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.
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.
Serial Femtosecond Crystallography (SFX) is the most commonly used method for the emerging structure determination at X-ray free-electron lasers (FELs). The high peak brilliance of the FEL and the possibility of using femtosecond pulses afford use of nano-to-micron sized crystals in a diffraction-before-destruction approach for the acquisition of high-resolution undamaged diffraction data [1]. The crystals are obliterated upon exposure to an FEL X-ray pulse so only a single snapshot can be collected per crystal, necessitating a constant supply of fresh crystals. The crystals are therefore injected in a liquid microjet [2], [3]. We show that this serial method of data collection and the associated data analysis can be successfully adapted to serial crystallography (SX) measurements at synchrotrons, enabling room temperature studies using the unattenuated beam. Given the continuous supply of fresh crystals, the full tolerable dose can be used for each single crystal exposure, permitting analysis of small or weakly scattering crystals. FEL X-ray pulses are much shorter than the fraction of a second exposure time at a synchrotron, so SFX injection conditions are modified in SX such as to slow down the typically fast travelling crystals. By embedding the crystals in a viscous material the crystals remain in the beam long enough to yield measurable diffraction and smearing out of the diffraction peaks due to crystal tumbling is avoided. We demonstrate the successful application of room temperature SX at the Swiss Light Source at ambient pressure. Our experimental setup allows collection of both still and rotation data. Recent progress using model systems will be presented, establishing this high throughput, high dose rate approach as a new route to structure determination of macromolecules in their native environment and at room temperature.
Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.
Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes.In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxidetoform the catalytic intermediate compound I(Fe IV = Oa nd ap orphyrin cation radical). Using serial femtosecond X-rayc rystallography (SFX), we have determined the pristine structures of the Fe III and Fe IV =Oredox states of aB-type DyP.These structures reveal aw ater-free distal heme site that, together with the presence of an asparagine,imply the use of the distal arginine as ac atalytic base.Acombination of mutagenesis and kinetic studies corroborate such ar ole.O ur SFX approach thus provides unique insight into howt he distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxideh eterolysis.
Type 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD N ) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH P -FimD N ternary complex. We report the structure of a new ternary complex (FimC-FimF t -FimD N ) with the subunit FimF t instead of FimH p . FimD N recognizes FimC-FimF t and FimC-FimH P very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a ''hot spot'' on the chaperone surface could guide the design of pilus assembly inhibitors.
The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K 6PG that ranges from 5-to 800-fold. There is also a less pronounced increase in K NADP , ranging from 3-to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E t (600-and 300-fold, respectively) as well as in V/K 6PG E t (10 5 -and 10 4 -fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E t was observed for the Y191A mutant enzyme, whereas 20-and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K 6PG E t range from 3-to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13 C-isotope effect ؊1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.6-Phosphogluconate dehydrogenase (6PGDH 2 ; EC 1.1.1.44) catalyzes the reversible oxidative decarboxylation of 6-phosphogluconate (6PG), producing ribulose 5-phosphate (Ru5P) and CO 2 with the concomitant reduction of NADP to NADPH. The kinetic mechanism is rapid equilibrium random on the basis of a complete kinetic characterization of the sheep liver enzyme and Candida utilis (1, 2). The pH dependence of kinetic parameters indicates a general acid-general base chemical mechanism (1, 2), and sitedirected mutagenesis (3, 4) studies suggest that Lys-183 and Glu-190 are likely the general base and the general acid, respectively. In this mechanism, the general base (Lys-183) is required to accept a proton from the 3-hydroxyl group of 6PG concomitant with hydride transfer from C-3 of 6PG to the coenzyme. Reduction of the nicotinamide ring is accompanied by rotation around the N-glycosidic bond such that the ring occupies the position formerly occupied by the 1-carboxylate of the substrate (5). The resulting 3-keto-6-phosphogluconate intermediate is decarboxylated to produce the enediol of Ru5P with the general base used to protonate the carboxyl oxygen. A general acid (Glu-190) is needed to facilitate the tautomerization of the enediol of ...
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