The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na+-pumping across the membrane. Earlier X-ray and cryo-electron microscopy structures of Na+-NQR from Vibrio cholerae suggested that the subunits harboring redox cofactors undergo conformational changes during catalytic turnover. However, these proposed rearrangements have not yet been confirmed. Here, we have identified at least five distinct conformational states of Na+-NQR using: mutants that lack specific cofactors, specific inhibitors or low-sodium conditions. Molecular dynamics simulations based on these structural insights indicate that 2Fe-2S reduction in NqrD/E plays a crucial role in triggering Na+ translocation by driving structural rearrangements in the NqrD/E subunits, which subsequently influence NqrC and NqrF positioning. This study provides structural insights into the mechanism of Na+ translocation coupled to electron transfer in Na⁺-NQR.

Scorpionato-dithiocarbamate Ni(II) complexes [(TpPh,Me)NiS2CNR2], where TpPh,Me = hydrotris(3-phenyl-5-methylpyrazol-1-yl)borate and NR2 = NMe2, NEt2, NPh2, carbazolyl, are advanced as structural and spectroscopic models for the active site of nickel-dependent superoxide dismutase in its reduced state. The TpPh,Me ligand can adopt variable κ2- and κ3-coordination modes in the synthetic complexes that give rise to several ligand field conformations: square-planar, elongated square-pyramidal, square-pyramidal, and trigonal-bipyramidal. The first two are red and diamagnetic (S = 0), and the latter two are green and paramagnetic (S = 1). The different spin states are facilitated by a range of resonance structures within the dithiocarbamate coligand. These different geometric and spin isomers have been captured in the solid state and structurally characterized by X-ray crystallography. Electronic structures of a simplified [(Tp)NiS2CNMe2] model in relevant conformations, spin and oxidation states were calculated using Density Functional Theory and compared to the enzyme. Reversible one-electron redox couples were observed at biologically relevant potentials. We propose that the diamagnetic, square-planar and elongated square-pyramidal conformations at Ni(II) are relevant to the base-off and base-on conformations, respectively, reported for NiSOD.

Time-resolved X-ray solution scattering (TR-XSS) is a synchrotron-based methodology that enables real-time structural characterization under near-native conditions to provide insight into dynamic and transient structural changes inaccessible to static high-resolution methods such as cryo-electron microscopy (cryo-EM) or X-ray crystallography. Here, we present a workflow for light-triggered TR-XSS experiments that spans data collection, data processing, kinetic analysis, and structural refinement, with accompanying Python scripts. A calcium-transporting P-type ATPase membrane protein (LMCA1) is used as an illustrative example, but the protocol is broadly applicable to diverse protein systems. This workflow offers a practical framework for collecting TR-XSS synchrotron data and subsequent data analysis and interpretation.

The dynamic properties of biomolecules are important to the biogenesis of essential building blocks, the maintenance of biological homeostasis, and the cellular responses to external stimuli critical for survival. Structural studies define biomolecular functions, and their dynamic behavior provides mechanistic insights into these processes. Real-time tracing of dynamic biomolecular structural changes and interactions is highly desirable. Current methodologies such as cryo-EM and X-ray crystallography could provide detailed structural information; however, the images are rather static. Insufficient temporal resolution precludes the observation of crucial transient intermediates. Furthermore, sample preparation and non-physiological imaging environments can compromise the native state of biomolecules. On the other hand, other tools, such as FRET and NMR, could detect dynamic changes of targets at high temporal resolution but lack real-time observation. Atomic force microscopy (AFM) enables nanoimaging and biophysical characterization of biomolecules but is limited by slow scanning speeds and strong tapping forces. Later, high-speed AFM (HS-AFM) with high spatiotemporal resolution and gentle tapping forces, emerged as an ideal nanoimaging approach for studying the functions of delicate biological samples. Building on these developments, we and others have contributed to expand HS-AFM toward biomedical applications, including the direct visualization of disease-relevant organelles and nanostructures under near-physiological conditions. In this review, we examine HS-AFM applications in biomedical science, emphasizing real-time nanoimaging of structural dynamics across biological systems relevant to infectious diseases, infertility, cancer, and neurodegeneration. We also critically discuss the technical limitations of HS-AFM and mitigation strategies.

