Atomic Resolution X-ray Research Articles

Pt single-atom electrocatalysts at Cu2O nanowires for boosting electrochemical sensing toward glucose

In electrochemical biosensors, rational design and synthesis of high-performance electrochemical glucose sensors based on emerging single-atom catalysts (SACs) are paramount. Herein, a facile approach is proposed for dispersing single-atom doping of cuprous oxide (Cu2O) nanowires with Pt on a copper foam substrate (Pt1/Cu2O@CF) via the electrochemical deposition process. The specific nanostructure of the single-atom catalyst has been elaborately revealed with the aid of atomic resolution scanning transmission electron microscopy (STEM) and X-ray absorption fine structure spectroscopy (XAS). The as-fabricated Pt1/Cu2O@CF biosensor with satisfactory scalability exhibits a low limit of detection (1 μM), ultrahigh sensitivity (31.55 mA mM−1 cm−2), excellent selectivity, and robust reliability toward glucose. The first principles simulations reveal that Pt SAC is beneficial to the adsorption of glucose on the surface and further facilitates the electron transfer for the deprotonation process, resulting in high glucose sensing performance. This work sheds light on the applications of SACs for designing ultrasensitive electrochemical biosensors.

Investigating ultrafast structural dynamics using high repetition rate x-ray FEL radiation at European XFEL

European XFEL is an international facility providing hard and soft x-ray free-electron laser radiation for user experiments with a wide range of scientific applications. Its superconducting linear accelerator enables high repetition rate experiments with a broad range of x-ray pulse delivery patterns. The combination of time-resolved experiments, providing access to the time-domain from sub-femtoseconds to milliseconds, with atomic resolution x-ray geometric and electronic structure determination methods is responsible for the bulk of scientific applications of European XFEL. In addition, the extreme x-ray intensities and coherence properties open new methods for studying matter out of equilibrium. After start of operation in 2017, the facility now harvests scientific applications with impact to the challenge areas climate and energy, health, environment and sustainability, and digitalization. Extensions of European XFEL aim to increase performance and capabilities for new scientific applications. An upgrade of the facility in the early 2030s will increase the applicability of European XFEL to solid materials and provide dedicated instruments for improved conditions in specific research fields.

Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

Evolution in X-ray analysis from micro to atomic scales in aberration-corrected scanning transmission electron microscopes.

X-ray analysis is one of the most robust approaches to extract quantitative information from various materials and is widely used in various fields ever since Raimond Castaing established procedures to analyze electron-induced X-ray signals for materials characterization '70 years ago'. The recent development of aberration-correction technology in a (scanning) transmission electron microscopes (S/TEMs) offers refined electron probes below the Å level, making atomic-resolution X-ray analysis possible. In addition, the latest silicon drift detectors allow complex detector arrangements and new configurational designs to maximize the collection efficiency of X-ray signals, which make it feasible to acquire X-ray signals from single atoms. In this review paper, recent progress and advantages related to S/TEM-based X-ray analysis will be discussed: (i) progress in quantification for materials characterization including the recent applications to light element analysis, (ii) progress in analytical spatial resolution for atomic-resolution analysis and (iii) progress in analytical sensitivity toward single-atom detection and analysis in materials. Both atomic-resolution analysis and single-atom analysis are evaluated theoretically through multislice-based calculation for electron propagation in oriented crystalline specimen in combination with X-ray spectrum simulation.

Single atom surface engineering: A new strategy to boost electrochemical activities of Pt catalysts

Pt-based catalysts are widely applied in several catalytic electrochemical reactions for energy storage and conversion. The improvement of specific activity of Pt is typically achieved by introducing the transition metal to obtain the alloy structure. Different from the traditional alloy structure, herein, we report Pt catalyst modified with Co single atoms obtained by atomic layer deposition (ALD). The as-prepared catalysts show much higher mass activity and excellent stability compared to commercial Pt/C catalysts towards the hydrogen evolution reaction (HER) and oxygen reduction reaction (ORR). The atomic resolution TEM images and X-ray absorption spectroscopy (XAS) indicate the formation of atomically dispersed Co on Pt. First principle calculations reveal that the Co atom affects the electronic structure of the Pt catalysts, which resulted in the high HER and ORR performance. This work provides a new approach for the rational design of highly active and stable Pt-based catalysts, which hold great potential for application in various catalytic reactions.

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme.

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE.

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme.

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97-Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

Atomic resolution imaging of cation ordering in niobium\u2013tungsten complex oxides

Energy dispersive X-ray emission imaging at atomic resolution is a powerful tool to solve order–disorder problems in complex metal oxide crystals, supplementing conventional X-ray or neutron diffraction. Here, we use this method, based on scanning transmission electron microscopy, to investigate cation ordering in ternary metal oxides 4Nb2O5·9WO3 and 2Nb2O5·7WO3, which have recently attracted attention as energy storage materials in lithium-ion batteries. Their crystal structures are a tetragonal tungsten bronze-type and its hybrid with a ReO3-type ‘block structure’, respectively. Our study reveals the presence of chemical ordering of metal ions in these materials, which have previously been assumed to be solid-solutions. In particular, we show that the two types of cations, Nb and W, are well ordered in their lattices, and that the Nb ions tend to occupy one third of the pentagonal channel sites. These results demonstrate that atomic resolution X-ray emission imaging is an effective alternative approach for the study of locally ordered crystal structures.

Co-segregation of Mg and Zn atoms at the planar η1-precipitate/Al matrix interface in an aged Al–Zn–Mg alloy

Using aberration-corrected scanning transmission electron microscopy, the interface between η1-precipitates and Al matrix in the aged Al–2.1Zn–1.7Mg (at.%) alloy has been investigated. Atomic-resolution X-ray dispersive spectroscopy mapping reveals the occurrence of co-segregation of Mg and Zn atoms in the {002}α habit plane of the η1(MgZn2) precipitates. Site-specific Mg atoms segregate outside the concave of an outermost zig-zag Zn-rich layer of the η1 habit plane. Zn segregation surround the segregated Mg atomic columns was also observed. Hybrid molecular dynamics (MD) and Monte Carlo (MC) atomistic simulations with the accuracy of density functional theory indicate the site-specific segregation is energetically favourable.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

How the Destabilization of a Reaction Intermediate Affects Enzymatic Efficiency: The Case of Human Transketolase.

Atomic resolution X-ray crystallography has shown that an intermediate (the X5P-ThDP adduct) of the catalytic cycle of transketolase (TK) displays a significant, putatively highly energetic, out-of-plane distortion in a sp2 carbon adjacent to a lytic bond, suggested to lower the barrier of the subsequent step, and thus was postulated to embody a clear-cut demonstration of the intermediate destabilization effect. The lytic bond of the subsequent rate-limiting step was very elongated in the X-ray structure (1.61 Å), which was proposed to be a consequence of the out-of-plane distortion. Here we use high-level QM and QM/MM calculations to study the intermediate destabilization effect. We show that the intrinsic energy penalty for the observed distortion is small (0.2 kcal·mol–1) and that the establishment of a favorable hydrogen bond within X5P-ThDP, instead of enzyme steric strain, was found to be the main cause for the distortion. As the net energetic effect of the distortion is small, the establishment of the internal hydrogen bond (−0.6 kcal·mol–1) offsets the associated penalty. This makes the distorted structure more stable than the nondistorted one. Even though the energy contributions determined here are close to the accuracy of the computational methods in estimating penalties for geometric distortions, our data show that the intermediate destabilization effect provides a small contribution to the observed reaction rate and does not represent a catalytic effect that justifies the many orders of magnitude which enzymes accelerate reaction rates. The results help to understand the intrinsic enzymatic machinery behind enzyme’s amazing proficiency.

GaAs1-xBix growth on Ge: anti-phase domains, ordering, and exciton localization

The dilute bismide alloy GaAs1-xBix has drawn significant attention from researchers interested in its fundamental properties and the potential for infrared optoelectronics applications. To extend the study of bismides, molecular-beam heteroepitaxy of nominally 1.0 eV bandgap bismide on Ge substrates is comprehensively investigated. Analysis of atomic-resolution anti-phase domain (APD) images in the direct-epitaxy revealed a high-density of Ga vacancies and a reduced Bi content at their boundaries. This likely played a key role in the preferential dissolution of Bi atoms from the APD interiors and Bi spiking in Ge during thermal annealing. Introduction of GaAs buffer on offcut Ge largely suppressed the formation of APDs, producing high-quality bismide with single-variant CuPtB-type ordered domains as large as 200 nm. Atomic-resolution X-ray imaging showed that 2-dimensional Bi-rich (111) planes contain up to x = 9% Bi. The anomalously early onset of localization found in the temperature-dependent photoluminescence suggests enhanced interactions among Bi states, as compared to non-ordered samples. Growth of large-domain single-variant ordered GaAs1-xBix films provides new prospects for detailed analysis of the structural modulation effects and may allow to further tailor properties of this alloy for optoelectronic applications.

Direct observation and impact of co-segregated atoms in magnesium having multiple alloying elements

Modern engineering alloys contain multiple alloying elements, but their direct observation when segregated at the atomic scale is challenging because segregation is susceptible to electron beam damage. This is very severe for magnesium alloys, especially when solute atoms segregate to form single atomic columns. Here we show that we can image segregation in magnesium alloys with atomic-resolution X-ray dispersive spectroscopy at a much lower electron voltage. We report a co-segregation pattern at twin boundaries in a magnesium alloy with both larger and smaller solutes forming alternating columns that fully occupy the twin boundary, in contrast to previous observations of half occupancy where mixed-solute columns alternate with magnesium. We further show that the solute co-segregation affects the twin migration mechanism and increases the twin boundary pinning. Our work demonstrates that the atomic-scale analysis of the structure and chemistry of solute segregation in metallic alloys with complex compositions is now possible.

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.

Fast inside-source X-ray fluorescent holography.

Atomic resolution X-ray holography can be realized by using the atoms of the sample as inside sources or inside detectors. However, until now there were only very few experiments in which the atoms played the role of inside sources. The reason is twofold: (i) technically, inside-detector experiments are much easier and faster; (ii) by using atoms as inside detectors one can measure holograms at many energies on the same sample, which helps the reconstruction. This paper shows that, using new technical developments, inside-source holograms can be taken much faster than inside-detector holograms and, by applying a sophisticated evaluation method, high-quality reconstruction from a single-energy hologram can also be obtained.

Highly Pure Soluble Chimeras of the Intracellular Domain of Anionic Pentameric Ligand-Gated Ion Channels

The past few years have seen remarkable advances in structural information gleaned from atomic resolution X-ray crystallographs of important anionic and cationic pentameric ligand-gated ion channels (pLGIC) from the Cys-loop superfamily. The highly conserved extracellular domain (ECD) and transmembrane domain (TMD), and the diverse intracellular domain (ICD) constitute each subunit of eukaryotic pLGICs. The ECD and TMD of different Cys-loop receptors are prime targets for several clinical drugs. These drugs are thus notorious for binding indiscriminately to undesired receptors within the superfamily resulting in a wide range of unwanted side-effects. Intriguingly, the marked variability of ICDs between different subtypes can be exploited to aid in the development of newer drugs with refined specific binding. However, the ICD of any Cys-loop receptor has yet to be fully characterized. The two prominent anion-conducting pLGICs, glycine-α1 (Gly-α1) and γ-amino butyric acid type A-ρ1 (GABAA-ρ1) receptors, of the Cys-loop superfamily are critical in mediating postsynaptic inhibition in the central nervous system. Dysfunction of these pLGICs has been implicated in numerous serious neuropsychiatric and neurological diseases. The goal of our current project was to develop a strategy for protein expression and purification of the ICD of Gly-α1 as well as GABAA-ρ1 receptors. Here, we generated chimeras containing maltose-binding protein (MBP) fused to the N-terminus of the ICD of either Gly-α1 receptor or that of GABAA-ρ1 receptor and separated by a short alanine linker. We expressed these chimeras in E. coli and were able to purify both proteins of interest to homogeneity by employing 3-step purification. Further characterization to determine size using size exclusion chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) revealed the oligomeric state. Experiments are underway to gain detailed structural insights.

Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations

BackgroundApolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. MethodsThe HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. ResultsTwo final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. ConclusionsThe generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini. General significanceThe novel full-length structures of apoA-I dimers deepens the understanding to the structure-function relationship of nascent HDL with significance for the prevention of lipoprotein-related disease. The biased simulation method used in this study provides a powerful and convenient modelling tool with applicability for structural studies and modelling of other proteins and protein complexes.

From Nanometer to Atomic Resolution X-ray EDS analysis of Al in Ni-rich Layered Oxide Li-Ion Cathodes

Journal Article From Nanometer to Atomic Resolution X-ray EDS analysis of Al in Ni-rich Layered Oxide Li-Ion Cathodes Get access P Mukherjee, P Mukherjee Department of Materials Science & Engineering, Rutgers University, Piscataway, NJ 08854 Search for other works by this author on: Oxford Academic Google Scholar P Lu, P Lu Sandia National Laboratories, Albuquerque, NM 87185 Search for other works by this author on: Oxford Academic Google Scholar N Faenza, N Faenza Energy Storage Research Group, Rutgers University, North Brunswick, NJ 08902 Search for other works by this author on: Oxford Academic Google Scholar N Pereira, N Pereira Energy Storage Research Group, Rutgers University, North Brunswick, NJ 08902 Search for other works by this author on: Oxford Academic Google Scholar G Amatucci, G Amatucci Energy Storage Research Group, Rutgers University, North Brunswick, NJ 08902 Search for other works by this author on: Oxford Academic Google Scholar F Cosandey F Cosandey Department of Materials Science & Engineering, Rutgers University, Piscataway, NJ 08854 Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 386–387, https://doi.org/10.1017/S1431927617002616 Published: 04 August 2017

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation.

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.