Search

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, 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 temperature 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., 視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.

IUCrJ 8(6), 905 - 920 (2021). doi:10.1107/S2052252521008046, 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 [$����/��$ = 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 (����/�� = 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., Published by Chester

Using a number of model and real-life practical cases, the use of sample supports made of a thin perforated cyclic olefin co­polymer membrane for serial crystallography that enable low-background serial data collection is described., Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

We present the first lasing results of SwissFEL, a hard X-ray free-electron laser (FEL) that recently came into operation at the Paul Scherrer Institute in Switzerland. SwissFEL is a very stable, compact and cost-effective X-ray FEL facility driven by a low-energy and ultra-low-emittance electron beam travelling through short-period undulators. It delivers stable hard X-ray FEL radiation at 1-A wavelength with pulse energies of more than 500 μJ, pulse durations of ~30 fs (root mean square) and spectral bandwidth below the per-mil level. Using special configurations, we have produced pulses shorter than 1 fs and, in a different set-up, broadband radiation with an unprecedented bandwidth of ~2%. The extremely small emittance demonstrated at SwissFEL paves the way for even more compact and affordable hard X-ray FELs, potentially boosting the number of facilities worldwide and thereby expanding the population of the scientific community that has access to X-ray FEL radiation. The first lasing results at SwissFEL, an X-ray free-electron laser, are presented, highlighting the facility’s unique capabilities. A general comparison to other major facilities is also provided.

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets., IUCrJ, 7 (6), ISSN:2052-2525

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump. ISSN:0036-8075 ISSN:1095-9203

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., IUCrJ, 7 (6), ISSN:2052-2525

Use of the charge-integrating JUNGFRAU detector for fluorescence detection is investigated, and its applications in macromolecular crystallography at synchrotron and X-ray free-electron laser sources are demonstrated., Detection of heavy elements, such as metals, in macromolecular crystallography (MX) samples by X-ray fluorescence is a function traditionally covered at synchrotron MX beamlines by silicon drift detectors, which cannot be used at X-ray free-electron lasers because of the very short duration of the X-ray pulses. Here it is shown that the hybrid pixel charge-integrating detector JUNGFRAU can fulfill this function when operating in a low-flux regime. The feasibility of precise position determination of micrometre-sized metal marks is also demonstrated, to be used as fiducials for offline prelocation in serial crystallography experiments, based on the specific fluorescence signal measured with JUNGFRAU, both at the synchrotron and at SwissFEL. Finally, the measurement of elemental absorption edges at a synchrotron beamline using JUNGFRAU is also demonstrated.

A microcrystal-prelocation method is demonstrated using low-dose 2D full-field propagation-based X-ray phase-contrast imaging on samples with an essentially flat geometry for automated serial crystallography data collection at a microfocus macromolecular crystallography beamline., Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

In this paper, the design and functionalities of the high-throughput TELL sample exchange system for macromolecular crystallography is presented. TELL was developed at the Paul Scherrer Institute with a focus on speed, storage capacity and reliability to serve the three macromolecular crystallography beamlines of the Swiss Light Source, as well as the SwissMX instrument at SwissFEL.

A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the `diffract-before-destroy' regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si3N4windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution.

Strategies for sample delivery of macromolecular crystals at X-ray free-electron lasers are reviewed, covering injection methods, fixed-target approaches and hybrid methods., Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drugs development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets but the complex stability varies according to a pocket specific network of interactions. The following values have no corresponding Zotero field:auth-address: 1 Institut Européen de Chimie et Biologie , Université de Bordeaux-CNRS UMR 5248, CBMN , 2, rue Robert Escarpit , 33607 Pessac , France. 2 Swiss Light Source (SLS) , Paul-Scherrer-Institute (PSI) , 5232 Villigen , Switzerland. 3 Université de Strasbourg , CNRS, Architecture et Réactivité de l'ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS , 15 rue René Descartes , F-67000 Strasbourg , France. 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) , Département de Biologie Structurale et Génomique , 1 rue Laurent Fries , BP10142, 67404 Illkirch , France. 5 Biologie et Signalisation Cellulaire, ESBS , UMR7242 CNRS/Université de Strasbourg , Bld S. Brant , 67412 Illkirch Cedex , France.accession-num: 30912430

Vitamin A deficiency has a widespread occurrence globally and is considered as one of the world's most serious health risk factors. Potential solutions to address this deficiency include dietary diversification or supplementation, but food fortification is generally accepted as the most cost-effective solution. The main issue with food fortification of this vitamin is related to its high instability in food matrices. Dilution of vitamin A in triglycerides is a natural and appropriate way to stabilize this compound. We show here that vitamin A palmitate stability increases with increasing concentration of triglycerides. Moreover, we found that vitamin A palmitate displays improved stability in more saturated oils. Using various temperatures, and Arrhenius plots of experiments performed at storage temperatures between 30 °C and 60 °C for oils varying by their saturation and crystallinity, we demonstrate that crystallization is not responsible for this phenomenon. Additionally, we show by centrifugation that vitamin A is preferably solubilized in the liquid phase compared to the crystalline phase, explaining that triglyceride crystallization does not stabilize vitamin A palmitate. It is proposed that unsaturated fats generate more oxidation products such as radicals and peroxides, leading to a quicker degradation of vitamin A.

