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This work introduces a simplified deposition procedure for multidimensional (2D/3D) perovskite thin films, integrating a phenethylammonium chloride (PEACl)-treatment into the antisolvent step when forming the 3D perovskite. This simultaneous deposition and passivation strategy reduces the number of synthesis steps while simultaneously stabilizing the halide perovskite film and improving the photovoltaic performance of resulting solar cell devices to 20.8%. Using a combination of multimodal in situ and additional ex situ characterizations, it is demonstrated that the introduction of PEACl during the perovskite film formation slows down the crystal growth process, which leads to a larger average grain size and narrower grain size distribution, thus reducing carrier recombination at grain boundaries and improving the device's performance and stability. The data suggests that during annealing of the wet film, the PEACl diffuses to the surface of the film, forming hydrophobic (quasi-)2D structures that protect the bulk of the perovskite film from humidity-induced degradation.

Nucleation and crystallization arising from liquid to solid phase are involved in a multitude of processes in fields ranging from materials science to biology. Controlling the thermodynamics and kinetics of growth is advantageous to help tune the formation of complex morphologies. Here, we harness wide-angle X-ray scattering and vibrational spectroscopy to elucidate the mechanism for crystallization and growth of the metal-organic framework Co-MOF-74 within microscopic volumes enclosed in a capillary and an attenuated total reflection microchip reactor. The experiments reveal molecular and structural details of the growth processes, while the results of plane wave density functional calculations allow identification of lattice and linker modes in the formed crystals. Synthesis of the metal-organic framework with microscopic volumes leads to monodisperse and micron-sized crystals, in contrast to those typically observed under bulk reaction conditions. Reduction in the volume of reagents within the microchip reactor was found to accelerate the reaction rate. The coupling of spectroscopy with scattering to probe reactions in microscopic volumes promises to be a useful tool in the synthetic chemist's kit to understand chemical bonding and has potential in designing complex materials.

It is well known that molecules confined very close to a surface arrange into molecular layers. Because solid-liquid interfaces are ubiquitous in the chemical, biological and physical sciences, it is crucial to develop methods to easily access molecular layers and exploit their distinct properties by producing molecular layered crystals. Here we report a method based on crystallization in ultra-thin puddles enabled by gas blowing, which allows to produce molecular layered crystals with thickness down to the monolayer onto a surface, making them directly accessible for characterization and further processing. By selecting four molecules with different types of polymorphs, we observed exclusive crystallization of polymorphs with Van der Waals interlayer interactions, which have not been observed with traditional confinement methods. In conclusion, the gas blowing approach unveils the opportunity to perform materials chemistry under confinement onto a surface, enabling the formation of distinct crystals with selected polymorphism. Molecules arranged in close proximity to a surface form molecular layers, exhibiting distinct properties. However, the creation of these layers is challenging. Here, the authors present a technique for generating molecular layers through crystallization induced by gas blowing onto a surface. [ABSTRACT FROM AUTHOR]

Understanding nanostructure ripening mechanisms is desirable for gaining insight on the growth and potential applications of nanoscale materials. However, the atomic pathways of nanostructure ripening in solution have rarely been observed directly. Here, we report defect-mediated ripening of Cd-CdCl2 core-shell nanoparticles (CSN) revealed by in-situ atomic resolution imaging with liquid cell transmission electron microscopy. We find that ripening is initiated by dissolution of the nanoparticle with an incomplete CdCl2 shell, and that the areas of the Cd core that are exposed to the solution are etched first. The growth of the other nanoparticles is achieved by generating crack defects in the shell, followed by ion diffusion through the cracks. Subsequent healing of crack defects leads to a highly crystalline CSN. The formation and annihilation of crack defects in the CdCl2 shell, accompanied by disordering and crystallization of the shell structure, mediate the ripening of Cd-CdCl2 CSN in the solution.

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.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

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.

