Recent lbnl_bs_mbib items

Exploring the impact of nucleotide length on lipid nanoparticle structure and properties

Wed, 10 Jun 2026 00:00:00 +0000

Lipid nanoparticles (LNPs) are versatile carriers for nucleic acid (NA) therapeutics, including ASOs, siRNA, mRNA, and poly-IC. While lipid composition is known to influence LNP properties, the impact of NA length on morphology and internal structure is less understood, particularly during the stages of carrier–cargo assembly. Here, we examine NA length and lipid composition immediately after mixing using high-throughput SAXS, dynamic light scattering, and cryogenic electron microscopy. All LNPs form ordered NA/lipid compartments, with longer NAs promoting inverse hexagonal (HII) phases and larger intercompartment distances. In contrast, short NAs, especially in formulations with SM102 ionizable lipid, favor lamellar phases. SAXS peak deconvolution quantifies ordered versus disordered phases via a Robustness of Ordered Phase factor, which correlates with particle size and encapsulation efficiency. Formulations with MC3 ionizable and DOPE helper lipids exhibit the most stable HII-phase...

Nucleus softens during herpesvirus infection.

Fri, 5 Jun 2026 00:00:00 +0000

Nuclear mechanics is remodeled not only by extracellular forces but also by internal modifications, such as those induced by viral infections. During herpes simplex virus type 1 infection, the nuclear structures undergo drastic reorganization, but little is known about how nuclear mechanobiology changes as a result. We show that the nucleus softens dramatically during the infection. To understand the phenomenon, we used advanced microscopy and computational modeling. We discovered that the enlarged viral replication compartment had a low biomolecular density, partially explaining the observed nuclear softening. The mobility of the nuclear lamina decreased, which suggests increased rigidity and an inability to induce softening. However, computational modeling supported by experimental data showed that reduced outward forces, such as cytoskeletal pull and intranuclear osmotic pressure acting both on and within the nucleus, can explain the decreased nuclear stiffness. Our findings...

Automated segmentation of soft X-ray tomography: Native cellular structure with submicron resolution at high-throughput for whole-cell quantitative imaging in yeast.

Thu, 4 Jun 2026 00:00:00 +0000

Soft X-ray tomography (SXT) is an invaluable tool for quantitatively analyzing cellular structures at suboptical isotropic resolution. However, it has traditionally depended on manual segmentation, limiting its scalability for large datasets. Here, we leverage a deep learning-based autosegmentation pipeline to segment and label cellular structures in hundreds of cells across three Saccharomyces cerevisiae strains. This task-based pipeline uses manual iterative refinement to improve segmentation accuracy for key structures, including the cell body, nucleus, vacuole, and lipid droplets, enabling high-throughput and precise phenotypic analysis. Using this approach, we quantitatively compared the three-dimensional (3D) whole-cell morphometric characteristics of wild-type, VPH1-GFP, and vac14 strains, uncovering detailed strain-specific cell and organelle size and shape variations. We show the utility of SXT data for precise 3D curvature analysis of entire organelles...

Design and commissioning of a new synchrotron beamline dedicated to X‐ray footprinting mass spectrometry

Thu, 21 May 2026 00:00:00 +0000

The structural biology method of X-ray footprinting mass spectrometry (XFMS) is available at two national synchrotron beamlines in the USA: one at the Advanced Light Source (ALS) on the West Coast and the other at the National Synchrotron Light Source II on the East Coast. XFMS is a solution-state technique that utilizes oxidative modifications of proteins at micromolar concentrations in aqueous buffer to extract structural information. X-rays are employed to generate hydroxyl radicals in situ, which covalently modify specific protein side chains. These modifications are subsequently quantified using liquid chromatography and mass spectrometry. Ratiometric changes in modification levels between two protein states (e.g. with and without ligand) generate a relative solvent accessibility map of the protein pairs, which serves to reveal structural features. Up until recently, the XFMS capability was available as part of a shared program at the ALS without a dedicated beamline. In...

pyDiSCaMB: enabling the use of multipolar scattering factors in Phenix

Fri, 15 May 2026 00:00:00 +0000

Multipolar scattering models, such as the transferable aspherical atom model, account for atomic chemical interactions and provide a more accurate representation of experimental data. However, the simpler independent atom model (IAM), which assumes non-interacting atoms, is the only model available in the most widely used macromolecular refinement programs. This is primarily because IAM offers a hard-to-beat combination of computational efficiency and modelling power at typical macromolecular resolutions. By contrast, more accurate multipolar modelling has historically been limited due to its computational cost and the absence of an interface between software capable of calculating structure factors and gradients based on multipolar models and software designed for macromolecular refinement. This work introduces pyDiSCaMB, a Python software package designed to integrate between the computational crystallography toolbox (cctbx) and the quantum crystallography library DiSCaMB (Densities...

EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks

Fri, 24 Apr 2026 00:00:00 +0000

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH)2] domains to clamp double-stranded (ds) DNA at reversed DNA replication forks for fork protection. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable "wrist" enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication...

Structural Conservation of the A1 Binding Site in Photosystem I across Cyanobacteria and Green Algae

Fri, 24 Apr 2026 00:00:00 +0000

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy was used to obtain (A₁ ^- - A₁) FTIR difference spectra from photosystem I (PSI) samples isolated from eight phylogenetically diverse cyanobacterial strains and one green alga, totaling 13 PSI preparations. These included samples from cells grown under far-red light and PSI in monomeric, dimeric, trimeric, and tetrameric states. Spectral profiles were shown to be independent of oligomeric state. Remarkably, all (A₁ ^- - A₁) FTIR difference spectra exhibited high similarity, underscoring the robustness of the technique and indicating minimal experimental variability. This congruence reveals a highly conserved environment for the phylloquinone cofactor at the A₁ binding site across diverse taxa. Conserved bands associated with the A₀ pigment further suggest structural continuity from A₀ to A₁....

ALS mutations disrupt self-association between the ubiquilin STI1 hydrophobic groove and internal placeholder sequences

Fri, 24 Apr 2026 00:00:00 +0000

Ubiquilins are molecular chaperones that play multifaceted roles in proteostasis, with point mutations in UBQLN2 leading to altered phase-separation properties and amyotrophic lateral sclerosis (ALS). Our mechanistic understanding of this essential process has been hindered by a lack of structural information on the STI1 domain, which is essential for ubiquilin chaperone activity and phase separation. Here, we present the first crystal structure of a ubiquilin-family STI1 domain bound to a transmembrane domain (TMD), and show that ALS mutations disrupt the STI1-TMD interaction. We further demonstrate that ubiquilins contain multiple conserved internal sequences that bind to the STI1 domain, including the PXX-repeat region that is a hotspot for ALS mutations. We propose that these placeholder sequences prevent solvent exposure of the STI1 hydrophobic groove and contribute to the multivalency that drives ubiquilin phase-separation. Together, this work provides a new paradigm for...

Deep-learning methods for contrast enhancement and artifact reduction in cryo-electron tomography: a systematic analysis of the state of the art and proposed improvements.

Wed, 22 Apr 2026 00:00:00 +0000

Cryo-electron tomography (cryo-ET) has emerged as the preferred technique for visualizing the organization of macromolecular complexes in situ and resolving their structures at subnanometre resolution [Tegunov et al. (2021), Nat. Methods, 18, 186-193]. Despite improvements in data quality as a result of advances in detector technology, microscope stability and stage precision, the analysis and interpretation of tomograms remains challenging due to a low signal-to-noise ratio and reconstruction artifacts stemming from experimental constraints in specimen tilt during data collection resulting in a missing wedge in the Fourier space. Recently, self-supervised deep-learning methods have been proposed for contrast enhancement and reduction of resolution anisotropy in reconstructed tomograms. Here, we evaluate several state-of-the-art deep-learning methods which aim to improve the interpretability of cryo-ET reconstructions, with a focus on their performance on downstream tasks of template...

Validated ligand geometries for macromolecular refinement restraints and molecular-mechanics force fields.

Wed, 22 Apr 2026 00:00:00 +0000

In macromolecular structure refinement, the low observation-to-parameter ratio and the lack of high-resolution data are countered by using a priori information in the form of restraints. Having accurate geometries of the chemical entities in the sample is paramount for generating accurate chemical restraints and, therefore, accurate macromolecular structures. In particular, it is desirable to have accurate restraints for known and novel ligand entities. Quantum mechanics (QM) can minimize the energy of a ligand by adjusting its geometry, and these geometries can be used to generate restraints for macromolecular refinement. This article describes a library of approximately 37 000 small molecules extracted from the Chemical Component Dictionary in the Protein Data Bank and minimized by density-functional QM. The library includes restraint files for use in crystallography or cryo-EM refinement, along with files suitable for molecular-dynamics simulation. Because the geometries are...

PARP1-HPF1 structure and dynamics on nicked DNA suggest a mechanism for acute and localized ADP-ribosylation

Tue, 21 Apr 2026 00:00:00 +0000

PARP1 detection of DNA strand breaks allosterically leads to PARP1 synthesis of poly(ADP-ribose) modifications that signal DNA damage. HPF1 engages activated PARP1 to control modification site selection. Understanding of the mechanism of DNA break detection and catalytic activation is incomplete, due largely to limited structural information for full-length PARP1. Here, single-particle cryo-EM provides views of the full complement of PARP1 domains engaging a DNA single-strand break in the presence of HPF1 and a fragment of binding partner Timeless. Cryo-EM, single-molecule DNA dynamics, and small-angle X-ray scattering analysis indicate that PARP1 remains dynamic even when the multi-domain structure is organized on a DNA break, with the minimal catalytic region displaying high mobility relative to domains engaging damage. We propose that the organization of PARP1 domains on a DNA break releases a tethered, constitutively active catalytic region to modify molecules in a radius...