This article reports the incorporation of a fast continuous grid scan with both still and oscillation images into the Swiss Light Source macromolecular crystallography beamlines and its application in visualization of protein crystals with scanning transmission X-ray microscopy., A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities.

We present a process sequence for the fabrication of ultrathin perforated membranes suspended in polymer frames. Nanoimprint lithography was used to imprint high-density pillar arrays into a double layer consisting of 300 nm of water-soluble PVP and 2–3 μm COC sequentially spin coated onto a silicon wafer. After an RIE step to remove the residual layer and 3D-printing of PLA frames onto the structured COC film, the assemblies were released from the wafer by dissolving the sacrificial PVP bottom layer in water. The suspended membranes were developed as supports for serial protein crystallography, where small amounts of protein microcrystals are deposited onto a membrane and separated from most of their mother liquor through the sieving function of the membranes (blotting). After plunge freezing in liquid nitrogen crystallographic data are collected sequentially from the individual crystals, using X-rays from a synchrotron or femtosecond X-ray pulses of an X-ray free electron laser. Preliminary experiments confirmed that the supports allow for collection of good quality crystallographic data at high resolution, taking advantage of adequate liquid blotting, high stability and low X-ray interaction.

In situ data collection for macromolecular crystallography has undergone rapid development in the last decade. From routine crystal hit screening in X-ray-friendly SBS-format plates to serial data collection in dedicated setups, its field of application has expanded significantly. The in situ approach has benefitted from progress in beamline instrumentation and automation and from new modes of sample delivery, inspired by serial crystallography using X-ray free-electron lasers. In this review, the various in situ methods available at synchrotron facilities are described with a view to enabling users to make informed decisions regarding their applicability in structural biology or drug discovery projects.

The SwissFEL X-ray Free Electron Laser (XFEL) facility started construction at the Paul Scherrer Institute (Villigen, Switzerland) in 2013 and will be ready to accept its first users in 2018 on the Aramis hard X-ray branch. In the following sections we will summarize the various aspects of the project, including the design of the soft and hard X-ray branches of the accelerator, the results of SwissFEL performance simulations, details of the photon beamlines and experimental stations, and our first commissioning results.

Upon the addition of minute quantities of water into a phosphatidylcholine (PC) solution in certain organic solvents, PC micelles elongate into giant reverse wormlike micelles that entangle and form highly viscous microemulsions, called lecithin organogels. We investigated the microrheological properties of concentrated PC-cyclohexane reverse wormlike micellar systems by diffusive wave spectroscopy (DWS) in apolar medium, combined with bulk shear rheology. We applied DWS to our oil-continuous system by using hydrophobic poly(hydroxystearic acid)-grafted PMMA particles as monodisperse tracer particles. Relevant parameters such as the micellar scission energy and persistence length were extracted from the microrheology data and interpreted according to the sphere-to-rod-to-sphere structural transition. On the basis of these quantities, we calculated the bending and saddle-splay moduli of the PC-covered water-cyclohexane interface. This approach represents a new method for the quantitative estimation of these fundamental parameters, which are thought to underpin the self-assembly of surfactants.

Errors in the article by Opara, Martiel, Arnold, Braun, Stahlberg, Makita, David & Padeste [J. Appl. Cryst. (2017), 50, 909–918] are corrected.

Lipid self-assembled structures (SASs) have recently gained considerable interest for their potential applications, especially for sustained nutrient release and protein crystallization. An additional property, which is underexploited, is their ability to control chemical reactions in food products. Here, we concentrate on SASs formed by phospholipids (PLs) and monoglycerides (MGs), those compounds being the most natural surfactants and therefore, the best compatible with food products, in view of providing new functionalities through the formation of SASs. In this work, the phase behaviour of these amphiphiles when mixed with oil and water is described and compared. Subsequently, we address the influence of these structures to the oxidation and Maillard-type reactions. Finally, we show that SASs formed by MGs can strongly increase the yield of key aroma impact compounds generated by Maillard-type reactions when compared with the reaction performed in aqueous precursor solutions. Various SASs are compared. In particular, addition of oil to a reversed bicontinuous structure formed by MG leads to a reversed microemulsion, which, considering its low viscosity, is particularly suitable for food products and act as a very efficient reactor system. The influence of oil and precursors on phase behaviour is discussed and related to the efficiency of the Maillard reactions. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