A nonlinear least-squares method for refining a parametric expression describing the estimated errors of reflection intensities in serial crystallographic (SX) data is presented. This approach, which is similar to that used in the rotation method of crystallographic data collection at synchrotrons, propagates error estimates from photon-counting statistics to the merged data. Here, it is demonstrated that the application of this approach to SX data provides better SAD phasing ability, enabling the autobuilding of a protein structure that had previously failed to be built. Estimating the error in the merged reflection intensities requires the understanding and propagation of all of the sources of error arising from the measurements. One type of error, which is well understood, is the counting error introduced when the detector counts X-ray photons. Thus, if other types of random errors (such as readout noise) as well as uncertainties in systematic corrections (such as from X-ray attenuation) are completely understood, they can be propagated along with the counting error, as appropriate. In practice, most software packages propagate as much error as they know how to model and then include error-adjustment terms that scale the error estimates until they explain the variance among the measurements. If this is performed carefully, then during SAD phasing likelihood-based approaches can make optimal use of these error estimates, increasing the chance of a successful structure solution. In serial crystallography, SAD phasing has remained challenging, with the few examples of de novo protein structure solution each requiring many thousands of diffraction patterns. Here, the effects of different methods of treating the error estimates are estimated and it is shown that using a parametric approach that includes terms proportional to the known experimental uncertainty, the reflection intensity and the squared reflection intensity to improve the error estimates can allow SAD phasing even from weak zinc anomalous signal.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the μs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Spin wave logic circuits using quantum oscillations of spins (magnons) as carriers of information have been proposed for next generation computing with reduced energy demands and the benefit of easy parallelization. Current realizations of magnonic devices have micrometer sized patterns. Here we demonstrate the feasibility of biogenic nanoparticle chains as the first step to truly nanoscale magnonics at room temperature. Our measurements on magnetosome chains (ca 12 magnetite crystals with 35 nm particle size each), combined with micromagnetic simulations, show that the topology of the magnon bands, namely anisotropy, band deformation, and band gaps are determined by local arrangement and orientation of particles, which in turn depends on the genotype of the bacteria. Our biomagnonic approach offers the exciting prospect of genetically engineering magnonic quantum states in nanoconfined geometries. By connecting mutants of magnetotactic bacteria with different arrangements of magnetite crystals, novel architectures for magnonic computing may be (self-) assembled.

Water occupies typically 50% of a protein crystal and thus significantly contributes to the diffraction signal in crystallography experiments. Separating its contribution from that of the protein is, however, challenging because most water molecules are not localized and are thus difficult to assign to specific density peaks. The intricateness of the protein-water interface compounds this difficulty. This information has, therefore, not often been used to study biomolecular solvation. Here, we develop a methodology to surmount in part this difficulty. More specifically, we compare the solvent structure obtained from diffraction data for which experimental phasing is available to that obtained from constrained molecular dynamics (MD) simulations. The resulting spatial density maps show that commonly used MD water models are only partially successful at reproducing the structural features of biomolecular solvation. The radial distribution of water is captured with only slightly higher accuracy than its angular distribution, and only a fraction of the water molecules assigned with high reliability to the crystal structure is recovered. These differences are likely due to shortcomings of both the water models and the protein force fields. Despite these limitations, we manage to infer protonation states of some of the side chains utilizing MD-derived densities.

Direct evidence of complex prebiotic chemistry from a water-rich world in the outer solar system is provided by the 4.5-billion-year-old halite crystals hosted in the Zag and Monahans (1998) meteorites. This study offers the first comprehensive organic analysis of the soluble and insoluble organic compounds found in the millimeter-sized halite crystals containing brine inclusions and sheds light on the nature and activity of aqueous fluids on a primitive parent body. Associated with these trapped brines are organic compounds exhibiting wide chemical variations representing organic precursors, intermediates, and reaction products that make up life's precursor molecules such as amino acids. The organic compounds also contain a mixture of C-, O-, and N-bearing macromolecular carbon materials exhibiting a wide range of structural order, as well as aromatic, ketone, imine, and/or imidazole compounds. The enrichment in 15N is comparable to the organic matter in pristine Renazzo-type carbonaceous chondrites, which reflects the sources of interstellar 15N, such as ammonia and amino acids. The amino acid content of the Zag halite deviates from the meteorite matrix, supporting an exogenic origin of the halite, and therefore, the Zag meteorite contains organics synthesized on two distinct parent bodies. Our study suggests that the asteroidal parent body where the halite precipitated, potentially asteroid 1 Ceres, shows evidence for a complex combination of biologically and prebiologically relevant molecules.