Background: Infections caused by multidrug-resistant organisms pose a significant global health challenge due to the limited therapeutic options available. Synergistic drug combinations are often employed to enhance treatment efficacy. Because peptidoglycan is unique to prokaryotes, many studies focus on combinations that include beta-lactam antibiotics. To address bacterial strains that produce beta-lactamases, a range of beta-lactamase inhibitors has been developed. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) regularly update clinical guidelines to include standards for new beta-lactam/beta-lactamase inhibitor combinations. Objective: This work provides an overview of beta-lactam and beta-lactamase inhibitor combinations, together with updated clinical guidelines and an analysis of the relevant Protein Data Bank (PDB) entries related to these synergistic combinations. First, the latest EUCAST and CLSI guidelines are reviewed to identify the most recent beta-lactam/beta-lactamase inhibitor combinations used in therapy. Next, PDB entries related to these combinations are examined and compared with new compounds under investigation. EUCAST and CLSI guidelines are valuable for guiding therapeutic strategies, not only for interpreting antibiograms. The newest beta-lactamase inhibitors include avibactam, relebactam, vaborbactam, and enmetazobactam. X-ray crystallography studies have enhanced understanding of inhibitor interactions with major beta-lactamases in Gram-negative pathogens—serine beta-lactamases and metallo-beta-lactamases. Conclusion: Investigating synergistic combinations of beta-lactams and beta-lactamase inhibitors is promising because beta-lactams target peptidoglycan and may enable new therapeutic strategies.

The removal of perfluoroalkyl substances (PFAS) from water is critical to protect human health and the environment. However, removing short-chain PFAS remains a significant challenge, and a molecular-level understanding of their binding is lacking. Here, we utilise a metal-organic cage (MOC 1) as a model "pore" to elucidate the host-guest chemistry of short- and long-chain PFAS in water. X-ray crystallography of six 1·(PFAS)n complexes reveals a broad range of PFAS are encapsulated as anionic aggregates, with the degree of guest-guest aggregation decreasing as the fluoroalkyl chain length increases. 1H and 19F NMR spectroscopy, together with isothermal titration calorimetry reveal the cage host displays unusually large, entropy-driven association constants in water (log K ≥ 5) which remain high for short-chain PFAS. Doping mesoporous silica 60A with only ∼1 wt% of the cage results in a host-in-host adsorbent that removes >98% of short- and long-chain PFAS at environmentally relevant concentrations under flow-through conditions. The adsorbent exhibits rapid PFAS uptake with high selectivity over common water-borne anions and full regenerability. These findings translate host-guest chemistry into an effective materials platform for PFAS remediation, including short-chain species that evade conventional removal methods.

The obesity epidemic is increasingly linked to environmental factors like endocrine disrupting chemicals (EDCs). Bisphenol A (BPA), a known EDC, has been suspected to be linked to adiposity through activation of peroxisome proliferator activated receptor gamma (PPARγ), a key regulator of adipogenesis. Though many BPA alternatives have been introduced as substitutes, their effects on metabolic health remain unclear. This study aimed to investigate the mechanistic interactions of 11 BPA alternatives with PPARγ and their adipogenic potential. Using a PPARγ reporter assay, we assessed the binding affinity and activation potential of BPA alternatives, followed by X-ray crystallography of two potent activators, 4-benzyloxyphenyl 4-hydroxyphenyl sulfone (BPS4BE) and bisphenol PH (BPPH). Additionally, adipogenesis was assessed via a human mesenchymal stem cells (hMSCs) differentiation assay. Results revealed that the alternatives BPPH and BPS4BE potently activated PPARγ (BMD20 (μM): 0.23 and 0.34 respectively). Both significantly induced adipogenesis and a positive correlation was found between PPARγ activation and adipogenic differentiation. Crystallography revealed distinct binding modes for BPPH and BPS4BE compared to rosiglitazone, indicating partial agonism. These findings raise significant concerns about the safety of BPA alternatives and underscore the need for structure-based risk assessment to ensure safer substitutes.