Matrix polysaccharides affect preferred orientation of cellulose crystals in primary cell walls

Tue, 21 Apr 2026 00:00:00 +0000

The spatial organization and interactions of constituent components influence cell growth and determine physical and chemical properties of the cell wall, including its rigidity, flexibility, and degradability. Elucidating the interactions between cell wall polysaccharides is crucial for advancing our knowledge of how cell walls are assembled and for designing approaches to efficiently break down cell walls to produce renewable energy and biomaterials. Here, we investigated the effect of defects in the biosynthesis of cell wall components on the nanoscale organization of cellulose in primary cell walls through grazing incidence wide angle X-ray scattering (GIWAXS) measurements of hypocotyls of wild type Arabidopsis thaliana and of cellulose, pectin, and xyloglucan (hemicellulose) deficient mutants. GIWAXS reveals changes in lattice spacings, coherence lengths, and relative crystalline content for cellulose between wild type and mutant plants. In addition, X-ray pole figures constructed...

Coherent and Dynamic Small Polaron Delocalization in CuFeO2

Thu, 16 Apr 2026 00:00:00 +0000

Small polarons remain a bottleneck in realizing efficient transition metal oxide devices. Routes to engineer small polaron coupling to electronic states and lattice modes to control carrier localization remain unclear. Here, we measure small polaron formation in CuFeO₂ using transient extreme ultraviolet reflection spectroscopy and compare to theoretical predictions in realistically parametrized Holstein models, demonstrating that polaron localization depends on coupling to high-frequency versus low-frequency phonon bath components. We measure small polaron formation on a comparable ∼100 fs timescale to other Fe(III) compounds. Dynamic delocalization of the polaron follows formation through a coherent lattice expansion between Fe-O layers and charge-sharing with surrounding Fe(IV) states. Simulations reveal two major factors dictate polaron formation timescales: phonon density and reorganization energy distributions between acoustic and optical modes, matching experimental...

Can ferric-oxyl excited states explain elongated iron-oxygen bonds in heme peroxidase catalytic intermediates?

Wed, 15 Apr 2026 00:00:00 +0000

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...

Tulane virus protease as a structural surrogate for inhibitor screening of human norovirus proteases.

Tue, 31 Mar 2026 00:00:00 +0000

Human norovirus (HuNoV) is a significant cause of gastroenteritis worldwide, affecting people of all age groups. There are currently no vaccines or drugs available, leaving susceptible populations vulnerable to severe or protracted illness. A HuNoV cultivation system is pivotal for screening norovirus antivirals. While the human intestinal enteroid cultivation system allows robust replication of multiple HuNoV strains, it presents technical and cost barriers. Tulane virus (TV), a surrogate for HuNoV, replicates well in monkey kidney cell lines and is closely related to norovirus in cellular biology. Here, we determined the structures of TV protease (TV-Pro) alone and in complex with rupintrivir, a picornavirus inhibitor that also inhibits HuNoV proteases (HuNoV-Pro). Our data validate TV as an efficient surrogate system for rapid screening of HuNoV protease inhibitors. The TV protease structure exhibits significant backbone similarity to the GI.1 HuNoV protease in the substrate-binding...

Fyn–Saracatinib Complex Structure Reveals an Active State-like Conformation

Mon, 30 Mar 2026 00:00:00 +0000

Fyn is a Src-family tyrosine kinase implicated in synaptic dysfunction and neuroinflammation across multiple neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Saracatinib (AZD0530) is a potent Src-family inhibitor that has been explored as a repurposed therapeutic; however, its clinical utility is limited by poor kinase selectivity caused by high sequence conservation within Src-family ATP-binding sites. Here, we combine surface plasmon resonance (SPR) and X-ray crystallography to define saracatinib recognition by the Fyn kinase domain (KD). SPR single-cycle kinetics shows that saracatinib binds the isolated Fyn KD and full-length Fyn with low-nanomolar affinity, whereas dasatinib binds with subnanomolar affinity and markedly slower dissociation. We determined the crystal structure of the Fyn KD-saracatinib complex at 2.22 Å resolution. The kinase adopts an active-like conformation with the DFG motif and αC-helix in the 'in' state and...

