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Understanding how ice nucleates and grows into larger crystals is of crucial importance for many research fields. The purpose of this study was to shed light on the phase and structure of ice once a nucleus is formed inside a metastable water droplet. Wide-angle X-ray scattering (WAXS) was performed on micron-sized droplets evaporatively cooled to temperatures where homogeneous nucleation occurs. We found that for our weak hits ice grows more cubic compared to the strong hits that are completely hexagonal. Due to efficient heat removal caused by evaporation, we propose that the cubicity of ice at the vicinity of the droplet’s surface is higher than for ice formed within the bulk of the droplet. Moreover, the Bragg peaks were classified based on their geometrical shapes and positions in reciprocal space, which showed that ice grows heterogeneously with a significant population of peaks indicative of truncation rods and crystal defects. Frequent occurrences of the (100) reflection with extended in-planar structure suggested that large planar ice crystals form at the droplet surface, then fracture into smaller domains to accommodate to the curvature of the droplets. Planar faulting due to misaligned domains would explain the increased cubicity close to the droplet surface.

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 affects the dynamics and climate feedback of clouds 12 , 13 and can accelerate cloud freezing through secondary ice production 14 – 17 . Droplet freezing occurs at several timescales 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. On the basis of 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, whereas diffraction from the remaining liquid became similar to that from quasi-liquid layers on premelted ice 19 , 20 . The ice had a strained hexagonal crystal structure just after freezing, which is an early metastable state that probably 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. Optical microscopy and X-ray diffraction are used to study the freezing of water droplets in vacuum, leading to the development of a seven-stage model of freezing and the mapping of ice structures and crystal order.

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals—50 unit cells per edge, on average—has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation. The structure of the bacterial toxin BinAB, which is used to combat mosquito-borne diseases, reveals pH-sensitive switches and carbohydrate-binding modules that may contribute to the larvicidal function of the toxin. Structure of the mosquito larvicide BinAB Mosquitoes continue to be the insects most injurious to human health, because they act as a vector for several infectious diseases including malaria, dengue fever, West Nile encephalitis, and Zika virus. The Lysinibacillus sphaericus binary toxin BinAB is distributed worldwide to combat mosquito-borne diseases, because these proteins are toxic to their targets, but harmless to humans and other animals. These authors used serial-femtosecond crystallography at an X-ray free-electron laser source to solve the structure of BinAB crystals. The structure reveals four pH-sensitive switches that facilitate crystal dissolution in the larval midgut as well as three carbohydrate-binding modules in BinA that may assist in directing heterodimers to the cell surface.

Explosions are spectacular and intriguing phenomena that expose the dynamics of matter under extreme conditions. We investigated, using time-resolved imaging, explosions induced by ultraintense X-ray laser pulses in water drops and jets. Our observations revealed an explosive vaporization followed by high-velocity interacting flows of liquid and vapour, and by the generation of shock trains in the liquid jets. These flows are different from those previously observed in laser ablation, owing to a simpler spatial pattern of X-ray absorption. We show that the explosion dynamics in our experiments is consistent with a redistribution of absorbed energy, mediated by a pressure or shock wave in the liquid, and we model the effects of explosions, including their adverse impact on X-ray laser experiments. X-ray laser explosions have predictable dynamics that may prove useful for controlling the state of pure liquids over broad energy scales and timescales, and for triggering pressure-sensitive molecular dynamics in solutions. X-ray-induced explosions in water drops, examined using time-resolved imaging, show interacting high-speed liquid and vapour flows. This type of X-ray absorption dynamics is predictable and may be used for inducing particular dynamical liquid states.

A computational approach and software tool, cctbx.xfel, enables the determination of accurate macromolecular structure factors using a relatively small number of serial femtosecond crystallography diffraction snapshots. X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

A concentric-flow microfluidic electrokinetic sample injector enables efficient delivery of microcrystals in their mother liquor for serial femtosecond X-ray crystallography with minimal sample consumption. We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

X-ray free-electron lasers (XFELs) open up new possibilities for X-ray crystallographic and spectroscopic studies of radiation-sensitive biological samples under close to physiological conditions. To facilitate these new X-ray sources, tailored experimental methods and data-processing protocols have to be developed. The highly radiation-sensitive photosystem II (PSII) protein complex is a prime target for XFEL experiments aiming to study the mechanism of light-induced water oxidation taking place at a Mn cluster in this complex. We developed a set of tools for the study of PSII at XFELs, including a new liquid jet based on electrofocusing, an energy dispersive von Hamos X-ray emission spectrometer for the hard X-ray range and a high-throughput soft X-ray spectrometer based on a reflection zone plate. While our immediate focus is on PSII, the methods we describe here are applicable to a wide range of metalloenzymes. These experimental developments were complemented by a new software suite, cctbx.xfel. This software suite allows for near-real-time monitoring of the experimental parameters and detector signals and the detailed analysis of the diffraction and spectroscopy data collected by us at the Linac Coherent Light Source, taking into account the specific characteristics of data measured at an XFEL.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally toxic oligomeric pores. The small size of the crystals, 50 unit cells per edge, on average, has impeded structural characterization by conventional means. Here, we report the structure of BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser (XFEL). The structure reveals tyrosine and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.