Search for: All records

Abstract Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme. 

Pump–probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump–probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented. 

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. 

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 theMycobacterium tuberculosisβ-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. 

The analysis of a single-particle imaging (SPI) experiment performed at the AMO beamline at LCLS as part of the SPI initiative is presented here. A workflow for the three-dimensional virus reconstruction of the PR772 bacteriophage from measured single-particle data is developed. It consists of several well defined steps including single-hit diffraction data classification, refined filtering of the classified data, reconstruction of three-dimensional scattered intensity from the experimental diffraction patterns by orientation determination and a final three-dimensional reconstruction of the virus electron density without symmetry constraints. The analysis developed here revealed and quantified nanoscale features of the PR772 virus measured in this experiment, with the obtained resolution better than 10 nm, with a clear indication that the structure was compressed in one direction and, as such, deviates from ideal icosahedral symmetry. 

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation – in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described. 

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX. 

Abstract Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3μm x 1.7μm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.