Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE.

X-ray Free Electron Lasers (XFELs) allow the collection of high-quality serial femtosecond crystallography data. The next generation of megahertz superconducting FELs promises to drastically reduce data collection times, enabling the capture of more structures with higher signal-to-noise ratios and facilitating more complex experiments. Currently, gas dynamic virtual nozzles (GDVNs) stand as the sole delivery method capable of best utilizing the repetition rate of megahertz sources for crystallography. However, their substantial sample consumption renders their use impractical for many protein targets in serial crystallography experiments. Here, we present a novel application of a droplet-on-demand injection method, which allowed operation at 47 kHz at the European XFEL (EuXFEL) by tailoring a multi-droplet injection scheme for each macro-pulse. We demonstrate a collection rate of 150 000 indexed patterns per hour. We show that the performance and effective data collection rate are comparable to GDVN, with a sample consumption reduction of two orders of magnitude. We present lysozyme crystallographic data using the Large Pixel Detector at the femtosecond x-ray experiment endstation. Significant improvement of the crystallographic statistics was made by correcting for a systematic drift of the photon energy in the EuXFEL macro-pulse train, which was characterized from indexing the individual frames in the pulse train. This is the highest resolution protein structure collected and reported at the EuXFEL at 1.38 Å resolution.

Transient X-Ray Absorption Near Edge Structure Spectroscopy Using Broadband Free-Electron Laser Pulses.

A new method for time-resolved X-ray absorption near edge structure (XANES) spectroscopy that enables faster data acquisition and requires smaller sample quantities for high-quality data, thus allowing the analysis of more samples in a shorter time is introduced. The method uses large bandwidth free electron laser pulses to measure laser-excited XANES spectra in transmission mode. A beam-splitting grating configuration allows simultaneous measurements of the spectra of the incoming X-ray Free Electron Laser (XFEL) pulses and transmission XANES, which is crucial for compensating the pulse-dependent intensity and spectrum fluctuations due to the self-amplified spontaneous emission operation. The implementation of this new methodology is applied on a liquid solution of ammonium iron(III) oxalate jet and is compared to previous results, showing great improvements in the speed of acquisition and spectral resolution, and the ability to measure a large 2-D spectral-time map quickly.

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 the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Accessing metal-specific orbital interactions in C-H activation with resonant inelastic X-ray scattering.

Photochemically prepared transition-metal complexes are known to be effective at cleaving the strong C-H bonds of organic molecules in room temperature solutions. There is also ample theoretical evidence that the two-way, metal to ligand (MLCT) and ligand to metal (LMCT), charge-transfer between an incoming alkane C-H group and the transition metal is the decisive interaction in the C-H activation reaction. What is missing, however, are experimental methods to directly probe these interactions in order to reveal what determines reactivity of intermediates and the rate of the reaction. Here, using quantum chemical simulations we predict and propose future time-resolved valence-to-core resonant inelastic X-ray scattering (VtC-RIXS) experiments at the transition metal L-edge as a method to provide a full account of the evolution of metal-alkane interactions during transition-metal mediated C-H activation reactions. For the model system cyclopentadienyl rhodium dicarbonyl (CpRh(CO)2), we demonstrate, by simulating the VtC-RIXS signatures of key intermediates in the C-H activation pathway, how the Rh-centered valence-excited states accessible through VtC-RIXS directly reflect changes in donation and back-donation between the alkane C-H group and the transition metal as the reaction proceeds via those intermediates. We benchmark and validate our quantum chemical simulations against experimental steady-state measurements of CpRh(CO)2 and Rh(acac)(CO)2 (where acac is acetylacetonate). Our study constitutes the first step towards establishing VtC-RIXS as a new experimental observable for probing reactivity of C-H activation reactions. More generally, the study further motivates the use of time-resolved VtC-RIXS to follow the valence electronic structure evolution along photochemical, photoinitiated and photocatalytic reactions with transition metal complexes.

Tailoring p-Type Behavior in ZnO Quantum Dots through Enhanced Sol-Gel Synthesis: Mechanistic Insights into Zinc Vacancies.