Substituent size versus metal binding of inhibitors with variants of influenza endonuclease

Tue, 10 Feb 2026 00:00:00 +0000

The influenza virus causes a significant burden of illness each year. Although vaccination is the most effective method to prevent seasonal influenza infection, viral escape mechanisms make vaccine composition difficult to predict. Antivirals are crucial for decreasing rates of morbidity and mortality from influenza viral infection. The newest anti-influenza drugs target the RNA-dependent RNA polymerase acidic N-terminal (PA_N) endonuclease, a critical component of influenza viral replication machinery. This study examines the structure of inhibitors of PA_N that utilize a hydroxypyridinone-based metal-binding pharmacophore (MBP). Specifically, this report explores how the size of substituent groups impacts the binding conformation and affinity of a series of compounds against both wild-type (WT) and resistance mutant strains, I38T and E23K. Co-crystal structures revealed that the distance between compounds and enzyme residue 38 was conserved to maintain strong...

Label-free structural imaging of plant roots and microbes using third-harmonic generation microscopy

Fri, 23 Jan 2026 00:00:00 +0000

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With...

AQuaRef: machine learning accelerated quantum refinement of protein structures

Fri, 23 Jan 2026 00:00:00 +0000

Cryo-EM and X-ray crystallography provide crucial experimental data for obtaining atomic-detail models of biomacromolecules. Refining these models relies on library-based stereochemical data, which, in addition to being limited to known chemical entities, do not include meaningful noncovalent interactions. Quantum mechanical (QM) calculations could alleviate these issues but are too expensive for large molecules. Here we present a novel AI-enabled Quantum Refinement (AQuaRef) based on AIMNet2 machine learned interatomic potential (MLIP) mimicking QM at substantially lower computational costs. By refining 41 cryo-EM and 30 X-ray structures, we show that this approach yields atomic models with superior geometric quality compared to standard techniques, while maintaining an equal or better fit to experimental data. Notably, AQuaRef aids in determining proton positions, as illustrated in the challenging case of short hydrogen bonds in the parkinsonism-associated human protein DJ-1...

Three rate-determining protein roles in photosynthetic O2-evolution addressed by time-resolved experiments on genetically modified photosystems

Thu, 22 Jan 2026 00:00:00 +0000

Light-driven water splitting by plants, algae and cyanobacteria is pivotal for global bioenergetics and biomass formation. A manganese cluster bound to the photosystem II proteins catalyzes the complex reaction at high rate, but the rate-determining factors are insufficiently understood. Here we trace the oxygen-evolution transition by time-resolved polarography and infrared spectroscopy for cyanobacterial photosystems genetically modified at two strategic sites, complemented by computational chemistry. Our results highlight three rate-determining roles of the protein environment of the metal cluster: acceleration of proton-coupled electron transfer, acceleration of substrate-water insertion after O2-formation, and balancing of rate-determining enthalpic and entropic contributions. Whereas in general the substrate-water insertion step may be unresolvable in time-resolved experiments, here it likely becomes traceable because of deceleration by genetic modification. Our results...

New targets and procedures for validating the valence geometry of nucleic acid structures.

Thu, 15 Jan 2026 00:00:00 +0000

A Working Group consisting of the co-authors of this paper was established in 2020 to re-evaluate the standard valence geometry used for the validation of nucleic acid structure models in the Protein Data Bank (PDB). This Working Group re-examined the dependence of Cambridge Structural Database (CSD) derived targets on base and sugar type, sugar pucker, and phosphate and glycosidic conformation, before comparing those targets with the geometry of a quality-filtered reference set of nucleic acid crystal structural models held in the PDB. This revealed that the valence bond and angle mean values are close to the CSD targets, but many parameters have highly non-Gaussian or even multimodal distributions. One explanation is the inconsistency of restraints used over time and by different refinement programs. The Working Group recommends a new validation scheme for use by the PDB. For this purpose, we have developed a new three-tier scale for outlier detection-graded as Preferred, Allowed,...

Acclimation to high and low diurnal light is flexible in Chlamydomonas reinhardtii

Wed, 14 Jan 2026 00:00:00 +0000

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods [S. Dupuis et al., Plant Cell 37, koaf086 (2025), 10.1093/plcell/koaf086]. Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL...

Phase Transformation and Water Adsorption Behavior of As‐Deposited and Annealed Ru Metal Thin Films Prepared by Atomic Layer Deposition

Wed, 14 Jan 2026 00:00:00 +0000

ABSTRACT Surfaces play a central role in catalytic processes, and understanding the transformation of ruthenium metal into ruthenium oxide during annealing is essential for tailoring functional catalytic interfaces. In this study, we systematically investigate ≈22 nm thick Ru metal films deposited by atomic layer deposition (ALD) at 300°C, focusing on their chemical composition, structural evolution, and surface hydration behavior following post‐deposition annealing in air from 400 to 600°C. Lab‐based and synchrotron X‐ray photoelectron spectroscopy (XPS) reveal a gradual conversion from metallic Ru to fully oxidized Ru 4+ with increasing annealing temperature, accompanied by a corresponding increase in lattice oxygen. X‐ray diffraction (XRD) shows amorphous Ru oxide phases at 400°C and 500°C that evolve into crystalline RuO 2 at 600°C, while atomic force microscopy (AFM) indicates enhanced grain growth and surface roughening upon annealing. Ambient‐pressure XPS (AP‐XPS)...

