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Serial crystallography data can be challenging to index, as each frame is processed individually, rather than being processed as a whole like in conventional X-ray single-crystal crystallography. An algorithm has been developed to index still diffraction patterns arising from small-unit-cell samples. The algorithm is based on the matching of reciprocal-lattice vector pairs, as developed for Laue microdiffraction data indexing, combined with three-dimensional pattern matching using a nearest-neighbors approach. As a result, large-bandpass data (e.g. 5–24 keV energy range) and monochromatic data can be processed, the main requirement being prior knowledge of the unit cell. Angles calculated in the vicinity of a few theoretical and experimental reciprocal-lattice vectors are compared, and only vectors with the highest number of common angles are selected as candidates to obtain the orientation matrix. Global matching on the entire pattern is then checked. Four indexing options are available, two for the ranking of the theoretical reciprocal-lattice vectors and two for reducing the number of possible candidates. The algorithm has been used to index several data sets collected under different experimental conditions on a series of model samples. Knowing the crystallographic structure of the sample and using this information to rank the theoretical reflections based on the structure factors helps the indexing of large-bandpass data for the largest-unit-cell samples. For small-bandpass data, shortening the candidate list to determine the orientation matrix should be based on matching pairs of reciprocal-lattice vectors instead of triplet matching.

A method is presented to perform time-resolved X-ray crystallography with a 63 ms time resolution using a fast pixel detector and partial oscillation data sets. This minimizes the number of crystals (, The recent development of serial crystallography has popularized time-resolved crystallography as a technique to determine the structure of protein-reaction intermediate states. However, most approaches rely on the availability of thousands to millions of microcrystals. A method is reported here, using monochromatic synchrotron radiation, for the room-temperature collection, processing and merging of X-ray oscillation diffraction data from

IUCrJ 7(5), 784 - 792 (2020). doi:10.1107/S2052252520008672, Macromolecular crystallography (MX) is the dominant means of determining the three-dimensional structures of biological macromolecules. Over the last few decades, most MX data have been collected at synchrotron beamlines using a large number of different detectors produced by various manufacturers and taking advantage of various protocols and goniometries. These data came in their own formats: sometimes proprietary, sometimes open. The associated metadata rarely reached the degree of completeness required for data management according to Findability, Accessibility, Interoperability and Reusability (FAIR) principles. Efforts to reuse old data by other investigators or even by the original investigators some time later were often frustrated. In the culmination of an effort dating back more than two decades, a large portion of the research community concerned with high data-rate macromolecular crystallography (HDRMX) has now agreed to an updated specification of data and metadata for diffraction images produced at synchrotron light sources and X-ray free-electron lasers (XFELs). This `Gold Standard' will facilitate the processing of data sets independent of the facility at which they were collected and enable data archiving according to FAIR principles, with a particular focus on interoperability and reusability. This agreed standard builds on the NeXus/HDF5 NXmx application definition and the International Union of Crystallography (IUCr) imgCIF/CBF dictionary, and it is compatible with major data-processing programs and pipelines. Just as with the IUCr CBF/imgCIF standard from which it arose and to which it is tied, the NeXus/HDF5 NXmx Gold Standard application definition is intended to be applicable to all detectors used for crystallography, and all hardware and software developers in the field are encouraged to adopt and contribute to the standard., Published by Chester

During my PhD studies at the ETH Zurich and the Paul Scherrer Institute I worked in the field of time-resolved serial femtosecond X-ray crystallography (TR-SFX), an emerging structural biology method that is performed at X-ray free-electron lasers (XFELs). The first advantage of XFELs is their provision of ultrafast 10-100 fs pulses, which allow radiation damage effects to be avoided, and obtaining structures from data collected at room temperature. The second advantage of XFELs comes upon installing a pump laser that induces protein conformational changes and allows obtaining crystallographic datasets at different times after protein activation. The protein that was subject to the study, Krokinobacter eikastus rhodopsin 2 (KR2), is the first discovered light-driven sodium pump. It was intriguing to elucidate the protein’s new sodium-pumping mechanism. It had been long debated if such a non-H+ cation pump could exist, because Na+ was supposed to be electrostatically repulsed from a positively charged retinal chromophore Schiff base. Due to the fact that outward pumping of Na+ silences neuronal signalling, KR2 is considered a next-generation tool for optogenetics.