The fundamental crystal science underpinning the industrial crystallisation of organic materials is reviewed from molecular, intermolecular (synthonic) and crystallographic perspectives. The main aspects that differentiate the crystal growth of these materials from more conventional commercial crystal growth of large single crystals and epitaxial layers for micro-electronic applications are highlighted. Building up on key concepts of intermolecular forces, crystallisation and crystal chemistry, the factors that govern the bulk structure, equilibrium external morphology and hence surface chemistry of crystals are reviewed. The non-equilibrium case of solution-phase crystallisation builds upon this, dealing with the core aspects of solubility, solution metastability, and hence crystallisability and how supersaturation relates to molecular assembly through the nucleation process and the subsequent faceting of the nuclei through the growth process into well-defined polyhedral crystalline forms. The practical implications are brought into sharp focus through a number of case-study examples whereby the crystallisation process can be engineered to produce crystals with pre- desired physico-chemical properties, notably crystal size, crystal structure, crystal morphology, purity and agglomerability. Abbreviations: 0D: Zero-dimension; 1D: One-dimension; 2D: Two-dimension; 3D: Three-dimension; ADDoPT: Advanced Digital Design Transforming Pharmaceutical Development and Manufacture; AE: Attachment energy; AFM: Atomic force microscopic; AIDS: Acquired Immuno-Deficiency Syndrome; API: Active Pharmaceutical Ingredient; ATR-FTIR: Attenuated total reflectance – Fourier transform infrared; B&S: Birth & Spread; BCF: Burton-Cabrera-Frank; BFDH: Bravais–Friedel–Donnay–Harker; Bud: Budesonide; CCDC: Cambridge Crystallographic Data Centre; CSD: Crystal size distribution; DSC: Differential scanning calorimetry; EPSRC: Engineering and Physical Sciences Research Council; EtOH: Ethanol; F: Flat faces; FP: Fluticasone propionate; FTIR: Fourier transform infrared; H-bonding: Hydrogen bonding; IN: Instantaneous nucleation; ISSCG-17: The 17th International Summer School on Crystal Growth; K: Kink faces; KBHR: Kashchiev – Borissova – Hammond – Roberts; KJR: Kevin J Roberts; LGA: L-glutamic acid; MCS: Manufacturing Classification System; MSG: Monosodium glutamate; MSZW: Metastable zone width; NA: Nornizar Anuar; PABA: Para-amino benzoic acid; PBC: Periodic Bond Chain; PN: Progressive nucleation; PXRD: Powder x-ray diffraction; R&D: Research and Development; RIG: Rough interface growth; ROY: Red-Orange-Yellow; S: Stepped faces; SB: Salbutamol; SDS: Sodium dodecyl sulphate; UiTM: Universiti Teknologi MARA; UV-Vis: Ultraviolet–visible spectroscopy; XRD: X-ray diffraction [ABSTRACT FROM AUTHOR]

Protein crystallography data collection at synchrotrons is routinely carried out at cryogenic temperatures to mitigate radiation damage. Although damage still takes place at 100 K and below, the immobilization of free radicals increases the lifetime of the crystals by approximately 100-fold. Recent studies have shown that flash-cooling decreases the heterogeneity of the conformational ensemble and can hide important functional mechanisms from observation. These discoveries have motivated increasing numbers of experiments to be carried out at room temperature. However, the trade-offs between increased risk of radiation damage and increased observation of alternative conformations at room temperature relative to cryogenic temperature have not been examined. A considerable amount of effort has previously been spent studying radiation damage at cryo-temperatures, but the relevance of these studies to room temperature diffraction is not well understood. Here, the effects of radiation damage on the conformational landscapes of three different proteins (T. danielli thaumatin, hen egg-white lysozyme and human cyclophilin A) at room (278 K) and cryogenic (100 K) temperatures are investigated. Increasingly damaged datasets were collected at each temperature, up to a maximum dose of the order of 107 Gy at 100 K and 105 Gy at 278 K. Although it was not possible to discern a clear trend between damage and multiple conformations at either temperature, it was observed that disorder, monitored by B-factor-dependent crystallographic order parameters, increased with higher absorbed dose for the three proteins at 100 K. At 278 K, however, the total increase in this disorder was only statistically significant for thaumatin. A correlation between specific radiation damage affecting side chains and the amount of disorder was not observed. This analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

