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.

Application of density matrix Wigner transforms for ultrafast macromolecular and chemical x-ray crystallography.

Time-Resolved Serial Femtosecond Crystallography (TR-SFX) conducted at X-ray Free Electron Lasers (XFELs) has become a powerful tool for capturing macromolecular structural movies of light-initiated processes. As the capabilities of XFELs advance, we anticipate that a new range of coherent control and structural Raman measurements will become achievable. Shorter optical and x-ray pulse durations and increasingly more exotic pulse regimes are becoming available at free electron lasers. Moreover, with high repetition enabled by the superconducting technology of European XFEL (EuXFEL) and Linac Coherent Light Source (LCLS-II) , it will be possible to improve the signal-to-noise ratio of the light-induced differences, allowing for the observation of vibronic motion on the sub-Angstrom level. To predict and assign this coherent motion, which is measurable with a structural technique, new theoretical approaches must be developed. In this paper, we present a theoretical density matrix approach to model the various population and coherent dynamics of a system, which considers molecular system parameters and excitation conditions. We emphasize the use of the Wigner transform of the time-dependent density matrix, which provides a phase space representation that can be directly compared to the experimental positional displacements measured in a TR-SFX experiment. Here, we extend the results from simple models to include more realistic schemes that include large relaxation terms. We explore a variety of pulse schemes using multiple model systems using realistic parameters. An open-source software package is provided to perform the density matrix simulation and Wigner transformations. The open-source software allows us to define any arbitrary level schemes as well as any arbitrary electric field in the interaction Hamiltonian.

The Carbonyl Group in ß2 of the Carotenoid Tunes the Photocycle Kinetics in Orange Carotenoid Protein.

Adaptation to rapid environmental changes is crucial for maintaining optimal photosynthetic efficiency and is ultimately key to the survival of all photosynthetic organisms. Like most of them, cyanobacteria protect their photosynthetic apparatus against rapidly increasing light intensities by nonphotochemical quenching (NPQ). In cyanobacteria, NPQ is controlled by Orange Carotenoid Protein (OCP) photocycle. OCP is the only known photoreceptor that uses carotenoid for its light activation. How carotenoid drives and controls this unique photoactivation process is still unknown. However, understanding and potentially controlling the OCP photocycle may open up new possibilities for improving photosynthetic biomass. Here we investigate the effect of the carbonyl group in the ß2 ring of the carotenoid on the OCP photocycle. We report microsecond to minute OCP light activation kinetics and Arrhenius plots of the two OCP forms: Canthaxanthin-bound OCP (OCPCAN) and echinenone-bound OCP (OCPECH). The difference between the two carotenoids is the presence of a carbonyl group in the ß2-ring located in the N-terminal domain of the protein. A combination of temperature-dependent spectroscopy, flash photolysis, and pump-probe transient absorption allows us to report the previously unresolved OCP intermediate associated primarily with the absorption bleach (OCPB). OCPB dominates the photokinetics in the µs to subms time range for OCPCAN and in the µs to ms range for OCPECH. We show that in OCPCAN the OCP photocycle steps are always faster than in OCPECH: from 2 to almost 20 times depending on the step. These results suggest that the presence of the carbonyl group in the ß2-ring of the carotenoid accelerates the OCP photocycle.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.

Photoacid Dynamics in the Green Fluorescent Protein.

The photoacid dynamics of fluorescent proteins include both electronic excited- and ground-state mechanisms of proton transfer. The associated characteristic timescales of these reactions range over many orders of magnitude, and the tunneling, barrier crossing, and relevant thermodynamics have in certain cases been linked to coherent nuclear motion. We review the literature and summarize the experiments and theory that demonstrate proton tunneling in the electronic ground state of the green fluorescent protein (GFP). We also discuss the excited-state proton-transfer reaction of GFP that takes place on the picosecond timescale. Although this reaction has been investigated using several vibrational spectroscopic methods, the interpretation remains unsettled. We discuss recent advances as well as remaining questions, in particular those related to the vibrational mode couplings that involve low-frequency modulations of chromophore vibrations on the timescale of proton transfer.

Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy.

The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm-1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.

Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization.

Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9'-C8' and C7'-C6' single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.

High Power Irradiance Dependence of Charge Species Dynamics in Hybrid Perovskites and Kinetic Evidence for Transient Vibrational Stark Effect in Formamidinium.

Hybrid halide perovskites materials have the potential for both photovoltaic and light-emitting devices. Relatively little has been reported on the kinetics of charge relaxation upon intense excitation. In order to evaluate the illumination power density dependence on the charge recombination mechanism, we have applied a femtosecond transient mid-IR absorption spectroscopy with strong excitation to directly measure the charge kinetics via electron absorption. The irradiance-dependent relaxation processes of the excited, photo-generated charge pairs were quantified in polycrystalline MAPbI3, MAPbBr3, and (FAPbI3)0.97(MAPbBr3)0.03 thin films that contain either methylamonium (MA) or formamidinium (FA). This report identifies the laser-generated charge species and provides the kinetics of Auger, bimolecular and excitonic decay components. The inter-band electron-hole (bimolecular) recombination was found to dominate over Auger recombination at very high pump irradiances, up to the damage threshold. The kinetic analysis further provides direct evidence for the carrier field origin of the vibrational Stark effect in a formamidinium containing perovskite material. The results suggest that radiative excitonic and bimolecular recombination in MAPbI3 at high excitation densities could support light-emitting applications.

Open hardware microsecond dispersive transient absorption spectrometer for linear optical response.

An open hardware design and implementation for a transient absorption spectrometer are presented that has microsecond time resolution and measures full difference spectra in the visible spectral region from 380 to 750 nm. The instrument has been designed to allow transient absorption spectroscopy measurements of either low or high quantum yield processes by combining intense sub-microsecond excitation flashes using a xenon lamp together with stroboscopic non-actinic white light probing using LED sources driven under high pulsed current from a capacitor bank. The instrument is sensitive to resolve 0.15 mOD flash-induced differences within 1000 measurements at 20 Hz repetition rate using an inexpensive CCD sensor with 200 µm pixel dimension, 40 K electrons full well capacity and a dynamic range of 1800. The excitation flash has 230 ns pulse duration and the 2 mJ flash energy allows spectral filtering while retaining high power density with focussing to generate mOD signals in the 10-4-10-1 ΔOD range. We present the full electronics design and construction of the flash and probe sources, the optics as well as the timing electronics and CCD spectrometer operation and modification for internal signal referencing. The performance characterisation and example measurements are demonstrated using microsecond TAS of Congo red dye, as an example of a low quantum yield photoreaction at 2% with up to 78% of molecules excited. The instrument is fully open hardware and combines inexpensive selection of commercial components, optics and electronics and allows linear response measurements of photoinduced reactions for the purpose of accurate global analysis of chemical dynamics.

Theory of two-dimensional spectroscopy with intense laser fields.

Two-dimensional vibrational and electronic spectroscopic observables of isotropically oriented molecular samples in solution are sensitive to laser field intensities and polarization. The third-order response function formalism predicts a signal that grows linearly with the field strength of each laser pulse, thus lacking a way of accounting for non-trivial intensity-dependent effects, such as saturation and finite bleaching. An analytical expression to describe the orientational part of the molecular response, which, in the weak-field limit, becomes equivalent to a four-point correlation function, is presented. Such an expression is evaluated for Liouville-space pathways accounting for diagonal and cross peaks for all-parallel and cross-polarized pulse sequences, in both the weak- and strong-field conditions, via truncation of a Taylor series expansion at different orders. The results obtained in the strong-field conditions suggest how a careful analysis of two-dimensional spectroscopic experimental data should include laser pulse intensity considerations when determining molecular internal coordinates.

Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210-1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Radical-Triggered Reaction Mechanism of the Green-to-Red Photoconversion of EosFP.

Reaction intermediates in the green-to-red photoconversion of the photochromic fluorescent protein EosFP have been observed using high-intensity continuous blue illumination. An intermediate was identified through light-induced accumulation that continues to convert the green form in subsequent darkness, putatively containing a tyrosyl radical, albeit with anomalously shifted features in both the electronic and FTIR spectra. Lowering the pH to 5.5 significantly delays the decay of this tyrosyl intermediate, which is accompanied by Stark-shifted features in the electronic spectra of reactants and products. Vibrational mode assignments for the high-frequency and fingerprint FTIR spectral regions of the reaction intermediates support a proposed sequence of events where the newly formed Cα═Cß ethylenic bond precedes modifications on the His-62 imidazole ring and confirms a C═O(NH2) product group on Phe-61. We propose a reaction mechanism that involves tyrosyl generation via singlet excited-state-mediated oxidation which subsequently triggers the covalent reactions by oxidation of the green chromophore.

Advances and opportunities in ultrafast X-ray crystallography and ultrafast structural optical crystallography of nuclear and electronic protein dynamics.

Both nuclear and electronic dynamics contribute to protein function and need multiple and complementary techniques to reveal their ultrafast structural dynamics response. Real-space information obtained from the measurement of electron density dynamics by X-ray crystallography provides aspects of both, while the molecular physics of coherence parameters and frequency-frequency correlation needs spectroscopy methods. Ultrafast pump-probe applications of protein dynamics in crystals provide real-space information through direct X-ray crystallographic structure analysis or through structural optical crystallographic analysis. A discussion of methods of analysis using ultrafast macromolecular X-ray crystallography and ultrafast nonlinear structural optical crystallography is presented. The current and future high repetition rate capabilities provided by X-ray free electron lasers for ultrafast diffraction studies provide opportunities for optical control and optical selection of nuclear coherence which may develop to access higher frequency dynamics through improvements of sensitivity and time resolution to reveal coherence directly. Specific selection of electronic coherence requires optical probes, which can provide real-space structural information through photoselection of oriented samples and specifically in birefringent crystals. Ultrafast structural optical crystallography of photosynthetic energy transfer has been demonstrated, and the theory of two-dimensional structural optical crystallography has shown a method for accessing the structural selection of electronic coherence.

Coherent two-dimensional electronic and infrared crystallography.

The two-dimensional electronic and infrared spectroscopy of oriented single crystals is sensitive to structure and point group symmetry. The third order response of crystals is generally different from measurements of isotropic solutions because each coherence path that contributes to the measured field scales to the ensemble average of the four-point correlation functions of the four field-dipole interactions involved in the respective Feynman paths. An analytical evaluation of 2D optical crystallography which depends on the crystal symmetry, laboratory orientation, and the orientation in the crystallographic frame is presented. Applying a symmetry operator in the basis of the allowed polarised radiation modes provides a method for evaluation of non-zero fourth rank tensor elements alternative to direct inspection methods. Uniaxial and biaxial systems are distinguished and the contributions to the rephasing and non-rephasing directions are evaluated for isolated and coupled oscillators. By exploiting coordinate analysis, the extension of non-linear electronic and infrared crystallography for coupled oscillators demonstrates the structural, directional, and symmetry dependent selection of coherences to the four-wave mixing signal.

Excited State Frequencies of Chlorophyll f and Chlorophyll a and Evaluation of Displacement through Franck-Condon Progression Calculations.

