SFX Experiments Research Articles

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and theinterpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Sample Delivery Systems for Serial Femtosecond Crystallography at the PAL-XFEL

Serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) enables the determination of room-temperature structures without causing radiation damage. Using an optical pump-probe or mix-and-injection, SFX enables the intermediate state visualization of a molecular reaction. In SFX experiments, serial and stable microcrystal delivery to the X-ray interaction point is vital for reasonable data collection and efficient beam time. The Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) facility established SFX instruments at a nanocrystallography and coherent imaging (NCI) experimental station. Various sample delivery methods, including injection, fixed-target scanning, and hybrid methods, have been developed and applied to collect XFEL diffraction data. Herein, we report the currently available sample delivery methods for SFX at the NCI experimental station at the PAL-XFEL. This article will help PAL-XFEL users access the SFX system for their experiments.

Preparation of RNA Microcrystals for Serial Femtosecond Crystallography Experiments.

Serial femtosecond crystallography (SFX) experiments using an X-ray free electron laser (XFEL) is a burgeoning method for time-resolved structural studies of biomacromolecules. As with any crystallography experiment, the most important component is quality sample preparation. Whereas dozens of SFX experiments, including batch crystallization methods, have been reported for proteins, very few have been reported for RNA. This chapter outlines standard procedures for preparing RNA microcrystalline samples suitable for SFX studies.

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its “off-time” between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.

Serial femtosecond crystallography

With the advent of X-ray Free Electron Lasers (XFELs), new, high-throughput serial crystallography techniques for macromolecular structure determination have emerged. Serial femtosecond crystallography (SFX) and related methods provide possibilities beyond canonical, single-crystal rotation crystallography by mitigating radiation damage and allowing time-resolved studies with unprecedented temporal resolution. This primer aims to assist structural biology groups with little or no experience in serial crystallography planning and carrying out a successful SFX experiment. It discusses the background of serial crystallography and its possibilities. Microcrystal growth and characterization methods are discussed, alongside techniques for sample delivery and data processing. Moreover, it gives practical tips for preparing an experiment, what to consider and do during a beamtime and how to conduct the final data analysis. Finally, the Primer looks at various applications of SFX, including structure determination of membrane proteins, investigation of radiation damage-prone systems and time-resolved studies.

Combination of an inject-and-transfer system for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) enables the determination of room-temperature crystal structures of macromolecules with minimized radiation damage and provides time-resolved molecular dynamics by pump-probe or mix-and-inject experiments. In SFX, a variety of sample delivery methods with unique advantages have been developed and applied. The combination of existing sample delivery methods can enable a new approach to SFX data collection that combines the advantages of the individual methods. This study introduces a combined inject-and-transfer system (BITS) method for sample delivery in SFX experiments: a hybrid injection and fixed-target scanning method. BITS allows for solution samples to be reliably deposited on ultraviolet ozone (UVO)-treated polyimide films, at a minimum flow rate of 0.5 nl min-1, in both vertical and horizontal scanning modes. To utilize BITS in SFX experiments, lysozyme crystal samples were embedded in a viscous lard medium and injected at flow rates of 50-100 nl min-1 through a syringe needle onto a UVO-treated polyimide film, which was mounted on a fixed-target scan stage. The crystal samples deposited on the film were raster scanned with an X-ray free electron laser using a motion stage in both horizontal and vertical directions. Using the BITS method, the room-temperature structure of lysozyme was successfully determined at a resolution of 2.1 Å, and thus BITS could be utilized in future SFX experiments.

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods.

3D printed devices and infrastructure for liquid sample delivery at the European XFEL.

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Å is reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

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.

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Pixel modelling - a new age in SFX data analysis.

A new program, diffBragg, employs per-pixel maximum likelihood optimization of X-ray pulse and crystal parameters to improve the accuracy of structure factor amplitudes attainable in SFX experiments.

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Advances in long-wavelength native phasing at X-ray free-electron lasers.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

Mixing and jetting analysis using continuous flow microfluidic sample delivery devices.

Serial femtosecond crystallography (SFX) methods used at X-ray free electron lasers (XFELs) offer a range of new opportunities for structural biology. A crucial component of SFX experiments is sample delivery. Microfluidic devices can be employed in SFX experiments to precisely deliver microcrystals to the X-ray beam and to trigger molecular dynamics via rapid mix-and-inject measurements. Here, for the first time, we have developed a process based on high-resolution photolithography using SU8 on glass to fabricate microfluidic mix-and-inject devices. In order to characterise these devices a broad range of flow rates are used and the mixing and jetting response of the devices monitored. We observe that a stable jet is formed using these devices when injecting DI-water. Three different jetting regimes, liquid column, ribbon, and cylindrical jet, were observed. Furthermore, fluorescence experiments confirm that rapid and uniform mixing of the two injected solutions is possible using these devices indicating that they could be used to probe molecular dynamics on sub-microsecond timescales.

Membrane protein megahertz crystallography at the European XFEL

The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Electric Triggering for Enhanced Control of Droplet Generation.

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Elucidation of the entire Kok cycle for photosynthetic water oxidation by the large-scale quantum mechanics/molecular mechanics calculations: Comparison with the experimental results by the recent serial femtosecond crystallography

The fully optimized geometrical structures of the CaMn4Ox (x = 5, 6) clusters in the Si (i = 0–3) states of the Kok cycle of photosynthetic water oxidation by the large-scale quantum mechanics/molecular mechanics (QM/MM) calculations were compared to recent experimental results based on serial femtosecond crystallography (SFX). The MnMn and CaMn distances obtained by the QM/MM calculations were found to be totally comparable to the SFX experiments, elucidating the entire Kok cycle involving the S4 transition state during the OO bond formation for water oxidation in the oxygen evolving complex (OEC) of photosystem II (PSII).

Theoretical and computational investigations of geometrical, electronic and spin structures of the CaMn4 OX (X = 5, 6) cluster in the Kok cycle Si (i = 0-3) of oxygen evolving complex of photosystem II.

The optimized geometries of the CaMn4 OX (X = 5, 6) cluster in the oxygen evolving complex (OEC) of photosystem II (PSII) by large-scale quantum mechanics (QM) and molecular mechanics (MM) calculations are compared with recent serial femtosecond crystallography (SFX) results for the Si (i = 0-3) states. The valence states of four Mn ions by the QM/MM calculations are also examined in relation to the experimental results by the X-ray emission spectroscopy (XES) for the Si intermediates. Geometrical and valence structures of right-opened Mn-hydroxide, Mn-oxo and Mn-peroxide intermediates in the S3 state are investigated in detail in relation to recent SFX and XES experiments for the S3 state. Interplay between theory and experiment indicates that the Mn-oxo intermediate is a new possible candidate for the S3 state. Implications of the computational results are discussed in relation to possible mechanisms of the oxygenoxygen bond formation for water oxidation in OEC of PSII.