X-ray diffraction of metastable structures from supercooled liquid hydrogen.

We report time resolved observations of the crystallization from liquid hydrogen, supercooled to temperatures below the melting point, using 11.2 keV X-ray diffraction from the Linac Coherent Light Source (LCLS). Changes to the metastable solid and liquid structure factors have been dynamically measured. This allows for a direct determination of the lowest energy crystal polymorphs, the stacking probabilities, as well as the liquid and solid densities and temperatures. Such measurements provide experimental evidence of an Arrhenius-like growth kinetics along the stacking direction during supercooling.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

Modeling diffuse scattering with simple, physically interpretable models.

Diffuse scattering has long been proposed to probe protein dynamics relevant for biological function, and more recently, as a tool to aid structure determination. Despite recent advances in measuring and modeling this signal, the field has not been able to routinely use experimental diffuse scattering for either application. A persistent challenge has been to devise models that are sophisticated enough to robustly reproduce experimental diffuse features but remain readily interpretable from the standpoint of structural biology. This chapter presents eryx, a suite of computational tools to evaluate the primary models of disorder that have been used to analyze protein diffuse scattering. By facilitating comparative modeling, eryx aims to provide insights into the physical origins of this signal and help identify the sources of disorder that are critical for reproducing experimental features. This framework also lays the groundwork for the development of more advanced models that integrate different types of disorder without loss of interpretability.

Melting domain size and recrystallization dynamics of ice revealed by time-resolved x-ray scattering.

The phase transition between water and ice is ubiquitous and one of the most important phenomena in nature. Here, we performed time-resolved x-ray scattering experiments capturing the melting and recrystallization dynamics of ice. The ultrafast heating of ice I is induced by an IR laser pulse and probed with an intense x-ray pulse which provided us with direct structural information on different length scales. From the wide-angle x-ray scattering (WAXS) patterns, the molten fraction, as well as the corresponding temperature at each delay, were determined. The small-angle x-ray scattering (SAXS) patterns, together with the information extracted from the WAXS analysis, provided the time-dependent change of the size and the number of liquid domains. The results show partial melting (~13%) and superheating of ice occurring at around 20 ns. After 100 ns, the average size of the liquid domains grows from about 2.5 nm to 4.5 nm by the coalescence of approximately six adjacent domains. Subsequently, we capture the recrystallization of the liquid domains, which occurs on microsecond timescales due to the cooling by heat dissipation and results to a decrease of the average liquid domain size.

Advancing structural biology through breakthroughs in AI.

The past century has witnessed an exponential increase in our atomic-level understanding of molecular and cellular mechanisms from a structural perspective, with multiple landmark achievements contributing to the field. This, coupled with recent and continuing breakthroughs in artificial intelligence methods such as AlphaFold2, and enhanced computational power, is enabling our understanding of protein structure and function at unprecedented levels of accuracy and predictivity. Here, we describe some of the major recent advances across these fields, and describe, as these technologies coalesce, the potential to utilise our enhanced knowledge of intricate cellular and molecular systems to discover novel therapeutics to alleviate human suffering.

Liquid-liquid phase separation in supercooled water from ultrafast heating of low-density amorphous ice.

Recent experiments continue to find evidence for a liquid-liquid phase transition (LLPT) in supercooled water, which would unify our understanding of the anomalous properties of liquid water and amorphous ice. These experiments are challenging because the proposed LLPT occurs under extreme metastable conditions where the liquid freezes to a crystal on a very short time scale. Here, we analyze models for the LLPT to show that coexistence of distinct high-density and low-density liquid phases may be observed by subjecting low-density amorphous (LDA) ice to ultrafast heating. We then describe experiments in which we heat LDA ice to near the predicted critical point of the LLPT by an ultrafast infrared laser pulse, following which we measure the structure factor using femtosecond x-ray laser pulses. Consistent with our predictions, we observe a LLPT occurring on a time scale < 100 ns and widely separated from ice formation, which begins at times >1 µs.

Observation of site-selective chemical bond changes via ultrafast chemical shifts.

The concomitant motion of electrons and nuclei on the femtosecond time scale marks the fate of chemical and biological processes. Here we demonstrate the ability to initiate and track the ultrafast electron rearrangement and chemical bond breaking site-specifically in real time for the carbon monoxide diatomic molecule. We employ a local resonant x-ray pump at the oxygen atom and probe the chemical shifts of the carbon core-electron binding energy. We observe charge redistribution accompanying core-excitation followed by Auger decay, eventually leading to dissociation and hole trapping at one site of the molecule. The presented technique is general in nature with sensitivity to chemical environment changes including transient electronic excited state dynamics. This work provides a route to investigate energy and charge transport processes in more complex systems by tracking selective chemical bond changes on their natural timescale.

Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography.

Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Following the Crystallization of Amorphous Ice after Ultrafast Laser Heating.

Using time-resolved wide-angle X-ray scattering, we investigated the early stages (10 µs-1 ms) of crystallization of supercooled water, obtained by the ultrafast heating of high- and low-density amorphous ice (HDA and LDA) up to a temperature T = 205 K ± 10 K. We have determined that the crystallizing phase is stacking disordered ice (Isd), with a maximum cubicity of χ = 0.6, in agreement with predictions from molecular dynamics simulations at similar temperatures. However, we note that a growing small portion of hexagonal ice (Ih) was also observed, suggesting that within our timeframe, Isd starts annealing into Ih. The onset of crystallization, in both amorphous ice, occurs at a similar temperature, but the observed final crystalline fraction in the LDA sample is considerably lower than that in the HDA sample. We attribute this discrepancy to the thickness difference between the two samples.