Recent advances in theoretical structure prediction methods and high-throughput computational techniques are revolutionizing experimental discovery of the thermodynamically stable inorganic materials. Metastable materials represent a new frontier for these studies, since even simple binary non-ground state compounds of common elements may be awaiting discovery. However, there are significant research challenges related to non-equilibrium thin film synthesis and crystal structure predictions, such as small strained crystals in the experimental samples and energy minimization based theoretical algorithms. Here, we report on experimental synthesis and characterization, as well as theoretical first-principles calculations of a previously unreported mixed-valent binary tin nitride. Thin film experiments indicate that this novel material is N-deficient SnN with tin in the mixed ii/iv valence state and a small low-symmetry unit cell. Theoretical calculations suggest that the most likely crystal structure has the space group 2 (SG2) related to the distorted delafossite (SG166), which is nearly 0.1 eV/atom above the ground state SnN polymorph. This observation is rationalized by the structural similarity of the SnN distorted delafossite to the chemically related Sn3N4 spinel compound, which provides a fresh scientific insight into the reasons for growth of polymorphs of metastable materials. In addition to reporting on the discovery of the simple binary SnN compound, this paper illustrates a possible way of combining a wide range of advanced characterization techniques with the first-principle property calculation methods, to elucidate the most likely crystal structure of the previously unreported metastable materials.

Self-poisoning is a kinetic trap that can impair or prevent crystal growth in a wide variety of physical settings. Here we use dynamic mean-field theory and computer simulation to argue that poisoning is ubiquitous because its emergence requires only the notion that a molecule can bind in two (or more) ways to a crystal; that those ways are not energetically equivalent; and that the associated binding events occur with sufficiently unequal probability. If these conditions are met then the steady-state growth rate is in general a non-monotonic function of the thermodynamic driving force for crystal growth, which is the characteristic of poisoning. Our results also indicate that relatively small changes of system parameters could be used to induce recovery from poisoning.

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (

Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the `diffraction before destruction' nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.

A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 μm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

Activation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain. TRAAK (also known as KCNK4) ion channels are neuronally expressed members of the two-pore domain K(+) (K2P) channel family and are mechanosensitive. They are involved in controlling mechanical and temperature nociception in mice. Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer--it is a membrane-tension-gated channel. However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and non-conductive conformations defined by the presence of permeant ions along the conduction pathway. In the non-conductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2-TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity--an expansion in cross-sectional area up to 2.7 nm(2) in the conductive state is expected to create a membrane-tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.

We derive a phase diagram for amorphous solids and liquid supercooled water and explain why the amorphous solids of water exist in several different forms. Application of large-deviation theory allows us to prepare such phases in computer simulations. Along with nonequilibrium transitions between the ergodic liquid and two distinct amorphous solids, we establish coexistence between these two amorphous solids. The phase diagram we predict includes a nonequilibrium triple point where two amorphous phases and the liquid coexist. Whereas the amorphous solids are long-lived and slowly aging glasses, their melting can lead quickly to the formation of crystalline ice. Further, melting of the higher density amorphous solid at low pressures takes place in steps, transitioning to the lower-density glass before accessing a nonequilibrium liquid from which ice coarsens.

As the cylindrical sp(2)-bonded carbon allotrope, carbon nanotubes (CNTs) have been widely used to reinforce bulk materials such as polymers, ceramics, and metals. However, both the concept demonstration and the fundamental understanding on how 1D CNTs reinforce atomically thin 2D layered materials, such as graphene, are still absent. Here, we demonstrate the successful synthesis of CNT-toughened graphene by simply annealing functionalized CNTs on Cu foils without needing to introduce extraneous carbon sources. The CNTs act as reinforcing bar (rebar), toughening the graphene through both π-π stacking domains and covalent bonding where the CNTs partially unzip and form a seamless 2D conjoined hybrid as revealed by aberration-corrected scanning transmission electron microscopy analysis. This is termed rebar graphene. Rebar graphene can be free-standing on water and transferred onto target substrates without needing a polymer-coating due to the rebar effects of the CNTs. The utility of rebar graphene sheets as flexible all-carbon transparent electrodes is demonstrated. The in-plane marriage of 1D nanotubes and 2D layered materials might herald an electrical and mechanical union that extends beyond carbon chemistry.