This work demonstrates the use of polymer thin films and graphene to support and maintain the hydration of protein microcrystals on fixed targets for serial femtosecond crystallography at X-ray free-electron lasers. Rapid encystment protein (REP24) provides a benchmark for this encapsulation approach., For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

BioMAX is the first macromolecular crystallography beamline at the first fourth-generation storage ring, the 3 GeV storage ring at MAX IV Laboratory. It is a highly automated, user-friendly, microfocus beamline that provides excellent performance for most macromolecular crystallography applications., BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5–25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s−1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.

Serial crystallography (SX) is an emerging technique to determine macromolecules at room temperature. SX with a pump–probe experiment provides the time-resolved dynamics of target molecules. SX has developed rapidly over the past decade as a technique that not only provides room-temperature structures with biomolecules, but also has the ability to time-resolve their molecular dynamics. The serial femtosecond crystallography (SFX) technique using an X-ray free electron laser (XFEL) has now been extended to serial synchrotron crystallography (SSX) using synchrotron X-rays. The development of a variety of sample delivery techniques and data processing programs is currently accelerating SX research, thereby increasing the research scope. In this editorial, I briefly review some of the experimental techniques that have contributed to advances in the field of SX research and recent major research achievements. This Special Issue will contribute to the field of SX research.

IUCrJ 5(5), 524 - 530 (2018). doi:10.1107/S2052252518009223, During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools., Published by Chester

MSOX (multiple serial structures from one crystal) serial crystallography experiments were carried out using controlled X-ray radiolysis and photon-counting detectors to determine sequences of low-dose yet high-resolution structures of copper nitrite reductase. Working at 190 K and at room temperature provides greater dynamic freedom, allowing more of the catalytic cycle to be observed than at the usual cryogenic temperature of 100 K. The approach demonstrates the potential to obtain MSOX structural movies at variable temperatures, thus providing an unparalleled level of structural information during catalysis for redox enzymes., High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate ‘structural movies’ of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a ‘top-hat’ geometry, which was rapidly transformed to a ‘side-on’ configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

Background: Membrane receptors play an important role in signal transduction across the cell membrane in all living organisms. Their structural studies have been enabled by multiple technological breakthroughs in their heterologous expression, stabilization, crystallization, and crystallographic data collection as well as in cryogenic electron microscopy (cryoEM). During the last decade, serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has enabled structure determination of previously inaccessible proteins, including several G-protein-coupled receptors (GPCR), that produce only micrometer-sized crystals, thus paving the way towards understanding their activation mechanism and rational drug discovery. In addition to experimental difficulties, membrane protein structure determination is also often accompanied by data processing challenges. In particular, the lipidic cubic phase that serves as a carrier for membrane protein microcrystals, as well as various XFEL beam-shaping devices may generate substantial background scattering that could complicate the structure factor extraction from the diffraction images. Methods: In this work, we tested an adaptation of the denoising algorithm via matrix decomposition to XFEL-SFX data. We benchmarked its performance using high-background data from PAL-XFEL and established its applicability to serial crystallography image denoising, as well as compared it to the CrystFEL-based image denoising algorithm. Results: We find that, although the decomposition-based image denoising does not outperform CrystFEL median subtraction, it performs better than the integration without any additional subtraction. We find the non-negative matrix factorization performing better than more traditional singular-value decomposition methods, both in terms of visual interpretability and final data quality. Conclusion: We hope that this work will draw attention to background subtraction methods in structural biology, and will pave the way towards processing of most challenging datasets in structural biology, in particularly, those collected from membrane proteins.

