Inorganic-organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties
. This proliferation has led to a ...characterization bottleneck: many hybrid materials are obligate microcrystals with low symmetry and severe radiation sensitivity, interfering with the standard techniques of single-crystal X-ray diffraction
and electron microdiffraction
. Here we demonstrate small-molecule serial femtosecond X-ray crystallography (smSFX) for the determination of material crystal structures from microcrystals. We subjected microcrystalline suspensions to X-ray free-electron laser radiation
and obtained thousands of randomly oriented diffraction patterns. We determined unit cells by aggregating spot-finding results into high-resolution powder diffractograms. After indexing the sparse serial patterns by a graph theory approach
, the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data
. We describe the ab initio structure solutions of mithrene (AgSePh)
, thiorene (AgSPh) and tethrene (AgTePh), of which the latter two were previously unknown structures. In thiorene, we identify a geometric change in the silver-silver bonding network that is linked to its divergent optoelectronic properties
. We demonstrate that smSFX can be applied as a general technique for structure determination of beam-sensitive microcrystalline materials at near-ambient temperature and pressure.
Abstract
Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the ...transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S
2
to S
3
transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.
We study the structural dynamics of liquid water by time-resolved anisotropic x-ray scattering under the optical Kerr effect condition. In this way, we can separate the anisotropic scattering decay ...of 160 fs from the delayed temperature increase of ∼ 0.1K occurring at 1 ps and quantify transient changes in the O-O pair distribution function. Polarizable molecular dynamics simulations reproduce well the experiment, indicating transient alignment of molecules along the electric field, which shortens the nearest-neighbor distances. In addition, analysis of the simulated water local structure provides evidence that two hypothesized fluctuating water configurations exhibit different polarizability.
Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering ...(CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.
Serial crystallography of membrane proteins often employs high‐viscosity injectors (HVIs) to deliver micrometre‐sized crystals to the X‐ray beam. Typically, the carrier medium is a lipidic cubic ...phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self‐assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X‐ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three‐way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co‐flowing gas conditions used to stabilize the LCP stream. A combination of laboratory‐based optical polarization microscopy and simulation studies was used to investigate these observations.
This is a study of the phase changes detected in monoolein samples under constant flow using a high‐viscousity injector. The sample behaviour was studied using X‐ray techniques while light microscopy and modelling studies were used to help interpret some of the effects observed in the data.
Most crystallographic data processing methods use pixel integration. In serial femtosecond crystallography (SFX), the intricate interaction between the reciprocal lattice point and the Ewald sphere ...is integrated out by averaging symmetrically equivalent observations recorded across a large number (10
-10
) of exposures. Although sufficient for generating biological insights, this approach converges slowly, and using it to accurately measure anomalous differences has proved difficult. This report presents a novel approach for increasing the accuracy of structure factors obtained from SFX data. A physical model describing all observed pixels is defined to a degree of complexity such that it can decouple the various contributions to the pixel intensities. Model dependencies include lattice orientation, unit-cell dimensions, mosaic structure, incident photon spectra and structure factor amplitudes. Maximum likelihood estimation is used to optimize all model parameters. The application of prior knowledge that structure factor amplitudes are positive quantities is included in the form of a reparameterization. The method is tested using a synthesized SFX dataset of ytterbium(III) lysozyme, where each X-ray laser pulse energy is centered at 9034 eV. This energy is 100 eV above the Yb
-III absorption edge, so the anomalous difference signal is stable at 10 electrons despite the inherent energy jitter of each femtosecond X-ray laser pulse. This work demonstrates that this approach allows the determination of anomalous structure factors with very high accuracy while requiring an order-of-magnitude fewer shots than conventional integration-based methods would require to achieve similar results.
Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons ...can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A
adenosine receptor (A
AR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A
AR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.
During X-ray exposure of a molecular solution, photons scattered from the same molecule are correlated. If molecular motion is insignificant during exposure, then differences in momentum transfer ...between correlated photons are direct measurements of the molecular structure. In conventional small- and wide-angle solution scattering, photon correlations are ignored. This report presents advances in a new biomolecular structural analysis technique, correlated X-ray scattering (CXS), which uses angular intensity correlations to recover hidden structural details from molecules in solution. Due to its intense rapid pulses, an X-ray free electron laser (XFEL) is an excellent tool for CXS experiments. A protocol is outlined for analysis of a CXS data set comprising a total of half a million X-ray exposures of solutions of small gold nanoparticles recorded at the Spring-8 Ångström Compact XFEL facility (SACLA). From the scattered intensities and their correlations, two populations of nanoparticle domains within the solution are distinguished: small twinned, and large probably non-twinned domains. It is shown analytically how, in a solution measurement, twinning information is only accessible
intensity correlations, demonstrating how CXS reveals atomic-level information from a disordered solution of like molecules.
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the ...roomtemperature structures of PS II in the four (semi)stable S-states, S₁, S₂, S₃, and S₀, showing that a water molecule is inserted during the S₂ → S₃ transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O₂ formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S₂ → S₃ transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 μs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 μs) during the S₂ → S₃ transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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