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Abstract 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.

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.

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.

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.

Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.

Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

Serial femtosecond crystallography using X-ray free-electron lasers has huge potential for time-resolved structural experiments. Here, the authors present a structure of the light-driven proton pump bacteriorhodopsin using these techniques.

Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae.

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.

Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.