The complex evolution of diffracted intensities from protein crystals during irradiation by intense Gaussian X-ray microbeams is measured and analysed. The analysis explains non-exponential intensity decays without invoking sequential damage models, yields a revised metric to quantify the damage state of the crystal after a given irradiation time, explains previous observations of a damage ‘lag’ phase and shows how ultra-intense X-ray microbeams allow the data collected per crystal at and near room temperature to be increased., Serial synchrotron-based crystallography using intense microfocused X-ray beams, fast-framing detectors and protein microcrystals held at 300 K promises to expand the range of accessible structural targets and to increase overall structure-pipeline throughputs. To explore the nature and consequences of X-ray radiation damage under microbeam illumination, the time-, dose- and temperature-dependent evolution of crystal diffraction have been measured with maximum dose rates of 50 MGy s−1. At all temperatures and dose rates, the integrated diffraction intensity for a fixed crystal orientation shows non-exponential decays with dose. Non-exponential decays are a consequence of non-uniform illumination and the resulting spatial evolution of diffracted intensity within the illuminated crystal volume. To quantify radiation-damage lifetimes and the damage state of diffracting crystal regions, a revised diffraction-weighted dose (DWD) is defined and it is shown that for Gaussian beams the DWD becomes nearly independent of actual dose at large doses. An apparent delayed onset of radiation damage seen in some intensity–dose curves is in fact a consequence of damage. Intensity fluctuations at high dose rates may arise from the impulsive release of gaseous damage products. Accounting for these effects, data collection at the highest dose rates increases crystal radiation lifetimes near 300 K (but not at 100 K) by a factor of ∼1.5–2 compared with those observed at conventional dose rates. Improved quantification and modeling of the complex spatio-temporal evolution of protein microcrystal diffraction in intense microbeams will enable more efficient data collection, and will be essential in improving the accuracy of structure factors and structural models.

Methods are described for processing XFEL data from individual crystalline fibrils. The methods are applied to data collected at the Linac Coherent Light Source from an amyloid-forming oligopeptide from the adenovirus shaft., Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Geometry correction is performed with separation of Euclidean and non-Euclidean movements with respect to the sample, refined against independent target functions derived from the data. This leads to substantial improvements in indexing rates and data quality indicators and refines to convergence., Geometry correction is traditionally plagued by mis-fitting of correlated parameters, leading to local minima which prevent further improvements. Segmented detectors pose an enhanced risk of mis-fitting: even a minor confusion of detector distance and panel separation can prevent improvement in data quality. The slip-and-slide algorithm breaks down effects of the correlated parameters and their associated target functions in a fundamental shift in the approach to the problem. Parameters are never refined against the components of the data to which they are insensitive, providing a dramatic boost in the exploitation of information from a very small number of diffraction patterns. This algorithm can be applied to exploit the adherence of the spot-finding results prior to indexing to a given lattice using unit-cell dimensions as a restraint. Alternatively, it can be applied to the predicted spot locations and the observed reflection positions after indexing from a smaller number of images. Thus, the indexing rate can be boosted by 5.8% using geometry refinement from only 125 indexed patterns or 500 unindexed patterns. In one example of cypovirus type 17 polyhedrin diffraction at the Linac Coherent Light Source, this geometry refinement reveals a detector tilt of 0.3° (resulting in a maximal Z-axis error of ∼0.5 mm from an average detector distance of ∼90 mm) whilst treating all panels independently. Re-indexing and integrating with updated detector geometry reduces systematic errors providing a boost in anomalous signal of sulfur atoms by 20%. Due to the refinement of decoupled parameters, this geometry method also reaches convergence.

