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Femtosecond XFEL crystallography is used to identify dynamic changes in the adenine riboswitch aptamer domain, with at least four states identified in real time, two in the apo form before binding and two with the ligand bound. Riboswitch RNA reaction-state structures The potential of nanocrystallography to offer insights into dynamics is now beginning to be realized. Yun-Xing Wang and colleagues have used femtosecond X-ray free-electron laser (XFEL) crystallography to study dynamic changes in the ligand-binding, or aptamer, domain of the Vibrio vulnificus adenine riboswitch. They identify at least four states, two in the apo form before binding and two with ligand bound, in real time. These results allow the modelling of a kinetic scheme that describes how ligand binding transmits a signal through the P1 helix. The large-scale conformational changes captured within the crystal are enabled by the time-resolved serial crystallography. Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform 1 , 2 , 3 . A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time 4 . Here we use femtosecond X-ray free electron laser (XFEL) pulses 5 , 6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Femtosecond X-ray laser pulses are used to probe the structure of liquid water in micrometre-sized droplets that have been cooled below the homogeneous ice nucleation temperature, revealing the existence of metastable bulk liquid water down to temperatures of 227 kelvin. Anomalous bevaviour of supercooled water Water's anomalous physical properties become markedly enhanced upon supercooling below the freezing point and even seem to diverge towards infinity at around 228 K. Two papers in this issue use contrasting techniques to study this little-explored 'no-man's land' of water where extremely fast ice formation has prohibited measurements of the liquid state. Jonas Sellberg et al . use femtosecond X-ray laser pulses to measure bulk liquid water structure in droplets evaporatively cooled to 227 K. Even at this temperature some droplets remained liquid on a millisecond timescale. Pushing this technique further can shed light on controversial scenarios that aim to describe and explain the many anomalous properties of water. Jeremy Palmer et al . use six advanced computational methods to demonstrate the existence of two metastable liquid phases of ST2 water at the same deeply supercooled condition, undergoing a liquid–liquid transition that meets stringent thermodynamic criteria and could explain the behavior of water in this regime. Water has a number of anomalous physical properties, and some of these become drastically enhanced on supercooling below the freezing point. Particular interest has focused on thermodynamic response functions that can be described using a normal component and an anomalous component that seems to diverge at about 228 kelvin (refs 1 , 2 , 3 ). This has prompted debate about conflicting theories 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 that aim to explain many of the anomalous thermodynamic properties of water. One popular theory attributes the divergence to a phase transition between two forms of liquid water occurring in the ‘no man’s land’ that lies below the homogeneous ice nucleation temperature ( T H ) at approximately 232 kelvin 13 and above about 160 kelvin 14 , and where rapid ice crystallization has prevented any measurements of the bulk liquid phase. In fact, the reliable determination of the structure of liquid water typically requires temperatures above about 250 kelvin 2 , 15 . Water crystallization has been inhibited by using nanoconfinement 16 , nanodroplets 17 and association with biomolecules 16 to give liquid samples at temperatures below T H , but such measurements rely on nanoscopic volumes of water where the interaction with the confining surfaces makes the relevance to bulk water unclear 18 . Here we demonstrate that femtosecond X-ray laser pulses can be used to probe the structure of liquid water in micrometre-sized droplets that have been evaporatively cooled 19 , 20 , 21 below T H . We find experimental evidence for the existence of metastable bulk liquid water down to temperatures of  kelvin in the previously largely unexplored no man’s land. We observe a continuous and accelerating increase in structural ordering on supercooling to approximately 229 kelvin, where the number of droplets containing ice crystals increases rapidly. But a few droplets remain liquid for about a millisecond even at this temperature. The hope now is that these observations and our detailed structural data will help identify those theories that best describe and explain the behaviour of water.

Upon exposure to ultra-intense, hard X-ray pulses, polyatomic molecules containing one heavy atom reach a much higher degree of ionization than do individual heavy atoms, contrary to previous assumptions. Ultrafast molecular response to intense X-rays X-ray free-electron lasers offer many new applications such as the ability to structurally probe fast biological processes. This requires the use of hard and intense X-ray pulses, but the behaviour of matter under such conditions has not been fully explored. Artem Rudenko et al . show that when exposing small polyatomic molecules that contain one heavy atom to hard X-ray pulses with ultra-high intensities, the response is qualitatively different from what is seen in experiments carried out under less extreme conditions. The observed ionization of the molecule is considerably enhanced compared to that of an individual heavy atom under the same conditions, owing to ultrafast charge transfer within the molecule that replenishes the electrons removed from the heavy atom, enabling further ionization. Being able to account for this effect will aid further use of X-ray free-electron lasers for studying biological systems. X-ray free-electron lasers enable the investigation of the structure and dynamics of diverse systems, including atoms, molecules, nanocrystals and single bioparticles, under extreme conditions 1 , 2 , 3 , 4 , 5 , 6 , 7 . Many imaging applications that target biological systems and complex materials use hard X-ray pulses with extremely high peak intensities (exceeding 10 20 watts per square centimetre) 3 , 5 . However, fundamental investigations have focused mainly on the individual response of atoms and small molecules using soft X-rays with much lower intensities 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 . Studies with intense X-ray pulses have shown that irradiated atoms reach a very high degree of ionization, owing to multiphoton absorption 8 , 12 , 13 , 18 , which in a heteronuclear molecular system occurs predominantly locally on a heavy atom (provided that the absorption cross-section of the heavy atom is considerably larger than those of its neighbours) and is followed by efficient redistribution of the induced charge 14 , 15 , 16 , 17 , 19 , 20 . In serial femtosecond crystallography of biological objects—an application of X-ray free-electron lasers that greatly enhances our ability to determine protein structure 2 , 3 —the ionization of heavy atoms increases the local radiation damage that is seen in the diffraction patterns of these objects 21 , 22 and has been suggested as a way of phasing the diffraction data 23 , 24 . On the basis of experiments using either soft or less-intense hard X-rays 14 , 15 , 16 , 17 , 18 , 19 , 25 , it is thought that the induced charge and associated radiation damage of atoms in polyatomic molecules can be inferred from the charge that is induced in an isolated atom under otherwise comparable irradiation conditions. Here we show that the femtosecond response of small polyatomic molecules that contain one heavy atom to ultra-intense (with intensities approaching 10 20 watts per square centimetre), hard (with photon energies of 8.3 kiloelectronvolts) X-ray pulses is qualitatively different: our experimental and modelling results establish that, under these conditions, the ionization of a molecule is considerably enhanced compared to that of an individual heavy atom with the same absorption cross-section. This enhancement is driven by ultrafast charge transfer within the molecule, which refills the core holes that are created in the heavy atom, providing further targets for inner-shell ionization and resulting in the emission of more than 50 electrons during the X-ray pulse. Our results demonstrate that efficient modelling of X-ray-driven processes in complex systems at ultrahigh intensities is feasible.

