Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Light-driven sodium pumps actively transport small cations across cellular membranes 1 .They are used by microbes to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. While resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved 2,3 , it is unclear how structural alterations over time allow sodium translocation against a concentration gradient. Using the Swiss X-ray Free Electron Laser 4 , we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. Highresolution 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 transient binding of a sodium ion close to the retinal within one millisecond. In the last structural intermediate at 20 ms 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.

This article describes the structure determination of a membrane protein by serial injection of microcrystals in lipidic cubic phases into a synchrotron microfocus beam. The method is discussed with respect to serial femtosecond crystallography at free-electron lasers.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.

We present active-state structures of the G protein-coupled receptor (GPCRs) rhodopsin carrying the disease-causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non-progressive form of RP. Our analysis shows that the CSNBcausing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q-K296 activation switch and the covalent binding of the inverse agonist 11-cis-retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.

Light-sensitive G protein-coupled receptors (GPCRs)—rhodopsins—absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that “reisomerize” upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more “activation-ready” conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure–function relationship of both photosensitive and nonphotosensitive class A GPCRs.

We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 μm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.