Journal of applied crystallography 50(4), 1075-1083(2017). doi:10.1107/S1600576717007506, A novel algorithm for indexing multiple crystals in snapshot X-ray diffraction images, especially suited for serial crystallography data, is presented. The algorithm, FELIX, utilizes a generalized parametrization of the Rodrigues–Frank space, in which all crystal systems can be represented without singularities. The new algorithm is shown to be capable of indexing more than ten crystals per image in simulations of cubic, tetragonal and monoclinic crystal diffraction patterns. It is also used to index an experimental serial crystallography dataset from lysozyme microcrystals. The increased number of indexed crystals is shown to result in a better signal-to-noise ratio, and fewer images are needed to achieve the same data quality as when indexing one crystal per image. The relative orientations between the multiple crystals indexed in an image show a slight tendency of the lysozme microcrystals to adhere on $(\overline {1}10)$ facets., Published by IUCr, Copenhagen

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCATand HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCATprotonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCATis seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

Responding to different light conditions is an essential process for many organisms on earth. Unicellular organisms are no exception to this and mechanisms for controlling cellular movement must often be sensitive to light. Light sensing proteins commonly have internally bound chromophores that, when activated by specific light wavelengths, propagate structural changes through a protein to produce an appropriate cellular response. Microbial rhodopsins are a family of transmembrane proteins that harness light to perform a range of functions. These rhodopsins have been found to act as ion pumps, channels and light sensing proteins. They all utilize similar chemistry through a covalently bound retinal to perform these diverse functions. In this thesis, time-resolved structural techniques are utilized to track the changes in sensory rhodopsin II (SRII) a photophobic blue-light sensor in archaea that protects the cell against harmful UV-radiation. SRII is bound in the membrane to a transducer protein (HtrII) that extends into the cell to affect a response. Time-resolved structural biology has undergone a period of rapid methodological development. Inspired by the data collection challenges presented by X-ray free electron lasers (XFELS), serial crystallography has proved remarkably effective in resolving protein dynamics in crystals by time-resolved studies. These methods have more recently been used at synchrotrons. Recent work has shown that time-resolved serial millisecond crystallography (TR-SMX) on membrane protein microcrystals growing in lipidic cubic phase (LCP) is possible at synchrotrons. This complements time- resolved X-ray solution scattering (TR-XSS) methods already employed at synchrotron sources to measure protein dynamics. In this thesis, we utilize both methods to gain new insight into SRII and SRII-HtrII dynamics and structure. The papers presented here outline new crystallization conditions for SRII and SRII-HtrII that do not require lipid reconstitution. At the Swiss Light Source, we measured a light-activated structure for SRII that provides a istructural explanation of the long-lived signalling states using TR-SMX. We also collected a low-resolution room temperature SRII-HtrII structure that reveals new features and which paves the way for time-resolved serial femtosecond crystallography (TR-SFX) measurements at XFELs., Solution X- ray scattering experiments were carried out on SRII and SRII-HtrII to observe complex dynamics. These revealed that the presence of transducer inhibits the EF-helix motion, providing evidence that this motion in involved in signal transduction.

Life in all living organisms depend on chemical processes performed by proteins, which are molecular machineries encoded in the DNA. Cytochrome c oxidase (CcO) is a membrane protein essential for cellular respiration, a process in which chemical energy enters the electron transport chain in the form of electrons and is transformed to ATP via a proton gradient. In the active site of CcO a redox-reaction takes place, where oxygen that we breathe is reduced to two water molecules at the same time as protons are pumped over the membrane. This thesis focuses on structural investigations performed by serial crystallography (SX) of CcO in lipidic cubic phase (LCP). The advances of bright X-ray sources in the form of X-ray Free Electron Lasers with short pulse durations and 4:th generation synchrotrons have evolved the structural biology field in the sense that high-quality data can be collected at room temperature with minimal X-ray induced radiation damage. These advancements enable the capturing of structural intermediates of proteins in a time-resolved manner. Well-diffracting microcrystals are of essence in serial crystallography experiments, and the study of membrane proteins in lipidic cubic phase has proven to be favorable compared to detergent-based crystallization. This thesis focuses on developing procedures to produce microcrystals in LCP, and its use in studies of ba3-type CcO from Thermus thermophilus as well as reaction centre from Blastrochloris viridis. The room-temperature structure of ba3-type CcO is determined in both the resting oxidized state and the reduced CO-bound state. The resting state structure reveals the active site ligand as a single oxygen species, and in comparisons to previously published structures of CcO we show structural differences between ba3-type CcO and bovine aa3-type CcO upon CO-binding. The work presented in this thesis provides the groundwork for future time-resolved CO-photolysis experiments of ba3-type CcO by the pump-probe approach, which may reveal more about the structural mechanisms that explain proton pumping in CcO.