Obtaining diffraction quality crystals remains one of the major bottlenecks in structural biology. The ability to predict the chances of crystallization from the amino-acid sequence of the protein can, at least partly, address this problem by allowing a crystallographer to select homologs that are more likely to succeed and/or to modify the sequence of the target to avoid features that are detrimental to successful crystallization. In 2007, the now widely used XtalPred algorithm [Slabinski et al. (2007), Protein Sci. 16, 2472-2482] was developed. XtalPred classifies proteins into five `crystallization classes' based on a simple statistical analysis of the physicochemical features of a protein. Here, towards the same goal, advanced machine-learning methods are applied and, in addition, the predictive potential of additional protein features such as predicted surface ruggedness, hydrophobicity, side-chain entropy of surface residues and amino-acid composition of the predicted protein surface are tested. The new XtalPred-RF (random forest) achieves significant improvement of the prediction of crystallization success over the original XtalPred. To illustrate this, XtalPred-RF was tested by revisiting target selection from 271 Pfam families targeted by the Joint Center for Structural Genomics (JCSG) in PSI-2, and it was estimated that the number of targets entered into the protein-production and crystallization pipeline could have been reduced by 30% without lowering the number of families for which the first structures were solved. The prediction improvement depends on the subset of targets used as a testing set and reaches 100% (i.e. twofold) for the top class of predicted targets.

G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

(Figure Presented) A nanoporous biodegradable polymer (polycaprolactone) is fabricated utilizing a zinc oxide nanotemplate (see figure). Chemical characterization verifies removal of the template, and preliminary tests on the cytotoxicity demonstrate basic biocompatibility. Diffusion of a model small molecule and a protein are shown to be first and zero order, respectively, indicating these nanoporous membranes may be useful for controlled release of protein-based therapeutics. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

We report a photonic crystal fiber (PCF) coupler having an ultrawide spectral bandwidth keeping single mode operation. The use of the PCF coupler in a fiber-based optical coherence tomography (OCT) system enables us to handle the wide spectral bands of various light sources, including superluminescent diodes (SLDs) at 1300 nm and 820 nm, Ti:sapphire lasers, and white-light sources. The multiband imaging performances of the PCF-based OCT system are demonstrated by obtaining dental images at 1300 nm and 820 nm with the same setup. In addition, we show that the PCF coupler could cover the spectrum over a one octave span and guide both the fundamental wave (1030 nm) and the second harmonic wave (515 nm) simultaneously.

The Escherichia coli's membrane protein OmpA has been identified as a potential biosurfactant due to their amphiphilic nature, and their capacity to stabilize emulsions of dodecane in water. In this study, the influence of surfactant type, concentration, preservation time and droplet size on the crystallization of n-dodecane and water, in oil-in-water emulsions stabilized with six rationally designed Escherichia coli's OmpA-based peptides was investigated. A differential scanning calorimetry (DSC) protocol was established using emulsions stabilized with Tween 20® and Tween 80®. A relationship between the surfactant concentration and the crystallization temperatures of n-dodecane and water was observed, where the crystallization temperatures seem to be dependent on the preservation time. A deconvolution analysis shows that the peak morphology possibly depends on the interactions at the interface because the enthalpic contributions of each Gaussian peak remained similar in emulsions stabilized with the same peptide. Adsorption results show that the main driver for adsorption and thus stabilization of emulsions is polar interactions (e.g. H-bonding) through the hydrophilic parts of the peptides. Those peptides with a preponderance of polar interaction groups distribution (i.e. NH2, COOH, imidazole) showed the highest interfacial activity under favorable pH conditions. This suggests that custom-made peptides whose hydrophilic/hydrophobic regions can be fine-tuned depending on the application can be easily produced with the additional advantage of their biodegradable nature. [ABSTRACT FROM AUTHOR]

In this article, to realize precise control of gas phase and surface reactions for high rate synthesis of high quality microcrystalline silicon (μc-Si) films, influences of source gas supply method and direct current (dc) substrate bias during film deposition process are investigated utilizing a microwave plasma source. It is found that the supply of SiH4 source gas near the substrate holder significantly enhances the deposition rate and mass density of deposited film and reduces film defect density while maintaining high crystallinity. On the other hand, a negative dc substrate bias induces film crystallization from the initial growth stage. An appropriate negative dc bias also leads to higher film cystallinity and density. Kinetics of the controlling gas phase and surface reactions through the optimizing source gas supply method and dc substrate bias is demonstrated. We claim that the high flux film precursor SiH3 and H along with less contribution of short-lifetime radicals on the film growing surface and ion-assist structural relaxation benefit the high-rate synthesis of high quality μc-Si films. [ABSTRACT FROM AUTHOR]