We present ground and excited state frequency calculations of the recently discovered extremely red-shifted chlorophyll f. We discuss the experimentally available vibrational mode assignments of chlorophyll f and chlorophyll a which are characterised by particularly large downshifts of 13¹-keto mode in the excited state. The accuracy of excited state frequencies and their displacements are evaluated by the construction of Franck-Condon (FC) and Herzberg-Teller (HT) progressions at the CAM-B3LYP/6-31G(d) level. Results show that while CAM-B3LYP results are improved relative to B3LYP calculations, the displacements and downshifts of high-frequency modes are underestimated still, and that the progressions calculated for low temperature are dominated by low-frequency modes rather than fingerprint modes that are Resonant Raman active.

Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography.

Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue 'Measurement of ultrafast electronic and structural dynamics with X-rays'.

Femtosecond infrared spectroscopy of chlorophyll f-containing photosystem I.

The recent discovery of extremely red-shifted chlorophyll f pigments in both photosystem I (PSI) and photosystem II has led to the conclusion that chlorophyll f plays a role not only in the energy transfer, but also in the charge separation processes [Nürnberg et al., Science, 2018, 360, 1210-1213]. We have employed ultrafast transient infrared absorption spectroscopy to study the contribution of far-red light absorbing chlorophyll f to energy transfer and charge separation processes in far-red light-grown PSI (FRL-PSI) from the cyanobacterium Chroococcidiopsis thermalis PCC 7203. We compare the kinetics and spectra of FRL-grown PSI excited at 670 nm and 740 nm wavelengths to those of white light-grown PSI (WL-PSI) obtained at 675 nm excitation. We report a fast decay of excited state features of chlorophyll a and complete energy transfer from chlorophyll a to chlorophyll f in FRL-PSI upon 670 nm excitation, as indicated by a frequency shift in a carbonyl absorption band occurring within a 1 ps timescale. While the WL-PSI measurements support the assignment of initial charge separation to A-1+˙A0-˙ [Di Donato et al., Biochemistry, 2011, 50, 480-490] from the kinetics of a distinct cation feature at 1710 cm-1, in the case of FRL-PSI, small features at 1715 cm-1 from the chlorophyll cation are present from sub-ps delays instead, supporting the replacement of the A-1 pigment with chlorophyll f. Comparisons of nanosecond spectra show that charge separation proceeds with 740 nm excitation, which selectively excites chlorophyll f, and modifications in specific carbonyl absorption bands assigned to P700+˙ minus P700 and A1-˙ minus A1 indicate dielectric differences of FRL-PSI compared to WL-PSI in one or both of the two electron transfer branches of FRL-PSI.

X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature.

The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.

PyLDM - An open source package for lifetime density analysis of time-resolved spectroscopic data.

Ultrafast spectroscopy offers temporal resolution for probing processes in the femto- and picosecond regimes. This has allowed for investigation of energy and charge transfer in numerous photoactive compounds and complexes. However, analysis of the resultant data can be complicated, particularly in more complex biological systems, such as photosystems. Historically, the dual approach of global analysis and target modelling has been used to elucidate kinetic descriptions of the system, and the identity of transient species respectively. With regards to the former, the technique of lifetime density analysis (LDA) offers an appealing alternative. While global analysis approximates the data to the sum of a small number of exponential decays, typically on the order of 2-4, LDA uses a semi-continuous distribution of 100 lifetimes. This allows for the elucidation of lifetime distributions, which may be expected from investigation of complex systems with many chromophores, as opposed to averages. Furthermore, the inherent assumption of linear combinations of decays in global analysis means the technique is unable to describe dynamic motion, a process which is resolvable with LDA. The technique was introduced to the field of photosynthesis over a decade ago by the Holzwarth group. The analysis has been demonstrated to be an important tool to evaluate complex dynamics such as photosynthetic energy transfer, and complements traditional global and target analysis techniques. Although theory has been well described, no open source code has so far been available to perform lifetime density analysis. Therefore, we introduce a python (2.7) based package, PyLDM, to address this need. We furthermore provide a direct comparison of the capabilities of LDA with those of the more familiar global analysis, as well as providing a number of statistical techniques for dealing with the regularization of noisy data.