The unique nucleating and phasing capabilities of the crystallophore, Tb-Xo4, are illustrated through challenging cases., Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome by the crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating and phasing properties. This article presents examples of new crystallization conditions induced by the presence of Tb-Xo4. These new crystalline forms bypass crystal defects often encountered by crystallographers, such as low-resolution diffracting samples or crystals with twinning. Thanks to Tb-Xo4’s high phasing power, the structure determination process is greatly facilitated and can be extended to serial crystallography approaches.

Time-resolved crystallography (TRX) enables the identification at a near atomic detail of the progressive structural changes of a protein fulfilling its function, thus leading to the establishment of genuine molecular movies. Laue (polychromatic) diffraction at 3rd-generation synchrotrons first managed to obtain such movies at a 100 ps time scale, and more recently, the 4th generation X-ray sources XFELs (X-ray Free Electron Lasers) achieved a 100 fs time scale using a monochromatic beam. The goal of this thesis was to investigate the possibility of performing TRX on a macromolecular monochromatic crystallography beamline, by taking advantage of the latest technological developments. We envisaged three different photosensitive proteins, for which I first developed various crystallogenesis strategies in order to control the size and shape of the crystals. I also considered various ways of presenting the crystals of the beam at room temperature, either a single crystal mounted in a loop and kept in a wet air stream, or microcrystals moving in a grease jet passing through the beam. We then investigated how specific radiation damage could affect the structures of intermediate states at room temperature, given how sensitive they can be at cryogenic temperature. We concluded that it constitutes a much lesser concern at room than at cryogenic temperature. After initial characterization of the various proteins and sample environments, we focused on a time-resolved crystallography approach on crystals of the plant photoreceptor domain AtPhot2LOV2 (the LOV2 domain of the blue-light photoreceptor photropin-2 from Arabidopsis thaliana). The dark state AtPhot2LOV2 converts in microseconds into the signalling state, or light state, which relaxes in hundreds of seconds. Using a fast pixel X-ray detector, we characterized the structural decay of the light state, and showed that it proceeds via a space group conversion over a 20 min time course. We then slowed down the rate of the light state in crystals by limiting the quantity of photons, and we were able to monitor the process of light state population build-up with a 63 ms time resolution using an approach recording full data sets on less than 100 crystals, which we called TR-SOX for time-resolved serial oscillation crystallography, which takes advantage of merging partial data sets. Overall, our work paves the way for time-resolved crystallography on MX monochromatic synchrotron beamlines on regular-sized crystals of proteins that undergo structural rearrangements on the millisecond time scale., La cristallographie résolue dans le temps (TRX) permet l’identification à l’échelle quasi atomique des changements structuraux progressifs au sein d’une protéine lorsqu’elle réalise sa fonction, permettant la constitution de véritables films moléculaires. La diffraction Laue (polychromatique) dans les synchrotrons de 3ème génération a d’abord permis d’obtenir de tels films à une échelle de temps de 100 ps, et plus récemment les sources de rayons X de 4ème génération, les lasers à électrons libres (XFELs, pour X-Ray Free Electron Lasers), ont permis d’atteindre une résolution temporelle de 100 fs en utilisant un faisceau monochromatique. L’objectif de cette thèse était d’explorer la possibilité d’effectuer des expériences de TRX au synchrotron sur les lignes de lumière monochromatiques dédiées à la cristallographie macromoléculaire en tirant parti des tout derniers développements technologiques. Nous avons envisagé de travailler sur trois protéines photosensibles différentes, pour lesquelles j’ai dans un premier temps développé différentes stratégies de cristallogenèse afin de contrôler la taille et la forme des cristaux. J’ai également testé différentes façons d’exposer les cristaux au faisceau de rayons X à température ambiante, soit des cristaux uniques montés sur une boucle et maintenu dans un flux d’humidité contrôlée, soit des microcristaux incorporés dans un flux de graisse passant à travers le faisceau de rayons X. Nous avons ensuite évalué comment les dommages radiatifs spécifiques pouvaient affecter les structures d’état intermédiaires de protéines à température ambiante, espèces particulièrement sensibles à température cryogénique. Nous avons conclu de cette étude que le dommage radiatif spécifique constitue un bien moindre problème à température ambiante qu’à température cryogénique. Après une caractérisation initiale des différentes protéines et des moyens de présentation au faisceau, nous avons concentré notre approche de cristallographie résolue dans le temps sur des cristaux d’un domaine de photorécepteur de plante, AtPhot2LOV2 (le domaine LOV2 du récepteur à la lumière bleue phototropine-2 de la plante Arabidopsis thaliana). L’état initial de AtPhot2LOV2 se convertit en quelques microsecondes en un état de signalisation, ou état photo-activé, qui se relaxe en quelques centaines de secondes. En utilisant un détecteur rapide de rayons X, nous avons caractérisé structuralement le phénomène de relaxation, et montré qu’elle se développe en une vingtaine de minutes via une conversion de groupe d’espace. Nous avons ensuite diminué la vitesse de formation de l’état photo-activé dans les cristaux en limitant la quantité de photons nécessaires à la photoconversion, et nous avons pu visualiser l’augmentation progressive de la population en l’état photo-activé dans le cristal avec une résolution temporelle de 63 ms, en utilisant une approche basée sur l’enregistrement de données de diffraction sur moins de 100 cristaux et tirant parti de la combinaison de jeux de données partiels, approche que nous avons nommée TR-SOX (Time-Resolved Serial Oscillation Crystallography). En résumé, notre travail ouvre la voie aux expériences de cristallographie résolue dans le temps sur les lignes de lumières monochromatiques de cristallographie macromoléculaire au synchrotron, sur des cristaux de taille usuelle pour des protéines subissant des réarrangements structuraux à l’échelle de la milliseconde.

