Search

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 was 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-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article titled "Engineering Supramolecular Hybrid Architectures with Directional Organofluorine Bonds" discusses the use of metal-organic chalcogenolates (MOChas) in crystal engineering. The authors, Aaron S. Brewster, James Nathan Hohman, and their colleagues, prepared microcrystalline powders of silver 2-, 3-, and 4-fluorobenzenethiolates and analyzed their structures using small-molecule serial femtosecond X-ray crystallography. This research allows for the examination of supramolecular synthons in the crystal structures of 3D MOChas. The article is authored by a diverse group of researchers and provides valuable insights into the field of crystal engineering. [Extracted from the article]

The modulation of main‐chain and side‐chain conformational heterogeneity and solvent structure in monoclinic lysozyme crystals by dehydration (related to water activity) and temperature is examined. Decreasing the relative humidity (from 99 to 11%) and decreasing the temperature both lead to contraction of the unit cell, to an increased area of crystal contacts and to remodeling of primarily contact and solvent‐exposed residues. Both lead to the depopulation of some minor side‐chain conformers and to the generation of new conformations. Side‐chain modifications and main‐chain r.m.s.d.s associated with cooling from 298 to 100 K depend on relative humidity and are minimized at 85% relative humidity (r.h.). Dehydration from 99 to 93% r.h. and cooling from 298 to 100 K result in a comparable number of remodeled residues, with dehydration‐induced remodeling somewhat more likely to arise from contact interactions. When scaled to equivalent temperatures based on unit‐cell contraction, the evolution of side‐chain order parameters with dehydration shows generally similar features to those observed on cooling to T = 100 K. These results illuminate the qualitative and quantitative similarities between structural perturbations induced by modest dehydration, which routinely occurs in samples prepared for 298 and 100 K data collection, and cryocooling. Differences between these perturbations in terms of energy landscapes and occupancies, and implications for variable‐temperature crystallography between 180 and 298 K, are discussed. It is also noted that remodeling of a key lysozyme active‐site residue by dehydration, which is associated with a radical decrease in the enzymatic activity of lysozyme powder, arises due to a steric clash with the residue of a symmetry mate. [ABSTRACT FROM AUTHOR]

The thermal contraction of aqueous cryoprotectant solutions on cooling to cryogenic temperatures is of practical importance in protein cryocrystallography and in biological cryopreservation. In the former case, differential contraction on cooling of protein molecules and their lattice relative to that of the internal and surrounding solvent may lead to crystal damage and the degradation of crystal diffraction properties. Here, the amorphous phase densities of aqueous solutions of glycerol and ethylene glycol at T = 77 K have been determined. Densities with accuracies of <0.5% to concentrations as low as 30%( w/ v) were determined by rapidly cooling drops with volumes as small as 70 pl, assessing their optical clarity and measuring their buoyancy in liquid nitrogen-argon solutions. The use of these densities in contraction matching of internal solvent to the available solvent spaces is complicated by several factors, most notably the exclusion of cryoprotectants from protein hydration shells and the expected deviation of the contraction behavior of hydration water from bulk water. The present methods and results will assist in developing rational approaches to cryoprotection and an understanding of solvent behavior in protein crystals. [ABSTRACT FROM AUTHOR]