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Supercooled water droplets are widely used to study supercooled water [1,2], ice nucleation [3-5], and droplet freezing [6-11]. Their freezing in the atmosphere impacts the dynamics and the climate feedback of clouds [12,13], and can accelerate cloud freezing via secondary ice production [14-17]. Droplet freezing occurs at multiple time and length scales [14,18], and is sufficiently stochastic to make it unlikely that two frozen drops are identical. Here we use optical microscopy and X-ray laser diffraction to investigate the freezing of tens of thousands of water microdrops in vacuum after homogeneous ice nucleation around 234–235 K. Based on drop images, we developed a seven-stage model of freezing and used it to time the diffraction data. Diffraction from ice crystals showed that long-range crystalline order formed in less than 1 ms after freezing, while diffraction from the remaining liquid became similar to the one from quasiliquid layers on premelted ice [19,20]. The ice had a strained hexagonal crystal structure just after freezing, which is an early metastable state that likely precedes the formation of ice with stacking defects [8,9,18]. The techniques reported here could help determine the dynamics of freezing in other conditions, such as drop freezing in clouds, or help understand rapid solidification in other materials. 

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A_2Areceptor (A_2AAR) at 2.0 Å resolution is determined and compared with previous A_2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize. 

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. 

Using X-ray free-electron lasers (XFELs), it is possible to determine three-dimensional structures of nanoscale particles using single-particle imaging methods. Classification algorithms are needed to sort out the single-particle diffraction patterns from the large amount of XFEL experimental data. However, different methods often yield inconsistent results. This study compared the performance of three classification algorithms: convolutional neural network, graph cut and diffusion map manifold embedding methods. The identified single-particle diffraction data of the PR772 virus particles were assembled in the three-dimensional Fourier space for real-space model reconstruction. The comparison showed that these three classification methods lead to different datasets and subsequently result in different electron density maps of the reconstructed models. Interestingly, the common dataset selected by these three methods improved the quality of the merged diffraction volume, as well as the resolutions of the reconstructed maps. 

An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits. 

The analysis of a single-particle imaging (SPI) experiment performed at the AMO beamline at LCLS as part of the SPI initiative is presented here. A workflow for the three-dimensional virus reconstruction of the PR772 bacteriophage from measured single-particle data is developed. It consists of several well defined steps including single-hit diffraction data classification, refined filtering of the classified data, reconstruction of three-dimensional scattered intensity from the experimental diffraction patterns by orientation determination and a final three-dimensional reconstruction of the virus electron density without symmetry constraints. The analysis developed here revealed and quantified nanoscale features of the PR772 virus measured in this experiment, with the obtained resolution better than 10 nm, with a clear indication that the structure was compressed in one direction and, as such, deviates from ideal icosahedral symmetry. 

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation – in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described. 

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.