Interior soft x-ray tomography with sparse global sampling

Wed, 14 Jan 2026 00:00:00 +0000

To investigate the feasibility of interior imaging reconstruction in soft X-ray tomography for higher-resolution cellular imaging, including whole-cell imaging, we develop an alignment and reconstruction algorithm that combines a small number of sparse whole-cell images with a high-resolution local interior scan. Based on numerical simulations, we demonstrate that combined reconstructions mitigate the depth-of-field limitation in high-resolution scans, enable radiation dose optimization, and yield quantitative X-ray absorption values with sparse sampling. We further validate our numerical approach using experimental data from two different cell types and show that the combined reconstruction reliably provides high spatial resolution within an interior region of interest of a whole cell. The resulting sparse reconstruction framework offers robust, faithful visualization of cellular organelles in soft X-ray tomography. This mesoscale imaging strategy allows one to ‘scout’ and zoom...

Supramolecular Support of Cuprophilic Network Bonding in 2‑D Copper n‑Alkanethiolates

Wed, 7 Jan 2026 00:00:00 +0000

The development of heterogeneous materials, catalysts, and semiconductors is often reliant on precise control of self-assembly and crystal packing. Many new materials are initially synthesized as microcrystalline powders, making them incompatible with typical methods of structure determination, such as single-crystal X-ray diffraction. This resultant lack of structural information has made thorough investigation into the effect of metal substitution on crystal structure in metal-organic chalcogenolates (MOChas) challenging. Here, we use small molecule serial femtosecond crystallography (smSFX) to present the structures of four copper n-alkanethiolates: CuSC4, CuSC5, CuSC6, and CuSC7. Divergent patterns of alkyl chain packing are identified from microcrystalline powders via smSFX. An odd-even effect in crystal packing has been identified and attributed to different orientations of symmetry elements in the even- and odd-numbered chains. This results in minute changes in the...

Probing and Tuning Strain‐Localized Exciton Emission in 2D Material Bubbles at Room Temperature

Thu, 11 Dec 2025 00:00:00 +0000

In monolayer transition metal dichalcogenides bubbles-nanoscale deformations typically exhibiting a dome-like shape-Excitons are confined by the strain effect, which exhibits extraordinary emission properties, such as single photon generation, enhanced light emission, and spectrally tunable excitonic states. While the strain profiles of these bubbles are extensively studied, this work provides an approach 1) to directly visualize the associated exciton properties in bubbles formed in WSe₂ monolayer, revealing an intrinsic emission wavelength shift of ≈40 nm, and 2) actively modify local strain, enabling further exciton emission tuning over a range of 50 nm. These are achieved by emission mapping and nanoindentation using a dielectric near-field probe, which enables the detection of local emission spectra and emission lifetimes within individual bubbles. Statistical analysis of 67 bubbles uncovers an emission wavelength distribution centered around 780 nm. Furthermore,...

A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells

Fri, 21 Nov 2025 00:00:00 +0000

The main protease enzyme (M^pro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that M^pro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported. To enable work on M^pro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) M^pro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing M^pro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with M^pro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that...

Comparing photosynthetic light harvesting of single photons and pseudothermal light under ultraweak illumination

Wed, 19 Nov 2025 00:00:00 +0000

Photosynthesis in vivo is driven by sunlight, an ultraweak incoherent thermal source. However, most experiments and theories have studied photosynthetic light harvesting driven by strong coherent laser sources. The quantum states of light are characterized by their photon statistics, in addition to classical properties such as intensity and frequency spectrum. Here, we report experiments that investigate how photon statistics affect a natural photosynthetic system and vice versa. We directly compare how single photons and pseudothermal light from spontaneous parametric down-conversion drive light harvesting in the light-harvesting 2 complex from a purple bacterium. We find that the fluorescence lifetime and quantum efficiency are unchanged while the fluorescence photon statistics are markedly different, resembling that of the incident light, implying that the dynamics do not fundamentally modify the photon statistics. This represents a step toward clarification of the similarities...

Optomechanical Tuning of Second Harmonic Generation Anisotropy in Janus MoSSe/MoS2 Heterostructures

Tue, 18 Nov 2025 00:00:00 +0000

Symmetry breaking in van der Waals materials enables the realization of quantum states and advanced device functionalities. Janus transition-metal dichalcogenides (TMDs) exhibit distinctive nonlinear optical properties due to their broken out-of-plane mirror symmetry. However, the dynamic control of second harmonic generation (SHG) anisotropy and resonance behavior via optical excitation remains elusive. In this work, we investigate the SHG response of Janus MoSSe/MoS₂ heterostructures with 2H and 3R stackings. We can tune the SHG response by varying the incident photon wavelength from 800 to 1000 nm, which shows a resonance-dependent enhancement in intensity and a deviation from 6-fold symmetry, indicating wavelength-dependent anisotropy. The ratio between maximum and minimum intensity in the armchair directions, associated with the SHG anisotropy, reaches a value of 1.73 at the excitation wavelength of 1000 nm. Group theory analysis and first-principles calculations...

Unlocking expanded flagellin perception through rational receptor engineering

Wed, 5 Nov 2025 00:00:00 +0000

The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologues possess narrow perception ranges, but four with expanded perception have been identified. Using diversity analyses, AlphaFold modelling and amino acid properties, key residues enabling expanded recognition were mapped to FLS2’s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) into a homologue with canonical perception. A similar approach enabled transfer of Agrobacterium perception from FLS2XL (Vitis riparia) into VrFLS2. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22’s C terminus, suggesting more alleles with expanded perception exist. Our experimental...

Ancient pangenomic origins of noncanonical NLR genes underlying the recent evolutionary rescue of a staple crop

Wed, 5 Nov 2025 00:00:00 +0000

The recent adaptation of the cereal crop sorghum to a global aphid outbreak was a fortuitous case of evolutionary rescue, but the pangenomic and molecular basis is not known. We show that RMES1 disrupts phloem feeding via activation of conserved immunity networks, with a growth-to-defense transition mediated by phytohormone signaling and activated by nucleotide-binding site-leucine-rich repeat receptor (NLR) resistance genes on a structural variant. The causative NLRs [resistance to Melanaphis sorghi 1A (RMES1A) and RMES1B] lack signaling domains and have adenosine triphosphatase mutations expected to abrogate function, suggesting that RMES1 NLRs regulate immunity via a noncanonical mechanism. The RMES1 NLR family is ancient, orthologous to phloem-feeding resistance genes in rice and syntenic across the grass superpangenome. Thus, gene birth-and-death processes at an ancient gene cluster created rare standing variation and provided the adaptive allele for...

Reuniting crystallography with real space: Ab initio structure elucidation with 4D-STEM

Tue, 14 Oct 2025 00:00:00 +0000

Structure elucidation via single-crystal methods has historically lacked experimental access to real-space information, instead relying exclusively on diffraction-space measurements of Bragg reflections. Here we exploit the dual-space imaging power of 4D scanning transmission electron microscopy to meaningfully integrate real-space information into the crystallographic workflow. We show that virtual apertures assembled by segmentation of high-angle annular dark-field images enable i) pixel-by-pixel separation of coherent Bragg signal from clusters of closely spaced nanocrystals and ii) selective extraction of integrated intensities from thinner subregions of individual specimens, facilitating retroactive tuning of multiple scattering artifacts. This strategy empowers us to simply pick and choose whichever nanoscale regions of interest generate the highest-quality diffraction patterns, allowing us to solve several independent structures of the metal-organic framework UiO-66 from...

Ancient pangenomic origins of noncanonical NLR genes underlying the recent evolutionary rescue of a staple crop

Mon, 13 Oct 2025 00:00:00 +0000

Computational design of potent and selective binders of BAK and BAX

Tue, 7 Oct 2025 00:00:00 +0000

Potent and selective binders of the key proapoptotic proteins BAK and BAX have not been described. We use computational protein design to generate high affinity binders of BAK and BAX with greater than 100-fold specificity for their target. Both binders activate their targets when at low concentration, driving pore formation, but inhibit membrane permeabilization when in excess. Crystallography shows that the BAK binder induces BAK unfolding, exposing the α6 helix and BH3 domain. Together, these data suggest that upon binding, BAK or BAX unfold; at high binder concentrations, self-association of the partially folded BAK or BAX proteins is blocked and the membrane remains intact, whereas at low concentrations, dimers form, and the membrane ruptures. Our designed binders modulate apoptosis via direct, specific interactions with BAK and BAX and reveal that for therapeutic strategies targeting BAK and BAX, inhibition requires saturating binder concentrations at the site of action.

An alternative pocket for binding the N‐degrons by the UBR1 and UBR2 ubiquitin E3 ligases

Tue, 9 Sep 2025 00:00:00 +0000

The UBR family of ubiquitin ligases binds to N-termini of their targets (known as N-degron) to induce their ubiquitination and degradation via a conserved domain known as UBR-box. UBR1 and UBR2 share the highest sequence homology among the family, and substantial structural studies were previously performed for substrate binding by the UBR-boxes of UBR1 and UBR2. Here, we describe a new pocket in the UBR-boxes of UBR1 and UBR2 for binding the second residues of N-degrons through determining five co-crystal structures of the UBR-boxes with various N-degron peptides. Together with binding affinities measured by fluorescence polarization, we show that the two highly homologous UBR-boxes can interact with the second residue of an N-degron differently. In addition, the UBR-boxes undergo different conformational changes when binding N-degrons. Furthermore, we demonstrate that the sidechain of the third amino acid of an N-degron has no contribution to binding the UBR-boxes. These findings...