The ultrafast evolution of microstructure is key to understanding high-pressure and strain-rate phenomena. However, the visualization of lattice dynamics at scales commensurate with those of atomistic simulations has been challenging. Here, we report femtosecond x-ray diffraction measurements unveiling the response of copper to laser shock-compression at peak normal elastic stresses of ~73 gigapascals (GPa) and strain rates of 10⁹ per second. We capture the evolution of the lattice from a one-dimensional (1D) elastic to a 3D plastically relaxed state within a few tens of picoseconds, after reaching shear stresses of 18 GPa. Our in situ high-precision measurement of material strength at spatial (< 1 micrometer) and temporal (< 50 picoseconds) scales provides a direct comparison with multimillion-atom molecular dynamics simulations.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

X-ray free-electron lasers can generate radiation pulses with extreme peak intensities at short wavelengths. This enables the investigation of laser–matter interactions in a regime of high fields, yet at a non-relativistic ponderomotive potential, where ordinary rules of light–matter interaction may no longer apply and nonlinear processes are starting to become observable. Despite small cross-sections, first nonlinear effects in the hard x-ray regime have recently been observed in solid targets, including x-ray-optical sum-frequency generation (XSFG), x-ray second harmonic generation (XSHG) and two-photon Compton scattering (2PCS). Nonlinear interactions of bound electrons in the x-ray range are fundamentally different from those dominating at optical frequencies. Whereas in the optical regime nonlinearities are predominantly caused by anharmonicities of the atomic potential in the chemical bonds, x-ray nonlinearities far above atomic resonances are expected to be due to nonlinear oscillations of quasi-free electrons, including inner-shell atomic electrons. While the quasi-free-electron model agrees reasonably well with the experimental data for XSFG and XSHG, 2PCS measurements have led to unexpected results: the energy of the nonlinearly scattered photons from non-relativistic electrons shows a substantial unexpected red shift in addition to the Compton shift that is well beyond that predicted by a nonlinear quantum electrodynamics model for free electrons. A potential explanation for the spectral broadening is based on a previously unexplored scattering process that involves the whole atom rather than just quasi-free electrons. A first simulation that includes the atomic binding potential was successful in describing a broadening of the spectrum of the nonlinearly scattered photons to longer wavelengths for soft x-rays. However, the same model does not show any broadening at hard x-ray wavelengths, which is in agreement with other simulation approaches. To this point no calculation has been able to reproduce the experimentally observed broadening. Here we present further experimental data of 2PCS for an extended parameter range using additional diagnostics. In particular, we present measurements of the electron momentum distribution during the interaction that strongly suggest that the spectral broadening is not caused by an increased plasma temperature. We extend our measurement of the magnitude of the red shift in beryllium to > 1.9 k e V in addition to the Compton shift expected for free electrons and expand the measurement of the angular distribution to include forward scattering angles. We also present first measurements of 2PCS from diamond.

The development of X-ray free-electron lasers (XFELs) has launched a new era in X-ray science by providing ultrafast coherent X-ray pulses with a peak brightness that is approximately one billion times higher than previous X-ray sources. The Linac Coherent Light Source (LCLS) facility at the SLAC National Accelerator Laboratory, the world’s first hard X-ray FEL, has already demonstrated a tremendous scientific impact across broad areas of science. Here, a few of the more recent representative highlights from LCLS are presented in the areas of atomic, molecular, and optical science; chemistry; condensed matter physics; matter in extreme conditions; and biology. This paper also outlines the near term upgrade (LCLS-II) and motivating science opportunities for ultrafast X-rays in the 0.25–5 keV range at repetition rates up to 1 MHz. Future plans to extend the X-ray energy reach to beyond 13 keV (<1 Å) at high repetition rate (LCLS-II-HE) are envisioned, motivated by compelling new science of structural dynamics at the atomic scale.

Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.

The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 6.3.1.2.), glutamate dehydrogenase (GDH3; EC 1.4.1.1.), and asparagine synthetase (AS; EC 6.3.1.1.) were up-regulated by water deficit but not by ABA, except for a gene encoding delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit