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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Random Forest Prediction of Crystal Structure from Electron Diffraction Patterns

Transmission electron microscopy (TEM) diffraction patterns are regularly used to determine the structure of crystalline materials. Electron diffraction is the most common method to solve for unknown or partially known crystal structures, as it provides direct and interpretable feedback on the orientation of crystal grains under the beam [1]. However, it remains a challenge to determine the crystal structure of a new material or even a new phase of an existing material. Analysis of such materials commonly requires manual exploration and comparison with simulated diffraction patterns. This is often a time consuming process with no obvious start point when many similar structures are possible, and this method cannot be used to determine crystal structure or orientation from structures not included in the diffraction libraries. Therefore, we have developed a machine learning model to determine the crystal structure of a material from its electron diffraction pattern.

36 MATERIALS SCIENCE↗
DOE OSTI2022

S K-edge XAS of Cu Ⅱ , Cu Ⅰ , and Zn Ⅱ oxidized Dithiolene complexes: Covalent contributions to structure and the Jahn-Teller effect

We report reduced dithiolene ligands are bound to high valent Mo centers in the active site of the oxotransferase family of enzymes. Related model complexes have been studied with great insight by Prof. Holm and his colleagues. This study focuses on the other limit of dithiolene chemistry: an investigation of the 2-electron oxidized dithiolene bound to low-valent late transition metal (TM) ions (Zn II , Cu I , and Cu II ). The bonding descriptions of the oxidized dithiolene [N,N-dimethyl piperazine 2,3-dithione (Me 2 Dt 0 )] complexes are probed using S K-edge X-ray absorption spectroscopy (XAS) and the results are correlated to density functional theory (DFT) calculations. These experimentally supported calculations are then extended to explain the different geometric structures of the three complexes. The Zn II (Me 2 Dt 0 ) 2 complex has only ligand-ligand repulsion so it is stabilized at the D 2d symmetry limit. The Cu I (Me 2 Dt 0 ) 2 complex has additional weak backbonding thus distorts somewhat from D 2d toward D 2h symmetry. The Cu II (Me 2 Dt 0 ) 2 complex has a strong σ donor bond that leads to both a large Jahn-Teller stabilization to D 2h and an additional covalent contribution to the geometry. The combined strong stabilization results in the square planar, D 2h structure. This study quantifies the competition between the ligand-ligand repulsion and the change in electronic structures in determining the final geometric structures of the oxidized dithiolene complexes, and provides quantitative insights into the Jahn-Teller stabilization energy and its origin.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structure of the human dopamine transporter and mechanisms of inhibition

Abstract The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement 1 . Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn 2+ are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 2 (β-CFT), the non-competitive inhibitor MRS7292 3 and Zn 2 + (ref. 4 ). We show how β-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn 2+ ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn 2+ restrains the movement of EL4 relative to EL2 and inhibits transport activity.

Science & Technology - Other Topics↗
DOE OSTI2026

Revealing Local Structures through Machine-Learning-Fused Multimodal Spectroscopy

Atomistic structures of materials offer valuable insights into their functionality. Determining these structures remains a fundamental challenge in materials science, especially for systems with defects. While both experimental and computational methods exist, each has limitations in resolving nanoscale structures. Core-level spectroscopies, such as X-ray absorption (XAS) or electron energy-loss spectroscopies (EELS), have been used to determine the local bonding environment and structure of materials. Recently, machine learning (ML) methods have been applied to extract structural and bonding information from XAS/EELS data. However, frameworks relying solely on a single data stream, defined as characterization data derived from a single element using one technique, are often insufficient because multiple local environments can yield similar spectral features, making it challenging to differentiate between competing structural hypotheses. Here, in this work, we address this challenge by integrating multimodal ab initio simulations, experimental data acquisition, and ML techniques for structure characterization. Our goal is to determine local structures and properties using EELS and XAS data from multiple elements and edges. To showcase our approach, we use various lithium nickel manganese cobalt (NMC) oxide compounds which are used for lithium ion batteries, including those with oxygen vacancies and antisite defects, as the sample material system. We successfully inferred local element content, ranging from lithium to transition metals, with quantitative agreement with experimental data. Beyond local element inference, we find that ML model based on multimodal spectroscopic data is able to determine whether local defects such as oxygen vacancy and antisites are present, a task which is impossible for single mode spectra or other experimental techniques. Furthermore, our framework is able to provide physical interpretability, bridging spectroscopy with the local atomic and electronic structures.

battery↗
DOE OSTI2021

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure–activity relationship (SAR) study of noncovalent compounds that bind in the enzyme’s substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor’s P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Role of Neutrals Versus Transport in Determining the Pedestal Density Structure: Final Technical Report

In fusion devices the plasma density plays a crucial role in determining the fusion reaction rate and has a direct impact on the fusion gain of a given device. This density is in general regulated by the particle sources and transport near the plasma edge, which give rise to an edge density pedestal. When predicting the performance of future devices, this density pedestal is often prescribed, rather than predicted, due to a lack of models which allow confident extrapolation. This project aims to advance these models through the focused validation of theoretical models related to the transport of fueling neutral particles, and through interpretive transport modeling in present day fusion plasmas, in which the penetration of neutrals is altered to better simulate future reactor-like conditions. Achievements in theory and model validation under this project have advanced our understanding of how much of the edge density profile is set by transport versus direct ionization, enabling interesting projections to future burning plasma devices.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2025

A Probabilistic Reasoner Based on Bayes Risk for Damage Detection in Structural Systems

Structural health monitoring (SHM) systems are used to inform operation of structural systems subject to loads and environments that may affect their integrity. SHM systems rely on continuous monitoring of the structure to determine its health state. These systems are often coupled with a model of the deployed structure to determine the consequences of changes in the system by forecasting the response to future states. These models, which may be thought of as digital twins, need to be updated to reflect the latest state of the structural system. This work makes use of an uncertainty-aware machine learning model that enforces distance preservation of the original input space to determine deviations from the training data input space distributions. This workflow enables domain shift detection to determine whether damage is present in the structure. The uncertainty metrics generated by this network are then used in a Bayes risk framework to design an optimal damage detector given cost and risk considerations. The approach is demonstrated on a computational example with simulated damage.

Najera-Flores, David [ATA Engineering, Inc.]↗
DOE OSTI2023

Solving complex nanostructures with ptychographic atomic electron tomography

Abstract Transmission electron microscopy (TEM) is essential for determining atomic scale structures in structural biology and materials science. In structural biology, three-dimensional structures of proteins are routinely determined from thousands of identical particles using phase-contrast TEM. In materials science, three-dimensional atomic structures of complex nanomaterials have been determined using atomic electron tomography (AET). However, neither of these methods can determine the three-dimensional atomic structure of heterogeneous nanomaterials containing light elements. Here, we perform ptychographic electron tomography from 34.5 million diffraction patterns to reconstruct an atomic resolution tilt series of a double wall-carbon nanotube (DW-CNT) encapsulating a complex ZrTe sandwich structure. Class averaging the resulting tilt series images and subpixel localization of the atomic peaks reveals a Zr 11 Te 50 structure containing a previously unobserved ZrTe 2 phase in the core. The experimental realization of atomic resolution ptychographic electron tomography will allow for the structural determination of a wide range of beam-sensitive nanomaterials containing light elements.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Tandem sialoglycan-binding modules in a Streptococcus sanguinis serine-rich repeat adhesin create target dependent avidity effects

Sialic acid–binding immunoglobulin-like lectins (Siglec)–like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ΦTRX, a more rigorous description of this sialic acid–recognition motif given recent findings.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Low Cost Glass-Ceramic Matrix Composite Heat Exchanger

As part of ARPA-E’s High Intensity Thermal Exchange through Materials and Manufacturing Processes (HITEMMP) program, this project sought to develop novel heat exchanger (HX) capabilities to enable efficient and power dense power generation cycles. This class of HX comes under the category of ceramic/composite materials with the higher temperature goal in the program of ≥1100 °C inlet temperature operation. The enabling capability of this effort is the use of glass-ceramic matrix composite (GCMC) material which provides the high temperature durability of a ceramic, the flaw tolerance of a composite, a significantly faster and lower cost manufacturing process than conventional matrix CMCs and very low porosity levels < 0.5%. For thin-walled HX structures and the need to minimize leakage, the low porosity differentiator is particularly important. RTRC has prior experience with this material system and in the current project advanced the component design and manufacturing methods into new territory to produce features required for effective heat exchange under high pressures. In this approach, silicon carbide fiber is fabricated into a fiber preform using various textile processes. Graphite tooling is used both during the build-up of the fiber preform (interior tooling) and after the fiber preform has been completed (exterior tooling). This tooling assembly is heated to high temperature in an environment that has been evacuated and backfilled with inert gas. A reservoir of specialty glass is present and once the desired temperature has been reached to achieve the desired glass viscosity, an actuator distributes the glass throughout the fiber preform using passageways which are part of the tooling design in a process known as glass transfer molding. After the tooling has been removed, the composite is heat treated to convert the amorphous glass to a crystalline ceramic, providing improved properties. The project was divided into three phases focusing on the following: 1) 10 kW HX design and coupon-level tube sheet fabrication, 2) 10 kW HX fabrication, 3) 50 kW HX fabrication. During Budget Period 1 (BP1), additional risks were encountered and the need for additional funds was agreed upon by ARPA-E program leadership. Due to a variety of factors, the contract modification required nominally 18 months to execute at which time the HITEMMP program was effectively concluding. Because of this and the time that would be required to perform BP2 tasks, it was decided to conclude the project at the end of BP1. During the design of the 10 kW HX, manufacturing constraints were learned and incorporated, leading to a revised configuration for the fiber preform and HX. Heat exchange and pressure drop predictions also played a role in modifying the original design concept to be a higher aspect ratio shell-and-tube HX, simplifying the manufacturing process and improving the heat exchanger performance. Good gravimetric and volumetric thermal power densities of 11.2 kW/kg and 10,200 kW/m3 for the entire HX were projected that involved thermo-structural Finite Element Analysis to determine the structural mass needed for the high operation pressures of 250 bar cold inlet and 80 bar hot inlet. Fiber preforms using textile processes were produced for multiple headered tube sheets. Additional challenges were encountered during the glass transfer molding step for which solutions were identified, but programmatics did not allow them to be implemented in BP1. While complete HX test articles were not fabricated, the benefits of this GCMC material for a variety of high temperature applications remain.

30 DIRECT ENERGY CONVERSION↗
DOE OSTI2024

Clustering Effects on the Structure of Ionomer Solutions: A Combined SANS and Simulations Study

Ionic assemblies, or clusters, determine the structure and dynamics of ionizable polymers and enable their many applications. Fundamental to attaining well-defined materials is controlling the balance between van der Waals interactions that govern the backbone behavior and the forces that drive the formation of ionic clusters. Here, using small angle neutron scattering and fully atomistic molecular dynamics simulations, the structure of a model ionomer, sulfonated polystyrene in toluene solutions, was investigated as the cluster cohesion was tweaked by the addition of ethanol. The static structure factor was measured by both techniques and correlated with the size of the ionic clusters as the polymer concentration was varied. The conjunction of SANS results and molecular insight from MD simulations enabled the determination of the structure in these inhomogeneous networks on multiple length scales. Additionally, we find that across the entire concentration range studied, a network driven by the formation of ionic clusters was formed, where the size of the clusters drives the inhomogeneity of these systems. Tweaking the ionic clusters through the addition of ethanol impacts the packing of the sulfonated groups, their shape, and their size distribution, which in turn, affects the structure of these networks.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Cross-species analysis of FcγRIIa/b (CD32a/b) polymorphisms at position 131: structural and functional insights into the mechanism of IgG- mediated phagocytosis in human and macaque

Introduction Antibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques ( Macaca mulatta ), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2. Methods We determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species by x-ray crystallography and linked these structures to affinity and activity using SPR and an ADCP assay. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc by x-ray crystallography. Results Through analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species- and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P 131 variant showing uniformly reduced IgG affinity. Conclusion These insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.

Tolbert, William D.↗
DOE OSTI2023

Computational and Experimental Determination of the Properties, Structure, and Stability of Peptoid Nanosheets and Nanotubes

Peptoids (N-substituted glycines) are a group of highly controllable peptidomimetic polymers. Amphiphilic diblock peptoids have been engineered to assemble crystalline nanospheres, nanofibrils, nanosheets, and nanotubes with biochemical, biomedical, and bioengineering applications. The mechanical properties of peptoid nanoaggregates and their relationship to the emergent self-assembled morphologies have been relatively unexplored and are critical for the rational design of peptoid nanomaterials. In this work, we consider a family of amphiphilic diblock peptoids consisting of a prototypical tube-former (Nbrpm6Nc6, a NH 2 -capped hydrophobic block of six N-((4-bromophenyl)methyl)glycine residues conjugated to a polar NH 3 (CH 2 ) 5 CO tail), a prototypical sheet-former (Nbrpe6Nc6, where the hydrophobic block comprises six N-((4-bromophenyl)ethyl)glycine residues), and an intermediate sequence that forms mixed structures ((NbrpeNbrpm)3Nc6). We combine all-atom molecular dynamics simulations and atomic force microscopy to determine the mechanical properties of the self-assembled 2D crystalline nanosheets and relate these properties to the observed self-assembled morphologies. We find good agreement between our computational predictions and experimental measurements of Young’s modulus of crystalline nanosheets. A computational analysis of the bending modulus along the two axes of the planar crystalline nanosheets reveals bending to be more favorable along the axis in which the peptoids stack by interdigitation of the side chains compared to that in which they form columnar crystals with π-stacked side chains. Here, we construct molecular models of nanotubes of the Nbrpm6Nc6 tube-forming peptoid and predict a stability optimum in good agreement with experimental measurements. A theoretical model of nanotube stability suggests that this optimum is a free energy minimum corresponding to a “Goldilocks” tube radius at which capillary wave fluctuations in the tube wall are minimized.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Rheo-electric measurements of carbon black suspensions containing polyvinylidene difluoride in N -methyl-2-pyrrolidone

Lithium-ion battery cathode slurries have a microstructure that depends sensitively on how they are processed due to carbon black's (CB) evolving structure when subjected coating flows. While polyvinylidene difluoride (PVDF), one of the main components of the cathode slurry, plays an important role in modifying the structure and rheology of CB, a quantitative understanding is lacking. In this work, we explore the role of PVDF in determining the structural evolution of Super C65 CB in N-methyl-2-pyrrolidinone (NMP) with rheo-electric measurements. We find that PVDF enhances the viscosity of NMP resulting in a more extensive structural erosion of CB agglomerates with increasing polymer concentration and molecular weight. We also show that the relative viscosity of all suspensions can be collapsed by the fluid Mason number (Mnf), which compares the hydrodynamic forces imposed by the medium to cohesive forces holding CB agglomerates together. Using simultaneous rheo-electric measurements, we find at high Mnf, the dielectric strength (Δε) scales with Mnf, and the power-law scaling can be quantitatively predicted by considering the self-similar break up of CB agglomerates. The collapse of the relative viscosity and scaling of Δε both suggest that PVDF increases the hydrodynamic force of the suspending medium without directly changing the CB agglomerate structure. These findings are valuable for optimizing the rheology of lithium ion battery cathode slurries. We also anticipate that these findings can be extended to understand the microstructure of similar systems under flow.

Mechanics↗
DOE OSTI2020

Coverage-Dependent Adsorption of Hydrogen on Fe(100): Determining Catalytically Relevant Surface Structures via Lattice Gas Models

Hydrogen adatoms are a critical surface species for several reactions catalyzed by Fe surfaces such as Fischer–Tropsch synthesis, ammonia synthesis, and the hydrodeoxygenation of biomass-derived oxygenates. Parameterizing the energetics for H/Fe in terms of both coverage and configuration space can significantly aid in the development of multiscale models as well as provide atomic level insight into the dominant surface structures present under realistic reaction conditions. Here, we construct a lattice gas model for H/Fe(100), where the lateral interactions are determined from first-principles using density functional theory. Using 950 symmetrically unique H/Fe(100) configurations, we generate a cluster expansion with a predictive accuracy in terms of surface energy of 3.8 meV/site over a coverage range from 0 to 3 monolayers. Ten electronic ground state structures are identified from this thorough scan (including the structures at 0 and 3 monolayers), which were subsequently used to generate ab initio phase diagrams under a range of temperatures and pressures. Under reaction conditions typical of Fischer–Tropsch synthesis, ammonia synthesis, and biomass oxygenate hydrodeoxygenation, we find that the 1.0 monolayer structure is dominant. Furthermore, examination of the total H–H lateral interactions for the H/Fe(100) electronic ground state structures shows that H/Fe(100) can be accurately modeled via a mean-field ideal lattice gas model for coverages less than 1.0 monolayers. Altogether, this work enables the incorporation of H–H lateral interactions on Fe(100) into multiscale models, via either mean-field or site-dependent techniques, and provides atomic insight into the catalytically relevant H/Fe(100) structures for a range of heterogeneous reactions.

09 BIOMASS FUELS↗
DOE OSTI2022

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Abstract Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram‐positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase‐mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae , identifying a number of putative β7–β8 loop‐mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1′ position of the substrate motif, underlined in LPXT G , in contrast to the strict P1′ Gly recognition of SrtA from Staphylococcus aureus . However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C‐terminus, and three others of β7–β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7–β8 loop‐mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Gao, Melody↗
DOE OSTI2023

In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon–carbon (C–C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. Here, we show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme–substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C–C bond coupling chemistry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Virology↗