A Bence Jones protein isolated in the early 1960s from a patient (initials KWR) suffering from plasma-cell dyscrasia was crystallized and its structure was analyzed in four different unit cells by X-ray diffraction. The final models of the molecule in all crystal forms were virtually the same, although the elbow angles relating the constant and variable domains of the Bence Jones dimers varied over a range of 10 degrees. The tetragonal form had an R factor of 22.6% and an R(free) of 28.3% at 2.2 A resolution. Phosphate or sulfate ions (depending on the crystallization conditions) were found in the antigen-combining sites in all crystals, as well as an unidentified ligand tightly bound in the hydrophobic 'deep pocket' beneath the antigen-binding site. The ligand was treated as a phenol molecule. Two trigonal crystal forms were among those solved. One was grown at pH 4.0 and the other was only obtained after sitting for more than eight months at room temperature. The latter crystal was composed of molecules that were degraded in their constant domains. Both low pH and proteolytic degradation of constant domains are known to promote the polymerization of some Bence Jones proteins into amyloid fibrils. Indeed, in both trigonal crystal forms the molecules are organized with pseudo-hexagonal symmetry about the unique crystallographic axes in a manner suggestive of such fibrils. The arrangement of Bence Jones dimers is also consistent with other observations regarding Bence Jones amyloid-fibril structure and current models.

The hairpin ribozyme is a small catalytic RNA comprising two helix-loop-helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 A resolution or better. In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 6(1) contacts, which comprise numerous backbone to 2'-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

Automation of iterative model building, density modification and refinement in macromolecular crystallography has made it feasible to carry out this entire process multiple times. By using different random seeds in the process, a number of different models compatible with experimental data can be created. Sets of models were generated in this way using real data for ten protein structures from the Protein Data Bank and using synthetic data generated at various resolutions. Most of the heterogeneity among models produced in this way is in the side chains and loops on the protein surface. Possible interpretations of the variation among models created by repetitive rebuilding were investigated. Synthetic data were created in which a crystal structure was modelled as the average of a set of ;perfect' structures and the range of models obtained by rebuilding a single starting model was examined. The standard deviations of coordinates in models obtained by repetitive rebuilding at high resolution are small, while those obtained for the same synthetic crystal structure at low resolution are large, so that the diversity within a group of models cannot generally be a quantitative reflection of the actual structures in a crystal. Instead, the group of structures obtained by repetitive rebuilding reflects the precision of the models, and the standard deviation of coordinates of these structures is a lower bound estimate of the uncertainty in coordinates of the individual models.

Recently, the hypothesis was advanced that protein crystallization could be driven by the inclusion of small molecules rich in hydrogen-bonding, hydrophobic and electrostatic bonding possibilities. Conventional organic and biologically active molecules would promote lattice formation by their mediation of intermolecular interactions in crystals. The results of an extensive series of crystallization experiments strongly supported the idea. Here, difference Fourier X-ray diffraction analyses of nine crystals grown in the experiments are presented, which convincingly demonstrate the validity of the hypothesis and illustrate some of the ways in which small molecules can participate in lattice interactions.

