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Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and the complex experimental infrastructure that are required remain serious obstacles to its widespread application. Here, millisecond mix-and-quench crystallography (MMQX) has been developed, which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxy-kinase (PEPCK) is observed with a time resolution of 40 ms. By lowering the entry barrier to time-resolved crystallography, MMQX should enable a broad expansion in structural studies of protein dynamics.

Diffraction data acquired from cryocooled protein crystals often include diffraction from ice. Analysis of ice diffraction from crystals of three proteins shows that the ice formed within solvent cavities during rapid cooling is comprised of a stacking-disordered mixture of hexagonal and cubic planes, with the cubic plane fraction increasing with increasing cryoprotectant concentration and increasing cooling rate. Building on the work of Thorn and coworkers [Thorn et al. (2017), Acta Cryst. D73, 729-727], a revised metric is defined for detecting ice from deposited protein structure-factor data, and this metric is validated using full-frame diffraction data from the Integrated Resource for Reproducibility in Macromolecular Crystallography. Using this revised metric and improved algorithms, an analysis of structure-factor data from a random sample of 89 827 PDB entries collected at cryogenic temperatures indicates that roughly 16% show evidence of ice contamination, and that this fraction increases with increasing solvent content and maximum solvent-cavity size. By examining the ice diffraction-peak positions at which structure-factor perturbations are observed, it is found that roughly 25% of crystals exhibit ice with primarily hexagonal character, indicating that inadequate cooling rates and/or cryoprotectant concentrations were used, while the remaining 75% show ice with a stacking-disordered or cubic character.

When protein crystals are abruptly cooled, the unit-cell, protein and solvent-cavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperature-dependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with small-amplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to ∼200 K.

The local Fourier-space relation between diffracted intensity I, diffraction wavevector q and dose D, , is key to probing and understanding radiation damage by X-rays and energetic particles in both diffraction and imaging experiments. The models used in protein crystallography for the last 50 years provide good fits to experimental I(q) versus nominal dose data, but have unclear physical significance. More recently, a fit to diffraction and imaging experiments suggested that the maximum tolerable dose varies as q -1 or linearly with resolution. Here, it is shown that crystallographic data have been strongly perturbed by the effects of spatially nonuniform crystal irradiation and diffraction during data collection. Reanalysis shows that these data are consistent with a purely exponential local dose dependence, = I 0(q)exp[-D/D e(q)], where D e(q) ∝ q α with α ≃ 1.7. A physics-based model for radiation damage, in which damage events occurring at random locations within a sample each cause energy deposition and blurring of the electron density within a small volume, predicts this exponential variation with dose for all q values and a decay exponent α ≃ 2 in two and three dimensions, roughly consistent with both diffraction and imaging experiments over more than two orders of magnitude in resolution. The B-factor model used to account for radiation damage in crystallographic scaling programs is consistent with α = 2, but may not accurately capture the dose dependencies of structure factors under typical nonuniform illumination conditions. The strong q dependence of radiation-induced diffraction decays implies that the previously proposed 20-30 MGy dose limit for protein crystallography should be replaced by a resolution-dependent dose limit that, for atomic resolution data sets, will be much smaller. The results suggest that the physics underlying basic experimental trends in radiation damage at T ≃ 100 K is straightforward and universal. Deviations of the local I(q, D) from strictly exponential behavior may provide mechanistic insights, especially into the radiation-damage processes responsible for the greatly increased radiation sensitivity observed at T ≃ 300 K.

Ice formation within protein crystals is a major obstacle to the cryocrystallographic study of protein structure, and has limited studies of how the structural ensemble of a protein evolves with temperature in the biophysically interesting range from ∼260 K to the protein-solvent glass transition near 200 K. Using protein crystals with solvent cavities as large as ∼70 Å, time-resolved X-ray diffraction was used to study the response of protein and internal solvent during rapid cooling. Solvent nanoconfinement suppresses freezing temperatures and ice-nucleation rates so that ice-free, low-mosaicity diffraction data can be reliably collected down to 200 K without the use of cryoprotectants. Hexagonal ice (Ih) forms in external solvent, but internal crystal solvent forms stacking-disordered ice (Isd) with a near-random stacking of cubic and hexagonal planes. Analysis of powder diffraction from internal ice and single-crystal diffraction from the host protein structure shows that the maximum crystallizable solvent fraction decreases with decreasing crystal solvent-cavity size, and that an ∼6 Å thick layer of solvent adjacent to the protein surface cannot crystallize. These results establish protein crystals as excellent model systems for the study of nanoconfined solvent. By combining fast cooling, intense X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets, including membrane proteins and large complexes, may be collected at ∼220-240 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice.

Understanding how chemical modifications alter the atomic‐scale organization of materials is of fundamental importance in materials engineering and the target of considerable efforts in computational prediction. Incorporating covalent and noncovalent interactions in designing crystals while "piggybacking" on the driving force of molecular self‐assembly has augmented efforts to understand the emergence of complex structures using directed synthesis. In this work, microcrystalline powders of the silver 2‐, 3‐, and 4‐fluorobenzenethiolates are prepared and their structures are resolved by small‐molecule serial femtosecond X‐ray crystallography. These three compounds enable the emergence and role of supramolecular synthons in the crystal structures of 3D metal‐organic chalcogenolates to be examined. The unique divergence in their optoelectronic, morphological, and structural behaviors is assessed. The extent of CHF interactions and their influence on the structure and the observed trends in the thermal stability of the crystals are quantified through theoretical calculations and thermogravimetric analysis. [ABSTRACT FROM AUTHOR]

X‐ray free electron laser (XFEL) microcrystallography and synchrotron single‐crystal crystallography are used to evaluate the role of organic substituent position on the optoelectronic properties of metal–organic chalcogenolates (MOChas). MOChas are crystalline 1D and 2D semiconducting hybrid materials that have varying optoelectronic properties depending on composition, topology, and structure. While MOChas have attracted much interest, small crystal sizes impede routine crystal structure determination. A series of constitutional isomers where the aryl thiol is functionalized by either methoxy or methyl ester are solved by small molecule serial femtosecond X‐ray crystallography (smSFX) and single crystal rotational crystallography. While all the methoxy examples have a low quantum yield (0‐1%), the methyl ester in the ortho position yields a high quantum yield of 22%. The proximity of the oxygen atoms to the silver inorganic core correlates to a considerable enhancement of quantum yield. Four crystal structures are solved at a resolution range of 0.8–1.0 Å revealing a collapse of the 2D topology for functional groups in the 2‐ and 3‐ positions, resulting in needle‐like crystals. Further analysis using density functional theory (DFT) and many‐body perturbation theory (MBPT) enables the exploration of complex excitonic phenomena within easily prepared material systems. [ABSTRACT FROM AUTHOR]

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming were examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions - consistent with crystallization of most free water - during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between ∼1000 °C/min and ∼600,000°C /min and during warming at rates between 20,000 and 150,000 °C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced., Significance Statement: Cryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.