Luminescent radicals, the vast majority of which are derivatives of tris(trichlorophenyl)methyl (TTM), are of significant recent interest because of the unique photophysical properties of the doublet excited state. Though they show high chemical stability, most trityl radicals show very poor photostability, which hinders their application as magnetic, optical and quantum-related materials. In this work, we use density functional theory to study the mechanism of photodegradation of TTM. We isolate the photodecomposition products and characterize them via mass spectrometry, NMR, EPR, UV-Vis absorption spectroscopy, cyclic voltammetry (CV), and X-ray crystallography. We show that the reaction proceeds by a 5-electron electrocyclization followed by an unusual 1,8-sigmatropic chloride shift, affording two fluorenyl radicals, which slowly oxidize and hydrolyze to form semiquinone products. We carefully examine the reported photostability of >80 substituted triarylmethyl radicals and demonstrate that other common triarylmethyl radicals, including benchmark luminescent derivatives with the highest photostability, the carbazole-appended TTMs, photodecompose through the same cyclization mechanism, and thus the DFT-calculated activation energy of cyclization can be used to guide the design of photostability in new luminescent triarylmethyl radicals.

Solution-phase synthesis of long rylenes remains challenging due to their strong tendency to aggregate. Introducing backbone twist can reduce aggregation and simultaneously generate potential helical chirality. Herein, we report a series of rylene molecules-up to octarylene-bearing both odd- and even-numbered naphthalene units and multiple n-butoxy substituents at the bay/ortho positions, synthesized via stepwise cross-coupling followed by controlled oxidative dehydrogenation. Peri-attached 4-(trifluoromethyl)phenyl groups further stabilize the extended scaffolds. The electron-rich n-butoxy groups not only induce pronounced backbone twisting but also impart strong electron-donating character. X-ray crystallography reveals that long rylenes with two or more bay- tetra(n-butoxy) motifs adopt alternating P/M helicity rather than a homochiral helical conformation. These molecules exhibit clear length-dependent optical and electrochemical properties, with absorption and emission spanning the visible to near-infrared region. Their electron-rich nature enables facile formation of stable radical cations and dications. Solid-state structures of representative radical cations show distinct intermolecular stabilizing interactions, while 1H NMR and ACID analyses of the dications reveal a systematic evolution from local to global aromaticity.

Development of an inhibitory monoclonal nanobody targeting Streptococcus pyogenes siderophore binding protein FtsB.

Preparation and characterization of a series of oligo(ethylene glycol)methylether functionalized alkynyl gold(i) complexes capped with AuPPh3 (1a–1d) or dppfAu2 (dppf, 1,1′-bis(diphenyphosphino)ferrocene) (2a–2d) have been accomplished. The structures of 1b and 1c were established by X-ray crystallography. Their in vitro antitumor activities were measured by the CCK8 method against A549 and HeLa cells. The studies indicated that the cytotoxic activity in vitro was fine-tuned by modification of both the gold(i) centers and the oligo(ethylene glycol)methylether ancillary ligands. Compared to the dppfAu2 series, the AuPPh3 series showed better cytotoxicity. Especially, complex 4-(OCH2CH2OCH2CH2OCH3)C6H4CCreated by potrace 1.16, written by Peter Selinger 2001-2019]]>CAuPPh3 (1d) displayed strong anticancer activity toward both cancer cells due to the strong inhibition of thioredoxin reductase (TrxR).