Anomalous temperature dependence of the experimental x-ray structure factor of supercooled water.

The structural changes of water upon deep supercooling were studied through wide-angle x-ray scattering at SwissFEL. The experimental setup had a momentum transfer range of 4.5 Å-1, which covered the principal doublet of the x-ray structure factor of water. The oxygen-oxygen structure factor was obtained for temperatures down to 228.5 ± 0.6 K. Similar to previous studies, the second diffraction peak increased strongly in amplitude as the structural change accelerated toward a local tetrahedral structure upon deep supercooling. We also observed an anomalous trend for the second peak position of the oxygen-oxygen structure factor (q2). We found that q2 exhibits an unprecedented positive partial derivative with respect to temperature for temperatures below 236 K. Based on Fourier inversion of our experimental data combined with reference data, we propose that the anomalous q2 shift originates from that a repeat spacing in the tetrahedral network, associated with all peaks in the oxygen-oxygen pair-correlation function, gives rise to a less dense local ordering that resembles that of low-density amorphous ice. The findings are consistent with that liquid water consists of a pentamer-based hydrogen-bonded network with low density upon deep supercooling.

Nonuniform Flow Dynamics Probed by Nanosecond X-Ray Speckle Visibility Spectroscopy.

We report observations of nanosecond nonuniform colloidal dynamics in a free flowing liquid jet using ultrafast x-ray speckle visibility spectroscopy. Utilizing a nanosecond double-bunch mode, the Linac Coherent Light Source free electron laser produced pairs of femtosecond coherent hard x-ray pulses. By exploring anisotropy in the visibility of summed speckle patterns which relates to the correlation functions, we evaluate not only the average particle flow rate in a colloidal nanoparticle jet, but also the nonuniform flow field within. The methodology presented here establishes the foundation for the study of nano- and atomic-scale inhomogeneous fluctuations in complex matter using x-ray free electron laser sources.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet.

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet. We explored this matter with an X-ray-pump/X-ray-probe experiment employing haemoglobin microcrystals transported via a liquid jet into the XFEL beam. Diffraction data were collected using a shock-wave-free single-pulse scheme as well as the dual-pulse pump-probe scheme. The latter, relative to the former, reveals significant degradation of crystal hit rate, diffraction resolution and data quality. Crystal structures extracted from the two data sets also differ. Since our pump-probe attributes were chosen to emulate EuXFEL operation at its 4.5 MHz maximum pulse rate, this prompts concern about such data collection.

Enhancement and maximum in the isobaric specific-heat capacity measurements of deeply supercooled water using ultrafast calorimetry.

Knowledge of the temperature dependence of the isobaric specific heat (Cp) upon deep supercooling can give insights regarding the anomalous properties of water. If a maximum in Cp exists at a specific temperature, as in the isothermal compressibility, it would further validate the liquid-liquid critical point model that can explain the anomalous increase in thermodynamic response functions. The challenge is that the relevant temperature range falls in the region where ice crystallization becomes rapid, which has previously excluded experiments. Here, we have utilized a methodology of ultrafast calorimetry by determining the temperature jump from femtosecond X-ray pulses after heating with an infrared laser pulse and with a sufficiently long time delay between the pulses to allow measurements at constant pressure. Evaporative cooling of ∼15-µm diameter droplets in vacuum enabled us to reach a temperature down to ∼228 K with a small fraction of the droplets remaining unfrozen. We observed a sharp increase in Cp, from 88 J/mol/K at 244 K to about 218 J/mol/K at 229 K where a maximum is seen. The Cp maximum is at a similar temperature as the maxima of the isothermal compressibility and correlation length. From the Cp measurement, we estimated the excess entropy and self-diffusion coefficient of water and these properties decrease rapidly below 235 K.

Femtosecond quantification of void evolution during rapid material failure.

Understanding high-velocity impact, and the subsequent high strain rate material deformation and potential catastrophic failure, is of critical importance across a range of scientific and engineering disciplines that include astrophysics, materials science, and aerospace engineering. The deformation and failure mechanisms are not thoroughly understood, given the challenges of experimentally quantifying material evolution at extremely short time scales. Here, copper foils are rapidly strained via picosecond laser ablation and probed in situ with femtosecond x-ray free electron (XFEL) pulses. Small-angle x-ray scattering (SAXS) monitors the void distribution evolution, while wide-angle scattering (WAXS) simultaneously determines the strain evolution. The ability to quantifiably characterize the nanoscale during high strain rate failure with ultrafast SAXS, complementing WAXS, represents a broadening in the range of science that can be performed with XFEL. It is shown that ultimate failure occurs via void nucleation, growth, and coalescence, and the data agree well with molecular dynamics simulations.

Experimental observation of the liquid-liquid transition in bulk supercooled water under pressure.

We prepared bulk samples of supercooled liquid water under pressure by isochoric heating of high-density amorphous ice to temperatures of 205 ± 10 kelvin, using an infrared femtosecond laser. Because the sample density is preserved during the ultrafast heating, we could estimate an initial internal pressure of 2.5 to 3.5 kilobar in the high-density liquid phase. After heating, the sample expanded rapidly, and we captured the resulting decompression process with femtosecond x-ray laser pulses at different pump-probe delay times. A discontinuous structural change occurred in which low-density liquid domains appeared and grew on time scales between 20 nanoseconds to 3 microseconds, whereas crystallization occurs on time scales of 3 to 50 microseconds. The dynamics of the two processes being separated by more than one order of magnitude provides support for a liquid-liquid transition in bulk supercooled water.