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The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisonsmore » between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.« less

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 tomore » 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.« less

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL^TIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88^TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88^TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL^TIR. We demonstrate via mutagenesismore » that the MyD88^TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88^TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.« less

Here, the new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamasemore » from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.« less

Here, the unique properties of nanodiamonds make them suitable for use in a wide range of applications, including as biomarkers for cellular tracking in vivo at the molecular level. The sustained fluorescence of nanodiamonds containing nitrogen-vacancy (N-V) centres is related to their internal structure and strain state. Theoretical studies predict that the location of the N-V centre and the nanodiamonds' residual elastic strain state have a major influence on their photoluminescence properties. However, to date there have been no direct measurements made of their spatially resolved deformation fields owing to the challenges that such measurements present. Here we apply themore » recently developed technique of Bragg coherent diffractive imaging (BCDI) to map the three-dimensional deformation field within a single nanodiamond of approximately 0.5 µm diameter. The results indicate that there are high levels of residual elastic strain present in the nanodiamond which could have a critical influence on its optical and electronic properties.« less

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered 'random'. This would then allow the supposedly incoherent contribution from the electronic motion to be treatedmore » as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C₆₀. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C₆₀ were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. Our paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.« less

X-ray free-electron lasers (XFELs) deliver x-ray pulses with a coherent flux that is approximately eight orders of magnitude greater than that available from a modern third-generation synchrotron source. The power density of an XFEL pulse may be so high that it can modify the electronic properties of a sample on a femtosecond time scale. Exploration of the interaction of intense coherent x-ray pulses and matter is both of intrinsic scientific interest and of critical importance to the interpretation of experiments that probe the structures of materials using high-brightness femtosecond XFEL pulses. We report observations of the diffraction of extremely intensemore » 32-fs nanofocused x-ray pulses by a powder sample of crystalline C₆₀. We find that the diffraction pattern at the highest available incident power significantly differs from the one obtained using either third-generation synchrotron sources or XFEL sources operating at low output power and does not correspond to the diffraction pattern expected from any known phase of crystalline C₆₀. We interpret these data as evidence of a long-range, coherent dynamic electronic distortion that is driven by the interaction of the periodic array of C₆₀ molecular targets with intense x-ray pulses of femtosecond duration.« less