Photochemical C-H activation reactions mediated by transition metal complexes often proceed via the formation of σ-complexes. In these intermediates, the metal center coordinates to a C-H σ-bond, and metal-ligand donation and back-donation interactions ultimately lead to C-H bond cleavage. Because metal-alkane σ-complexes are weakly bond and short-lived, current experimental methods provide limited access to the transient electronic-structure effects that control their reactivity. Here, we photochemically prepared three different types of Rh-alkane σ-complexes in solution and demonstrate how optical pump and X-ray probe spectroscopy gives access to their decisive valence-electron interactions. With femtosecond-resolution and Rh specific X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray scattering (RIXS) at the Rh L3-edge we access the orbital interactions between the Rh centers, the ancillary ligands and the alkane C-H σ-bonds of the Rh-alkane σ-complexes. Supported by theoretical calculations, we identify spectral fingerprints of Rh-alkane donation and back-donation interactions and find trends for the reactivity of the σ-complexes toward C-H activation. We uncover the particular importance of specific occupied molecular orbitals with specific symmetry in modulating reactivity by providing, channeling and directing electron density via the metal to and from the C-H bond. Elucidating the electronic factors that facilitate C-H bond activation provides a basis for future ligand substitutions aimed at enhancing the reactivity of metal-alkane σ-complexes with enhanced efficiency of the activation step.

The Alvra experimental station at the Swiss X-ray free-electron laser, SwissFEL, investigates ultrafast dynamics in chemical and biological systems using X-ray scattering and spectroscopy techniques. A key feature of Alvra is its unique capability to perform time-resolved X-ray emission and resonant inelastic X-ray scattering in the tender X-ray regime, currently not available at other XFEL endstations, combined with simultaneous access to X-ray absorption spectroscopy and X-ray solution scattering. Together with sub-35 fs time resolution enabled by advanced timing diagnostics, this positions Alvra as a versatile instrument for ultrafast chemical dynamics in the liquid phase. Here we cover the various technical aspects of the beamline and experimental station, and present some examples of experimental results measured during the first years of SwissFEL commissioning and user operation.

Ultrafast pump-probe serial femtosecond crystallography (SFX) experiments are typically performed with extremely high excitation laser fluence to maximize the occupancy of the light-induced state. This has been justified by assuming that a dominant fraction (up to 99%) of the pump laser light is scattered by the jetting medium, strongly reducing the pump laser fluence in the crystals which, therefore, do not absorb multiple photons and thus undergo the biologically relevant single-photon photoreaction. However, this notion is strongly contested in the field. To address the issue of pump laser scattering losses in high viscosity jets, we performed time-resolved SFX experiments on fatty acid photodecarboxylase crystals using high-viscosity extrusion (HVE) and both patterned and non-patterned sheet-on-sheet (SOS) fixed-target sample delivery systems on the Cristallina-MX instrument at SwissFEL. Tightly focused pump laser beams were used to mitigate light contamination (the undesired accidental illumination of neighboring crystals) in SOS chips, paving the way for their use in pump-probe investigations of lipidic-cubic-phase-grown membrane protein crystals. Determination of the light-induced reaction intermediate occupancies enabled a comparison of the influence of the delivery method on the pump laser fluence reaching the crystals. We show that optically transparent high-viscosity jets and chip crystal delivery methods result in photoproduct yields that approach 70-80% and 60-70%, respectively, of computationally derived yields that take into account excitation probabilities. These results demonstrate that clear viscous jets scatter pump laser light far less than has been claimed. Consequently, the use of excessive photoexcitation energy densities is neither needed nor justified in optically pumped time-resolved SFX experiments when using appropriately sized crystals.

Abstract Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades 1 , and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 Å resolution: from excited-state distortion of the flavin ring (10–100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100–500 ns), to the thioether bond itself, caught half-formed at 1 µs (half the molecules reacted, half still poised) and complete at 10–100 µs. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from ∼35 to ∼15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be ∼237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

Abstract Cyanobacteria have produced Earth’s oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity– dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCP O ) generates a metastable intermediate (OCP 1hν ) that either relaxes thermally or, upon absorption of a second photon within ∼1 s, converts to the active photoprotective state (OCP R ). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP 1hν , establish its functional relevance and capture structural snapshots along the OCP O →OCP 1hν and OCP 1hν →OCP R photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

Cytochrome c oxidase (CcO) utilizes the energy released as dioxygen is reduced to two water molecules to pump protons across an energy transducing biological membrane. X-ray structures of CcO have been known for three decades1-3 and all electron transfer and proton uptake and release steps have been characterized.4,5 However, it remains unclear how the reduction of dioxygen at the enzyme’s active-site achieves proton pumping. Here we apply time-resolved serial femtosecond X-ray crystallography to observe structural changes in the ba3-type CcO from Thermus thermophilus following the release of photocaged oxygen using a UV laser flash. In the resting oxidised state, chloride ions are observed to ligate CuB of the active site and the farnesyl oxygen of haem a3. Both ions are displaced when the enzyme is fully reduced. Water molecules are observed to order and disorder within the active-site 3.3 ms after photocaged oxygen is released, creating and disrupting water mediated pathways for proton transport through the enzyme. Requiring that the active-site charges sum to zero for every step of the catalytic cycle yields a step-by-step atomistic model for proton pumping by ba3-type CcOs.

The SwissFEL currently provides the hard X-ray endstations Alvra, Bernina, and Cristallina. Especially Alvra is a forerunner in the crystallographic community, having successfully performed many serial femtosecond crystallography experiments, using both, high-viscosity extruders (HVE) and GDVN jets as the sample delivery method [1-3]. With the recently commissioned SwissMX experiment based at Cristallina, there is now also a dedicated setup for fixed-target experiment, both in high-throughput and pump-probe mode, with a mixing setup on the horizon. However, available beamtime at FELs is sparse and entry barrier for new teams is high.To alleviate these issues and close the probe-time-gap in the millisecond-to-second regime, we build the VESPA endstation at the Swiss Light Source (SLS), dedicated to multi-time-resolved serial millisecond crystallography [4], acoustic levitation goniometry [5], and kilohertz data acquisition serial crystallography [6]. The latter allowed us to push the achievable time resolution at a synchrotron source to microseconds, without the need for choppers. In combination with different pump methods, including dedicated cw and nanosecond Lasers, as well as temperature control, and ligand mixing, this will enable our research community to investigate an even larger array of protein samples.We will introduce the recently formed PSI focus team for time resolved crystallography, which is dedicated to facilitating easy access to facilities and instruments, as well as providing training and support for research teams interested to get into the field. We will also present an overview of available techniques and expertise, including results from our experimental portfolio, at both SwissFEL and the SLS, and will present an outlook on novel techniques and instruments, especially in light of the upcoming SLS 2.0 upgrade.

Sheet-on-sheet (SOS) fixed-target chips are arguably the most versatile, cheapest and simplest sample-delivery method for ambient-temperature data acquisition using serial crystallography approaches at synchrotrons and X-ray free-electron lasers (XFELs). Their defining feature, the absence of any hard-patterned restrictions around crystals, is their strength as it removes limitations on crystal sizes or environments. However, it is also their weakness when it comes to limiting undesired effects on yet-to-be-irradiated crystals due to diffusing heat, radicals or gas originating from previous exposures. We explored whether SOS chips can be used for damage-free serial data collection on the new ID29 beamline at the ESRF-EBS, a fourth-generation synchrotron light source, as well as at the new Cristallina-MX station at SwissFEL. We collected serial data sets from microcrystals of the hemoprotein DtpAa, which was reported to have a highly radiation-sensitive iron–water bond length. The data sets differ in step size between exposures within and between lines of a serpentine-like data-acquisition scan. We observe no significant changes in the distance of the water ligand of the heme in the structures obtained from the ID29 SSX data. However, when compared with those collected at Cristallina-MX, the diffraction intensities collected at ID29 suggest global damage akin to Bragg termination occurring during the 90 µs exposure at ID29. Moreover, differences in the heme geometry and the proximal histidine–iron bond length point to local damage in all ID29 data sets regardless of the X-ray spacing. SFX data collected at Cristallina-MX show a phase transition of the DtpAa crystal lattice for X-ray step sizes of ≤20 µm. This phase transition might be caused by heating and/or hydrogen-gas-induced crystal dehydration. Vigilance remains required to safeguard against radiation damage at fourth-generation synchrotrons and XFELs.

In recent years, time-resolved serial crystallography has emerged as a transformative technique for unraveling the intricate dynamics of macromolecules at atomic resolution. By leveraging the high-intensity and ultra-short pulses of X-ray free electron lasers (XFELs) alongside the high brilliance of synchrotron light sources, this technique has enabled the observation of transient states in biomolecules as they catalyze chemical reactions.This presentation will highlight the advancements and applications of time-resolved serial crystallography in the study of macromolecular dynamic. We will discuss the light-sensitive membrane protein Nonlabens marinus halorhodopsin (NmHR) as an example of how this method enables us to capture the structural dynamics from femtoseconds to milliseconds after light activation. Through combining time-resolved studies at the X-ray free electron laser and synchrotron with spectroscopy and chemical simulation, we obtained a comprehensive understanding of the molecular mechanism that allows NmHR to catalyze ion transport across biological membranes. In addition to discussing the rich chemical information that can be obtained in time-resolved crystallographic studies, this talk will highlight how steady-state experiments can provide exciting structural insights while requiring only a limited amount of beamtime and a minimal setup.

Serial femtosecond crystallography (SFX) exploits extremely brief X-ray free-electron laser pulses to obtain diffraction data before destruction of the crystal. However, during the pulse X-ray-induced site-specific radiation damage can occur, leading to electronic state and/or structural changes. Here, we present a systematic exploration of the effect of single-pulse duration and energy (and consequently different dose rates) on site-specific radiation damage under typical SFX room-temperature experimental conditions. For the first time in SFX we directly measured the photon pulse duration, varying from less than 10 fs to more than 50 fs, and used three pulse energies to probe in-pulse damage in two radiation-sensitive proteins: the iron-heme peroxidase DtpAa and the disulfide-rich thaumatin. While difference-map features arising from radiation damage are observed, they do not lead to significant change in refined atomic coordinates or key bond lengths. Our work thus provides experimental verification that average atomic coordinates are not significantly perturbed by radiation damage in typical SFX experiments.