The control of the diffusion coefficient by the dimensionality d of the structure appears as a most promising lever to efficiently tune the release rate from lyotropic liquid crystalline (LLC) phases and dispersed particles towards sustained, controlled and targeted release. By using phosphatidylcholine (PC)- and monolinoleine (MLO)-based mesophases with various apolar structural modifiers and water-soluble drugs, we present a comprehensive study of the dimensional structural control of hydrophilic drug release, including 3-d bicontinuous cubic, 2-d lamellar, 1-d hexagonal and 0-d micellar cubic phases in excess water. We investigate how the surfactant, the oil properties and the drug hydrophilicity mitigate or even cancel the effect of structure variation on the drug release rate. Unexpectedly, the observed behavior cannot be fully explained by the thermodynamic partition of the drug into the lipid matrix, which points out to previously overlooked kinetic effects. We therefore interpret our results by discussing the mechanism of structural control of the diffusion rate in terms of drug permeation through the lipid membrane, which includes exchange kinetics. A wide range of implications follow regarding formulation and future developments, both for dispersed LLC delivery systems and topical applications in bulk phase.

There is a need for the development of low-energy dispersion methods tailored to the formation of phospholipid-based nonlamellar lyotropic liquid crystalline (LLC) particles for delivery system applications. Here, facile formation of nonlamellar LLC particles was obtained by simple mixing of a phosphatidylcholine (PC) liposome solution and an oil-in-water emulsion, with limonene or isooctane as an oil. The internal structure of the particles was controlled by the PC-to-oil ratio, consistently with the sequence observed in bulk phase. For the first time, reverse micellar cubosomes with Fm3̅m inner structure were produced. The size, morphology, and inner structure of the particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and freeze-fracture cryo scanning electron microscopy (cryo-SEM). These findings pave the way to new strategies in low-energy formulation of LLC delivery systems.

Submicron sized, structured lyotropic liquid crystalline (LLC) particles, so-called hexosomes and cubosomes, are generally obtained by high energy input dispersion methods, notably ultrasonication and high-pressure emulsification. We present a method to obtain dispersions of such LLC particles with a significantly reduced energy input, by evaporation of an auxiliary volatile solvent immiscible with water, e.g. cyclohexane or limonene. The inner structure of the particles can be precisely controlled by the addition of a nonvolatile oil, such as α-tocopherol or tetradecane consistently with bulk phase diagrams,. Two different lyotropic surfactants were employed, industrial grade monolinoleine (MLO) and soy bean phosphatidylcholine (PC). The lyotropic surfactant and oil phase modifier were first dissolved in the volatile solvent to give a liquid reverse micellar (L2) phase, which requires significantly less energy input to be dispersed in an aqueous solution of secondary emulsifier compared to the corresponding gel-like bulk mesophase. The auxiliary volatile solvent was then removed from the emulsion by evaporation at room temperature, yielding LLC particles of the desired inner structure, Pn3̅m, H2, or Fd3̅m. The obtained particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Our method enables fine-tuning of the final particle size through the volatile-to-nonvolatile volume ratio and processing conditions.

Phospholipids are ubiquitous cell membrane components and relatively well-accepted ingredients due to their natural origin. Phosphatidylcholine (PC) in particular offers a promising alternative to monoglycerides for lyotropic liquid crystalline (LLC) delivery system applications in the food, cosmetics and pharmaceutical industries, provided its strong tendency to form zero-mean curvature lamellar mesophases in water can be overcome. Higher negative curvatures are usually reached through the addition of a third lipid component, forming a ternary diagram phospholipid/water/oil. The initial part of this work summarizes the potential advantages and the challenges of phospholipid-based delivery system applications. In the next part, various ternary PC/water/oil systems are discussed, with a special emphasis on the PC/water/cyclohexane and PC/water/α-tocopherol systems. We report that R-(+)-limonene has a quantitatively similar effect as cyclohexane. The last part is devoted to the theoretical interpretation of the observed phase behaviors. A fruitful parallel is drawn with PC polymer-like reverse micelles, leading to a thermodynamic description in terms of interfacial bending energy. Investigations at the molecular level are reviewed to help in bridging the empirical and theoretical approaches. Predictive rules are finally derived from this wide-ranging overview, thereby opening the way to a future rational design of PC-based LLC delivery systems.

We report the formation of a reverse micellar cubic mesophase of symmetry Fm3m (Q(225)) in ternary mixtures of soy bean phosphatidylcholine (PC), water, and an organic solvent, including cyclohexane, (R)-(+)-limonene, and isooctane, studied by small-angle X-ray scattering (SAXS) and oscillatory shear rheology at room temperature. The mesophase structure consists of a compact packing of remarkably large reverse micelles in a face-centered cubic (fcc) lattice, a type of micellar packing not yet reported for reverse micellar mesophases. Form factor fitting in the pure L2 phase and in the Fm3m-L2 coexistence region yields quantitative estimations of the PC interface rigidity. The compact Fm3m structure results mainly from release of lipid tail frustration and hard-sphere interactions between monodisperse micelles, as suggested by a comparison with the Fd3m structure found in the PC/water/α-tocopherol system.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes. ISSN:0028-0836 ISSN:1476-4687