The ability to control crystallization reactions is required in a vast range of processes including the production of functional inorganic materials and pharmaceuticals and the prevention of scale. However, it is currently limited by a lack of understanding of the mechanisms underlying crystal nucleation and growth. To address this challenge, it is necessary to carry out crystallization reactions in well-defined environments, and ideally to perform in situ measurements. Here, a versatile microfluidic synchrotron-based technique is presented to meet these demands. Droplet microfluidic-coupled X-ray diffraction (DMC-XRD) enables the collection of time-resolved, serial diffraction patterns from a stream of flowing droplets containing growing crystals. The droplets offer reproducible reaction environments, and radiation damage is effectively eliminated by the short residence time of each droplet in the beam. DMC-XRD is then used to identify effective particulate nucleating agents for calcium carbonate and to study their influence on the crystallization pathway. Bioactive glasses and a model material for mineral dust are shown to significantly lower the induction time, highlighting the importance of both surface chemistry and topography on the nucleating efficiency of a surface. This technology is also extremely versatile, and could be used to study dynamic reactions with a wide range of synchrotron-based techniques.

Manipulation of high-density materials, such as crystals and liquid condensates, is of great importance for many applications, including serial crystallography, structural and molecular biology, chemistry, and medicine. In this work, we describe an acoustic technique to focus and harvest flowing crystals and liquid condensates. Moreover, we show, based on numerical simulations, that the acoustic waves can be used for size-based particle (crystals, droplets, etc.) separation. This is an essential technological step in biological research, medical applications, and industrial processes. The presented technology offers high precision, biocompatibility, ease of use and additionally, is non-invasive and inexpensive. With the recent advent of X-ray Free Electron Laser (XFEL) technology and the associated enormous importance of a thin jet of crystals, this technology might pave the way to a novel type of XFEL injector.