Microbial rhodopsins form a diverse family of light-sensitive seven-transmembrane helix retinal proteins that function as active proton or ion pumps, passive light-gated ion channels, and photosensors. To understand how light-sensing in archaea is initiated by sensory rhodopsins, we perform serial synchrotron X-ray crystallography (SSX) studies of light induced conformational changes in sensory rhodopsin II (NpSRII) from the archaea Natronomonas pharaonis, both collecting time-resolved SSX data and collecting SSX data during continuous illumination. Comparing light-induced electron density changes in NpSRII with those reported for bacteriorhodopsin (bR) reveals several common light-induced structural perturbations. Unlike bR, however, helix G of NpSRII does not unwind near the conserved lysine residue to which retinal is covalently bound and therefore transient water molecule binding sites do not arise immediately to the cytoplasmic side of retinal. These structural differences prolong the duration of the NpSRII photocycle relative to bR, allowing time for the light-initiated sensory signal to be amplified.

Metabotropic glutamate receptor 5 (mGlu5) is implicated in various neurodegenerative disorders, making it an attractive drug target. Although several ligand-bound crystal structures of mGlu5 exist, their apo-state crystal structure remains unknown. Here, we study mGlu5 structural changes using the photochemical affinity switch, alloswitch-1, in combination with time-resolved freeze-trapping methods. By X-ray crystallography, we demonstrated that isomerizing alloswitch-1 leads to its release from the binding pocket within a few seconds. The apo structure, determined at a resolution of 2.9 Å, is more comparable to the inactive state than to the active state. Our approach presents an accessible alternative to time-resolved serial crystallography for capturing thermodynamically stable transient intermediates. The mGlu5 apo-structure provides molecular insights into the ligand-free allosteric pocket, which can guide the design of new allosteric modulators.

Channelrhodopsins, light-gated cation channels, enable precise control of neural cell depolarization or hyperpolarization with light in the field of optogenetics. This study integrates time-resolved serial crystallography and atomistic molecular dynamics (MD) simulations to resolve the structural changes during C1C2 channelrhodopsin activation. Our observations reveal that within the crystal environment, C1C2 predominantly remains in a light-activated state with characteristics of the M390 intermediate. Here, rearrangement of retinal within its binding pocket partially opens the central gate toward the extracellular vestibule. These structural changes initiate channel opening but were insufficient to allow K+ flow. Adjusting protonation states to represent the subsequent N520 intermediate in our MD simulations induced further conformational changes, including rearrangements of transmembrane helices 2 and 7, that opened the inner gate and the putative ion-translocation pathway. This allowed spontaneous cation conduction with low conductance, aligning with experimental findings. Our findings provide critical structural insights into key intermediates of the channel opening mechanism, enhancing our understanding of ion conduction and selectivity in channelrhodopsins at an atomistic level.

Time-resolved serial crystallography at X-ray Free Electron Lasers offers the opportunity to observe ultrafast photochemical reactions at the atomic level. The technique has yielded exciting molecular insights into various biological processes including light sensing and photochemical energy conversion. However, to achieve sufficient levels of activation within an optically dense crystal, high laser power densities are often used, which has led to an ongoing debate to which extent photodamage may compromise interpretation of the results. Here we compare time-resolved serial crystallographic data of the bacteriorhodopsin K-intermediate collected at laser power densities ranging from 0.04 to 2493 GW/cm2 and follow energy dissipation of the absorbed photons logarithmically from picoseconds to milliseconds. Although the effects of high laser power densities on the overall structure are small, in the upper excitation range we observe significant changes in retinal conformation and increased heating of the functionally critical counterion cluster. We compare light-activation within crystals to that in solution and discuss the impact of the observed changes on bacteriorhodopsin biology. Time-resolved serial crystallography at XFELs reveals ultrafast photochemical reactions, but high laser densities can cause photodamage to biological samples. Here, the authors study the early K-intermediate in bacteriorhodopsin at high power, showing overall conformation remains robust over a wide range.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. To trigger such protein-ligand interaction dynamics within the human adenosine A 2A receptor, we designed seven photochemical affinity switches derived from the anti-Parkinson’s drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, this disrupts interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, followed by large-scale receptor rearrangements upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceutics.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. Here we show how to trigger and resolve protein-ligand interaction dynamics within the human adenosine A2A receptor. For this, we designed seven photochemical affinity switches derived from the anti-Parkinson's drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-to-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, are disrupted and rearrangement of the orthosteric binding pocket is invoked upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceuticals.

X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump–probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light–oxygen–voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump–probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Light–oxygen–voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

The first demonstration of 2 kHz time-resolved serial crystallography data acquisition at a fourth-generation synchrotron, using the JUNGFRAU 4M pixel detector.