The synthesis and control of properties of p-type ZnO is crucial for a variety of optoelectronic and spintronic applications; however, it remains challenging due to the control of intrinsic midgap (defect) states. In this study, we demonstrate a synthetic route to yield colloidal ZnO quantum dots (QD) via an enhanced sol-gel process that effectively eliminates the residual intermediate reaction molecules, which would otherwise weaken the excitonic emission. This process supports the creation of ZnO with p-type properties or compensation of inherited n-type defects, primarily due to zinc vacancies under oxygen-rich conditions. The in-depth analysis of carrier recombination in the midgap across several time scales reveals microsecond carrier lifetimes at room temperature which are expected to occur via zinc vacancy defects, supporting the promoted p-type character of the synthesized ZnO QDs.

Correlation of refractive index based and THz streaking arrival time tools for a hard X-ray free-electron laser.

To fully exploit ultra-short X-ray pulse durations routinely available at X-ray free-electron lasers to follow out-of-equilibrium dynamics, inherent arrival time fluctuations of the X-ray pulse with an external perturbing laser pulse need to be measured. In this work, two methods of arrival time measurement were compared to measure the arrival time jitter of hard X-ray pulses. The methods were photoelectron streaking by a THz field and a transient refractive index change of a semiconductor. The methods were validated by shot-to-shot correction of a pump-probe transient reflectivity measurement. An ultimate shot-to-shot full width at half-maximum error between the devices of 19.2 ± 0.1 fs was measured.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Disentangling the evolution of electrons and holes in photoexcited ZnO nanoparticles.

The evolution of charge carriers in photoexcited room temperature ZnO nanoparticles in solution is investigated using ultrafast ultraviolet photoluminescence spectroscopy, ultrafast Zn K-edge absorption spectroscopy, and ab initio molecular dynamics (MD) simulations. The photoluminescence is excited at 4.66 eV, well above the band edge, and shows that electron cooling in the conduction band and exciton formation occur in <500 fs, in excellent agreement with theoretical predictions. The x-ray absorption measurements, obtained upon excitation close to the band edge at 3.49 eV, are sensitive to the migration and trapping of holes. They reveal that the 2 ps transient largely reproduces the previously reported transient obtained at 100 ps time delay in synchrotron studies. In addition, the x-ray absorption signal is found to rise in ∼1.4 ps, which we attribute to the diffusion of holes through the lattice prior to their trapping at singly charged oxygen vacancies. Indeed, the MD simulations show that impulsive trapping of holes induces an ultrafast expansion of the cage of Zn atoms in <200 fs, followed by an oscillatory response at a frequency of ∼100 cm-1, which corresponds to a phonon mode of the system involving the Zn sub-lattice.

XFEL Microcrystallography of Self-Assembling Silver n-Alkanethiolates.

New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd-even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd-even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd-even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.

Tracking C-H activation with orbital resolution.

Transition metal reactivity toward carbon-hydrogen (C-H) bonds hinges on the interplay of electron donation and withdrawal at the metal center. Manipulating this reactivity in a controlled way is difficult because the hypothesized metal-alkane charge-transfer interactions are challenging to access experimentally. Using time-resolved x-ray spectroscopy, we track the charge-transfer interactions during C-H activation of octane by a cyclopentadienyl rhodium carbonyl complex. Changes in oxidation state as well as valence-orbital energies and character emerge in the data on a femtosecond to nanosecond timescale. The x-ray spectroscopic signatures reflect how alkane-to-metal donation determines metal-alkane complex stability and how metal-to-alkane back-donation facilitates C-H bond cleavage by oxidative addition. The ability to dissect charge-transfer interactions on an orbital level provides opportunities for manipulating C-H reactivity at transition metals.

Ultrafast Jahn-Teller Photoswitching in Cobalt Single-Ion Magnets.

Single-ion magnets (SIMs) constitute the ultimate size limit in the quest for miniaturizing magnetic materials. Several bottlenecks currently hindering breakthroughs in quantum information and communication technologies could be alleviated by new generations of SIMs displaying multifunctionality. Here, ultrafast optical absorption spectroscopy and X-ray emission spectroscopy are employed to track the photoinduced spin-state switching of the prototypical complex [Co(terpy)2 ]2+ (terpy = 2,2':6',2″-terpyridine) in solution phase. The combined measurements and their analysis supported by density functional theory (DFT), time-dependent-DFT (TD-DFT) and multireference quantum chemistry calculations reveal that the complex undergoes a spin-state transition from a tetragonally elongated doublet state to a tetragonally compressed quartet state on the femtosecond timescale, i.e., it sustains ultrafast Jahn-Teller (JT) photoswitching between two different spin multiplicities. Adding new Co-based complexes as possible contenders in the search for JT photoswitching SIMs will greatly widen the possibilities for implementing magnetic multifunctionality and eventually controlling ultrafast magnetization with optical photons.