Unraveling the ecological success of Iodidimonas in a bioreactor treating oil and gas produced water

Fri, 5 Sep 2025 00:00:00 +0000

Iodidimonas sp., a bacterium found in bioreactors treating oil and gas produced water as well as iodide-rich brines, has garnered attention for its unique ability to oxidize iodine. However, little is known about the metabolic capabilities that enable Iodidimonas sp. to thrive in certain unique ecological niches. In this study, we isolated, characterized, and sequenced three strains belonging to the Iodidimonas genus from the sludge of a membrane bioreactor used for produced water treatment. We investigated the genomic features of these isolates and compared them with the four publicly available isolate genomes from this genus, as well as a metagenome-assembled genome from the source bioreactor. Our Iodidimonas isolates had several genes associated with mitigating salinity, heavy metal, and organic compound stress, which likely help these bacteria to survive in produced water. Phenotyping tests revealed that while the isolates could utilize a wide variety...

Attosecond inner-shell lasing at ångström wavelengths

Tue, 19 Aug 2025 00:00:00 +0000

Since the invention of the laser, nonlinear effects such as filamentation1, Rabi cycling2,3 and collective emission4 have been explored in the optical regime, leading to a wide range of scientific and industrial applications5, 6, 7–8. X-ray free-electron lasers (XFELs) have extended many optical techniques to X-rays for their advantages of ångström-scale spatial resolution and elemental specificity9. An example is XFEL-driven inner-shell Kα1 (2p3/2 → 1s1/2) X-ray lasing in elements ranging from neon to copper, which has been used for nonlinear spectroscopy and development of new X-ray laser sources10, 11, 12, 13, 14, 15–16. Here we show that strong lasing effects similar to those in the optical regime can occur at 1.5–2.1 Å wavelengths during high-intensity (>1019 W cm−2) XFEL-driven Kα1 lasing of copper and manganese. Depending on the temporal XFEL pump pulse substructure, the resulting X-ray pulses (about 106−108 photons) can exhibit strong spatial inhomogeneities and spectral...

Discovery of an autoinhibited conformation in mesotrypsin reveals a strategy for selective serine protease inhibition

Thu, 14 Aug 2025 00:00:00 +0000

Selective inhibition of the more than 100 S1 family serine proteases is a long-standing challenge due to their active site similarity. Mesotrypsin, implicated in cancer progression, exemplifies these difficulties; no current inhibitors achieve selectivity over other human trypsins. We found an unexpected autoinhibited conformation of mesotrypsin via x-ray crystallography, revealing a cryptic pocket adjacent to the active site. Using high-throughput virtual screening targeting this cryptic pocket, we identified a conformationally selective small-molecule inhibitor that stabilizes the inactive state of mesotrypsin. This inhibitor demonstrates selectivity for mesotrypsin over other trypsins. Our findings challenge the accepted view of digestive trypsins as constitutively active enzymes lacking potential for allosteric regulation. Furthermore, analyses of other structures suggest that dynamic sampling of closed states with analogous allosteric cryptic pockets appears widespread among...

Enabling simultaneous photoluminescence spectroscopy and X-ray footprinting mass spectrometry to study protein conformation and interactions

Mon, 4 Aug 2025 00:00:00 +0000

X-ray footprinting mass spectrometry (XFMS) is a structural biology method that uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation in solution. However, while XFMS alone provides important structural information on biomolecules, as we move into the era of the interactome, hybrid methods are becoming increasingly necessary to gain a comprehensive understanding of protein complexes and interactions. Toward this end, we report the development of the first synergetic application of inline and real-time fluorescent spectroscopy at the Advanced Light Source's XFMS facility to study local protein interactions and global conformational changes simultaneously. To facilitate general use, we designed a flexible and optimum system for producing high-quality spectroscopy-XFMS hybrid data, with rapid interchangeable liquid jet or capillary sample delivery for multimodal inline spectroscopy, and several choices for optofluidic environments....

A Solvatochromic Near Infrared Fluorophore Sensitive to the Full Amyloid Beta Aggregation Pathway

Mon, 4 Aug 2025 00:00:00 +0000

Alzheimer's disease has long been associated with the aggregation of amyloid beta peptides (Aβ42) into macroscale plaques, although specific neurodegenerative agents have not been definitively identified. Much evidence has pointed to the soluble nanoscale oligomers that form early in the Aβ42 aggregation pathway, but there is little understanding of these structures, their mechanisms of formation, or how they grow into plaques. Here, we show that a solvatochromic fluorophore with near-infrared (NIR) emission can track synthetic Aβ42 aggregation through environment-sensitive spectral shifts from the earliest time points through plaque formation. This azide-functionalized phosphine oxide azetidine rhodol (Phazr-N₃) shows large polarity-dependent changes in fluorescence emission, with maxima shifting from 630 nm in toluene to 703 nm in aqueous buffer, and a maximum quantum yield of 62%. Upon induction of Aβ42 aggregation, we observe immediate solvatochromic changes in...