Transformation of agro-industrial products into value-added products, such as prebiotic oligosaccharides, is a key element of the emerging bioeconomy. Here, we characterized a new GH30_8 glucuronoxylanase from Bacillus pumilus (BpXyn30_8A) for its potential in producing xylooligosaccharides (XOS). BpXyn30_8A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue. Its X-ray structure, determined at 2.16 Å resolution, revealed high similarity to other glucuronoxylanases. Furthermore, BpXyn30_8A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn30A. Finally, fermentation assays showed that Bifidobacterium adolescentis metabolized neutral XOS to acetate and lactate, whereas acidic XOS were poorly utilized. These results highlight the potential of BpXyn30_8A as a valuable enzyme for the green transformation of plant biomass into prebiotic oligosaccharides with promising applications in human and animal nutrition, health, and biotechnology.

The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H2O2 have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (FeIV-OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (FeIII-O•-) character. Such states offer insight into the nature of ferryl heme.

In this work, the synthesis and structural characterization of one coordination polymer based on 2,5-thiophenedicarboxylic acid (H2tdc) and 1-(4-carboxybenzyl)-4,4′-bipyridinium (bcbpy), namely, {[Ca(tdc)(bcbpy)(H2O)](0.5DMF)}n, has been reported. The compound was well characterized by infrared spectra, powder X-ray diffraction and single crystal X-ray diffraction. X-ray crystallography reveals that central seven-coordinated Ca(II) ions in the compound are bridged by chelating-bridging carboxylate groups of two tdc2– anions to give a 2D layered structure. The bcbpy molecules further modify the two-dimensional structure by coordinating with Ca(II) ions via carboxylate groups at one end. Driven by photo-induced electron transfer under UV light, the compound exhibits reversible photochromic behavior from pale yellow to blue within a short time owing to the presence of electron-deficient viologen moieties. Moreover, the photomodulated luminescence property has been investigated. After 5 min of irradiation, the emission intensity of the compound at 430 nm decreased by 83.2% and the emission could be also recovered to the original state after being kept in the dark for 12 h.

Anti-major histocompatibility complex class I (MHC-I) mAbs can stimulate immune responses to tumors and infections by blocking suppressive signals delivered via various immune inhibitory receptors. To understand such functions, we determined the structure of a highly cross-reactive anti-human MHC-I mAb, B1.23.2, in complex with the MHC-I molecule HLA-B*44:05 by both cryo-electron microscopy (cryo-EM) and X-ray crystallography. Structural models determined by the two methods were essentially identical revealing that B1.23.2 binds a conserved region on the α21 helix that overlaps the killer immunoglobulin-like receptor (KIR) binding site. Structural comparison to KIR/HLA complexes reveals a mechanism by which B1.23.2 blocks inhibitory receptor interactions, leading to natural killer (NK) cell activation. B1.23.2 treatment of the human KLM-1 pancreatic cancer model in humanized (NSG-IL15) mice provides evidence of suppression of tumor growth. Such anti-MHC-I mAb that block inhibitory KIR/HLA interactions may prove useful for tumor immunotherapy.

Crystallographic fragment screening discovers novel micromolar active inhibitors and druggable hotspots of SARS-CoV-2 PLpro.

Hyaluronic acid microneedles enhance transdermal delivery of minoxidil derivatives with 1,2,4-oxadiazole-5-one unit for effective hair regeneration: Insights from X-ray crystallography and animal models

Unexpected Novel Five-Coordinate Bis(dithiophosphato)cobalt(II) Complexes Formed by Aqua Coordination: Synthesis, Structural Characterization, X-ray Crystallography, and Theoretical Calculations

γ-lactam and γ-lactone-containing sesterterpenoids from Colquhounia coccinea var. mollis with immunosuppressive activity.

Molecular and electronic structure of a ruthenium polypyridyl cyanide complex: Insights from X-ray crystallography and soft X-ray spectroscopies