Ultrafast Energy Transfer from Photoexcited Tryptophan to the Haem in Cytochrome c.

We report femtosecond Fe K-edge absorption (XAS) and nonresonant X-ray emission (XES) spectra of ferric cytochrome C (Cyt c) upon excitation of the haem (>300 nm) or mixed excitation of the haem and tryptophan (<300 nm). The XAS and XES transients obtained in both excitation energy ranges show no evidence for electron transfer processes between photoexcited tryptophan (Trp) and the haem, but rather an ultrafast energy transfer, in agreement with previous ultrafast optical fluorescence and transient absorption studies. The reported (J. Phys. Chem. B 2011, 115 (46), 13723-13730) decay times of Trp fluorescence in ferrous (∼350 fs) and ferric (∼700 fs) Cyt c are among the shortest ever reported for Trp in a protein. The observed time scales cannot be rationalized in terms of Förster or Dexter energy transfer mechanisms and call for a more thorough theoretical investigation.

Atomic-scale observation of solvent reorganization influencing photoinduced structural dynamics in a copper complex photosensitizer.

Photochemical reactions in solution are governed by a complex interplay between transient intramolecular electronic and nuclear structural changes and accompanying solvent rearrangements. State-of-the-art time-resolved X-ray solution scattering has emerged in the last decade as a powerful technique to observe solute and solvent motions in real time. However, disentangling solute and solvent dynamics and how they mutually influence each other remains challenging. Here, we simultaneously measure femtosecond X-ray emission and scattering to track both the intramolecular and solvation structural dynamics following photoexcitation of a solvated copper photosensitizer. Quantitative analysis assisted by molecular dynamics simulations reveals a two-step ligand flattening strongly coupled to the solvent reorganization, which conventional optical methods could not discern. First, a ballistic flattening triggers coherent motions of surrounding acetonitrile molecules. In turn, the approach of acetonitrile molecules to the copper atom mediates the decay of intramolecular coherent vibrations and induces a further ligand flattening. These direct structural insights reveal that photoinduced solute and solvent motions can be intimately intertwined, explaining how the key initial steps of light harvesting are affected by the solvent on the atomic time and length scale. Ultimately, this work takes a step forward in understanding the microscopic mechanisms of the bidirectional influence between transient solvent reorganization and photoinduced solute structural dynamics.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Šresolution when exposed to XFEL radiation, which is an improvement of 0.15 Šover previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Hole Dynamics in Photoexcited Hematite Studied with Femtosecond Oxygen K-edge X-ray Absorption Spectroscopy.

Hematite (α-Fe2O3) is a photoelectrode for the water splitting process because of its relatively narrow bandgap and abundance in the earth's crust. In this study, the photoexcited state of a hematite thin film was investigated with femtosecond oxygen K-edge X-ray absorption spectroscopy (XAS) at the PAL-XFEL in order to follow the dynamics of its photoexcited states. The 200 fs decay time of the hole state in the valence band was observed via its corresponding XAS feature.

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.

Femtosecond X-ray spectroscopy of haem proteins.

We discuss our recently reported femtosecond (fs) X-ray emission spectroscopy results on the ligand dissociation and recombination in nitrosylmyoglobin (MbNO) in the context of previous studies on ferrous haem proteins. We also present a preliminary account of femtosecond X-ray absorption studies on MbNO, pointing to the presence of more than one species formed upon photolysis.

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.

Spin cascade and doming in ferric hemes: Femtosecond X-ray absorption and X-ray emission studies.

The structure-function relationship is at the heart of biology, and major protein deformations are correlated to specific functions. For ferrous heme proteins, doming is associated with the respiratory function in hemoglobin and myoglobins. Cytochrome c (Cyt c) has evolved to become an important electron-transfer protein in humans. In its ferrous form, it undergoes ligand release and doming upon photoexcitation, but its ferric form does not release the distal ligand, while the return to the ground state has been attributed to thermal relaxation. Here, by combining femtosecond Fe Kα and Kß X-ray emission spectroscopy (XES) with Fe K-edge X-ray absorption near-edge structure (XANES), we demonstrate that the photocycle of ferric Cyt c is entirely due to a cascade among excited spin states of the iron ion, causing the ferric heme to undergo doming, which we identify. We also argue that this pattern is common to a wide diversity of ferric heme proteins, raising the question of the biological relevance of doming in such proteins.