust ptychographic X-ray speckle tracking with multilayer Laue lenses.

In recent years, X-ray speckle tracking techniques have emerged as viable tools for wavefront metrology and sample imaging applications, and have been actively developed for use at synchrotron light sources. Speckle techniques can recover an image free of aberrations and can be used to measure wavefronts with a high angular sensitivity. Since they are compatible with low-coherence sources they can be also used with laboratory X-ray sources. A new implementation of the ptychographic X-ray speckle tracking method, suitable for the metrology of highly divergent wavefields, such as those created by multilayer Laue lenses, is presented here. This new program incorporates machine learning techniques such as Huber and non-parametric regression and enables robust and quick wavefield measurements and data evaluation even for low brilliance X-ray beams, and the imaging of low-contrast samples. To realize this, a software suite was written in Python 3, with a C back-end capable of concurrent calculations for high performance. It is accessible as a Python module and is available as source code under Version 3 or later of the GNU General Public License.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Unsupervised learning approaches to characterizing heterogeneous samples using X-ray single-particle imaging.

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand-maximize-compress (EMC) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered.

Using diffraction losses of X-rays in a single crystal for determination of its lattice parameters as well as for monochromator calibration.

A way has been developed to measure the unit-cell parameters of a single crystal just from an energy scan with X-rays, even when the exact energy of the X-rays is not well defined due to an error in the pitch angle of the monochromator. The precision of this measurement reaches da/a ∼ 1 × 10-5. The method is based on the analysis of diffraction losses of the beam, transmitted through a single crystal (the so-called `glitch effect'). This method can be easily applied to any transmissive X-ray optical element made of single crystals (for example, X-ray lenses). The only requirements are the possibility to change the energy of the generated X-ray beam and some intensity monitor to measure the transmitted intensity. The method is agnostic to the error in the monochromator tuning and it can even be used for determination of the absolute pitch (or 2θ) angle of the monochromator. Applying the same method to a crystal with well known lattice parameters allows determination of the exact cell parameters of the monochromator at any energy.

Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature.

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis ß-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.

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.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.