We report a reversible liquid–solid transition upon heating of a simple solution composed of a-cyclodextrine (αCD), water, and 4-methylpyridine. These solutions are homogeneous and transparent at ambient temperature and solidify when heated to temperatures between 45° and 75°. Quasielastic and elastic neutron scattering show that molecular motions are slowed down in the solid and that crystalline order is established. The solution “freezes on heating.” This process is fully reversible, on cooling the solid melts. A rearrangement of hydrogen bonds is postulated to be responsible for the observed phenomenon. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The initial susceptibility of concentrated magnetic fluids (ferrocolloids) has been experimentally investigated at low temperatures. The results obtained indicate that the interparticle dipole-dipole interactions can increase the susceptibility by several times as compared to the Langevin value. It is shown that good agreement between recent theoretical models and experimental observations can be achieved by introducing a correction for coefficients in the series expansion of susceptibility in powers of density and aggregation parameter. A modified equation for equilibrium susceptibility is offered to sum over corrections made by Kalikmanov (Statistical Physics of Fluids, Springer-Verlag, Berlin, 2001) and by B. Huke and M. Lücke (Phys. Rev. E 67, 051403, 2003). The equation gives good quantitative agreement with the experimental data in the wide range of temperature and magnetic particles concentration. It has been found that in some cases the magnetic fluid solidification occurs at temperature several tens of kelvins higher than the crystallization temperature of the carrier liquid. The solidification temperature of magnetic fluids is independent of particle concentration (i.e., magneto-dipole interparticle interactions) and dependent on the surfactant type and carrier liquid. This finding allows us to suggest that molecular interactions and generation of some large-scale structure from colloidal particles in magnetic fluids are responsible for magnetic fluid solidification. If the magnetic fluid contains the particles with the Brownian relaxation mechanism of the magnetic moment, the solidification manifests itself as the peak on the “susceptibility-temperature” curve. This fact proves the dynamic nature of the observed peak: it arises from blocking the Brownian mechanism of the magnetization relaxation. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The supercooled plastically crystalline phase (glassy crystal) of cyanoadamantane was investigated by multidimensional 2H NMR (T>Tg). Although the orientationally disordered crystalline phase always coexisted with the orientationally ordered crystalline phase, we were able to single out the signal from the glassy crystal by selective excitation and it was possible to carry out line shape measurements and two-dimensional (2D) experiments (in frequency and time domain). The latter directly reveal sixfold jumps with an reorientation of the molecular C3 axis via 90° angles, thus reflecting the symmetry of the lattice. The motion around the C3 axis is found to be always fast. We can reproduce the line shape by random walk simulations properly taking into account these molecular motions. Both methods (line shape and 2D experiments) yield time constants which agree with those reported by other techniques. Refining the analysis a narrow distribution of correlation times is introduced to account for a weak stretching of the correlation function. We did not find any indication of a small angle process usually found in structural glasses. Thus, the motional process in the glassy crystal appears to be simple and quite different from that in structural glasses. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Special computational techniques are required to compute absolute crystal nucleation rates of colloidal suspensions. Using crystal nucleation of hard-sphere colloids as an example, we describe in some detail the novel computational tools that are needed to perform such calculations. In particular, we focus on the definition of appropriate order parameters that distinguish liquid from crystal, and on techniques to compute the kinetic prefactor that enters in the expression for the nucleation rate. In addition, we discuss the relation between simulation results and theoretical predictions based on classical nucleation theory. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

A genetically engineered humanized C(H)2-domain-deleted monoclonal antibody lacking any interchain-hinge disulfide bonds has been crystallized in the presence of detergent in a form suitable for X-ray diffraction analysis. The crystals were grown from 4 M formate along with Triton X-100 and had P2(1)2(1)2 space-group symmetry, with unit-cell parameters a = 83, b = 224, c = 167 A. The crystals diffract to beyond 2.8 A resolution. A disordered crystal form of larger size and more attractive habit was also grown from 4 M formate, but in the presence of the Anapoe series of detergents. Preliminary X-ray data, in conjunction with atomic force microscopy images, are consistent with asymmetric units consisting of two intact antibodies forming a circular dimeric ring. The crystallizing unit, which must contain a twofold axis, is a toroidal assembly of four antibodies (two dimeric rings). Competition between dimers and tetramers to enter the lattice, along with a unique kind of planar defect of packing, may be responsible for the unusually high defect density and the disorder of the X-ray diffraction pattern exhibited by the second crystal form. An approach to crystallizing proteins showing phase separation, particularly intact antibodies, that uses a preliminary detergent test set is described.

Atomic force microscopy (AFM) investigation revealed the sources of disorder and mechanisms of their formation in crystals of an icosahedral plant virus, Cucumber Mosaic Virus (CMV) and structure of the Herpes Simplex Virus (HSV-1). The combination of defects and local disorder in CMV crystals presented here are likely the physical bases for mosaicity in virus crystals, and may be largely responsible for their limited diffraction resolution. High-resolution images of intact, enveloped HSV-1 and the underlying capsid structure demonstrate capabilities of AFM to probe structures of large macromolecular assemblies.

Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non-perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystal's diffraction pattern.

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

The purification and crystallization of the stoichiometric complex of human RXRalpha ligand-binding domain (hRXRalpha LBD) bound to its natural ligand 9-cis retinoic acid (9-cisRA) are described. A three-step purification yields a pure and homogeneous complex. Based on the crystallization conditions of several other nuclear receptors, an exhaustive crystallization screening using carboxylic acids as precipitating agents was performed in association with the use of polyhydric alcohols acting as cosmotropic solutes. Crystals of the hRXRalpha LBD-9-cisRA complex grew in a tripartite mixture containing sodium formate, glycerol and propane-1,2-diol. Micro- and macroseeding were necessary to improve both the size and the quality of crystals in order to make them suitable for structure determination.

Using the sectioning effect of the two-photon fluorescence microscope, we studied the behavior of phospholipid giant unilamellar vesicles (GUVs) composed of pure diacylphosphatidylcholine phospholipids during the gel-to-liquid crystalline phase transition. We used the well-characterized excitation generalized polarization function (GP(ex)) of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN), which is sensitive to the changes in water content in the lipid vesicles, to monitor the phase transition in the GUVs. Even though the vesicles do not show temperature hysteresis at the main phase transition, we observed different behaviors of the vesicle shape, depending on how the GUV sample reaches the main phase transition. During the cooling cycles, we observed an increase in the vesicle diameter at the phase transition ( approximately 0.5-1%), followed by a decrease in the diameter when the vesicle reached the gel phase. During the heating cycles and close to the phase transition temperature, a surprising behavior is observed, showing a sequence of different vesicle shapes as follows: spherical-polygonal-ellipsoidal. We attribute these changes to the effect of lipid domain coexistence on the macroscopic structure of the GUVs. The "shape hysteresis" in the GUVs is reversible and largely independent of the temperature scan rate. In the presence of 30 mol% of cholesterol the events observed at the phase transition in the GUVs formed by pure phospholipids were absent.

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the 'bottoms' and 'tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Ceterach officinarum Willd is a plant widespread throughout Europe and used in southern Italy as a diuretic. Beliefs in the benefits of C. officinarum aqueous extract in the treatment of calcium oxalate kidney stones are widely held. Little is known, however, about the actual mechanism of its antilithiatic action. Our results in this in vitro study corroborate C. officinarum aqueous extract as a good source of antioxidants with a high antioxidant effects. Our results also demonstrate a major impact of C. officinarum aqueous extract on in vitro induced calcium oxalate crystallization kinetics and crystal morphology, showing its critical role in kidney stone formation and/or elimination. We show that progressively increasing doses of C. officinarum aqueous extract cause a sequence of effects. A powerful inhibitory action on calcium oxalate monohydrate (COM) growth and aggregation is first observed. C. officinarum aqueous extract also appears highly effective in stimulating nucleation increasing the number and reducing the size of COM crystals, which become progressively thinner, rounded and concave in a dose-dependent manner. These shape-modified COM crystals are known to be less adherent to renal tubular cells and more easily excreted through the urinary tract preventing kidney stone formation. Further, C. officinarum aqueous extract promotes the formation of calcium oxalate dihydrate (COD) rather than the monohydrate so that, at the highest concentrations used, only COD crystals are observed, in significant greater numbers with a clear reduction in their size, in a dose-dependent manner. Furthermore, AFM analyses allowed us to reveal the presence of C. officinarum component(s) on the surfaces of COD and modified COM crystals. The crystal surface adsorbed component(s) are shown to be similarly active as the total aqueous extract, suggesting a trigger factor which may direct crystal modification towards COD forms. In urolithiasis pathogenesis COD crystals are less dangerous than the COM forms due to their lower affinity for renal tubular cells. Our results are important in understanding the mechanisms which guide the modification induced by C. officinarum on the crystallization process. Based on these data, together with no adverse toxic effect being observed on the in vitro model of human intestinal enterocytes, C. officinarum aqueous extract could represent an attractive natural therapy for the treatment of urolithiasis. [ABSTRACT FROM AUTHOR]

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus. [ABSTRACT FROM AUTHOR]