Serial rotation electron diffraction enables fully automated screening and data acquisition on submicrometre-sized crystals using the continuous rotation method via combined image processing and control of the transmission electron microscopy hardware. A data processing pipeline revolving around hierarchical cluster analysis deals with the large multi-crystal data set for phase analysis and structure determination., Serial rotation electron diffraction (SerialRED) has been developed as a fully automated technique for three-dimensional electron diffraction data collection that can run autonomously without human intervention. It builds on the previously established serial electron diffraction technique, in which submicrometre-sized crystals are detected using image processing algorithms. Continuous rotation electron diffraction (cRED) data are collected on each crystal while dynamically tracking the movement of the crystal during rotation using defocused diffraction patterns and applying a set of deflector changes. A typical data collection screens up to 500 crystals per hour, and cRED data are collected from suitable crystals. A data processing pipeline is developed to process the SerialRED data sets. Hierarchical cluster analysis is implemented to group and identify the different phases present in the sample and to find the best matching data sets to be merged for subsequent structure analysis. This method has been successfully applied to a series of zeolites and a beam-sensitive metal–organic framework sample to study its capability for structure determination and refinement. Two multi-phase samples were tested to show that the individual crystal phases can be identified and their structures determined. The results show that refined structures obtained using automatically collected SerialRED data are indistinguishable from those collected manually using the cRED technique. At the same time, SerialRED has lower requirements of expertise in transmission electron microscopy and is less labor intensive, making it a promising high-throughput crystal screening and structure analysis tool.

IUCrJ 6(5), 927 - 937 (2019). doi:10.1107/S205225251900914X, Reliable sample delivery and efficient use of limited beam time have remained bottlenecks for serial crystallography (SX). Using a high-intensity polychromatic X-ray beam in combination with a newly developed charge-integrating JUNGFRAU detector, we have applied the method of fixed-target SX to collect data at a rate of 1 kHz at a synchrotron-radiation facility. According to our data analysis for the given experimental conditions, only about 3 000 diffraction patterns are required for a high-quality diffraction dataset. With indexing rates of up to 25%, recording of such a dataset takes less than 30 s., Published by Chester

Acta crystallographica / A 75(5), 694 - 704 (2019). doi:10.1107/S2053273319010593, 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., Published by Blackwell, Oxford [u.a.]

Direct protein crystallization on fixed-target sample holders allows highly efficient serial crystallography experiments and ligand-binding studies., Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, ‘naked’ crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

Acta crystallographica / D 75(2), 1-15 (2019). doi:10.1107/S205979831801238X, This article provides a step-by-step guide to the use of the CrystFEL software for processing serial crystallography data from an X-ray free-electron laser or a synchrotron light source. Whereas previous papers have described the theory and algorithms and their rationale, this paper describes the steps to be performed from a user perspective, including command-line examples., Published by Wiley-Blackwell, Oxford

A novel method is presented to screen for suitable crystallization conditions and produce large amounts of microcrystals of membrane proteins in lipidic cubic phase for serial crystallography experiments., Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

Current developments and challenges for serial sample delivery at synchrotrons and X-ray free-electron lasers are reviewed, including the new megahertz repetition-rate machines, with an emphasis on liquid injection and high-viscosity extrusion., The high peak brilliance and femtosecond pulse duration of X-ray free-electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high-throughput serial approach to sample delivery. To this end, a number of such sample-delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low-dose data collection at room temperature. Here, the current sample-delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high-viscosity extrusion, including their application for time-resolved experiments. The challenges associated with sample delivery at megahertz repetition-rate XFELs are also outlined.

A new approach for efficiently loading fixed targets for serial crystallography using acoustic dispensing is presented., Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.