ALS-ENABLE: creating synergy and opportunity at the Advanced Light Source synchrotron structural biology beamlines

Tue, 29 Jul 2025 00:00:00 +0000

ALS-ENABLE is an integrated NIH P30 resource at the Advanced Light Source synchrotron at Lawrence Berkeley National Laboratory in Berkeley, California, USA. The resource provides a single portal to the combined mature structural biology technologies of macromolecular crystallography, small-angle X-ray scattering and X-ray footprinting mass spectrometry, and includes beamlines 2.0.1, 3.3.1, 4.2.2, 5.0.1, 5.0.2, 5.0.3, 8.2.1, 8.2.2, 8.3.1 and 12.3.1. This paper describes the organizational structure and the technologies of ALS-ENABLE. A case study showcasing the main technologies of the resource applied to the characterization of the SpyCatcher-SpyTag protein system is presented.

Insights from designing an artificial cascade catalysis system using principles from substrate channeling in enzymes

Tue, 29 Jul 2025 00:00:00 +0000

Generalizing the key requirements of highly-selective, multi-step chemical conversions involving spatially separated reaction centers remains one of the grand challenges of chemistry. Much work towards this effort has focused on decomposing multi-step conversions into their constituent reactions, whose intermediates are successively upgraded in a chemical cascade via diffusion from center to center. This approach for synthesizing more complex molecules takes its cues from biochemical networks, where near-unit conversion of even complex carbohydrates is achieved by upgrading chemical precursors via enzymatic cascades. In this computational study we examine a simple cascade involving coupled Ag and Cu catalysts that sequentially converts CO₂ to CO and then CO₂ and CO to reduced products, generically named CO₂Product and COProduct. The system architecture is inspired by the phenomenon of biological substrate channeling, and components...

Nucleophilic Displacement Reactions of Silver-Based Metal–Organic Chalcogenolates

Mon, 28 Jul 2025 00:00:00 +0000

We report nucleophilic displacement reactions that can increase the dimensionality or coordination number of silver-based metal-organic chalcogenolates (MOChas). MOChas are crystalline ensembles containing one-dimensional (1D) or two-dimensional (2D) inorganic topologies with structures and properties defined by the choice of metal, chalcogen, and ligand. MOChas can be readily prepared from a variety of small-molecule ligands and metals or metal ions. Although MOChas offer ligand diversity, most reported examples use relatively small ligands, typically involving short alkyl chains, aryl rings, or molecular cages. This is because larger, more complex molecules often yield poor product morphologies with indeterminate structures. In this study, we overcame this limitation by employing a ligand exchange strategy whereby a 1D MOCha, silver(I) methyl 2-mercaptobenzoate (2MMB), is used as a silver source for preparing 2D examples. The reaction proceeds generally toward products composed...

Optimizing inference of segmentation on high-resolution images in MLExchange

Thu, 24 Jul 2025 00:00:00 +0000

MLExchange is a machine learning (ML) operations platform providing web user-interfaces (UIs) for data visualization and analysis pipelines at synchrotron facilities. Among these UIs is the segmentation app which helps synchrotron users utilize ML algorithms to automatically segment high-resolution scientific images with minimal manual annotation effort. In this work, we share code optimizations that significantly speed up the segmentation inference workflow of large data in short time. By optimizing the sequence of CPU-GPU data transfers and introducing CPU parallelization to key operations, we improve the per-device, per-image frame computational efficiency and observe close to 3×$$\times$$ speedup over the original segmentation inference workflow run time when utilizing a single GPU. Further adaptations enabling multi-GPU inference yield more than 40×$$\times$$ speedup with 100 GPUs compared to the optimized single GPU inference workflow. This acceleration of the segmentation...

The electrode–electrolyte interface of Cu via modulation excitation X-ray absorption spectroscopy

Thu, 24 Jul 2025 00:00:00 +0000

The development and application of modulation excitation X-ray absorption spectroscopy with sub-second resolution and better than 0.02% detection sensitivity unveils the kinetics of the anodic oxidation of Cu in bicarbonate electrolytes. Accessing the electrode–electrolyte interface under operating conditions and capturing time-resolved kinetics remain challenging in electrochemical studies. Copper's interfacial oxidation dynamics remain unclear despite extensive research. Modulation excitation X-ray absorption spectroscopy (ME-XAS) was used to probe Cu in 100 mM KHCO 3 with sub-second sensitivity, revealing that hydroxide forms 30 ± 10 ms before the appearance of Cu 2 O at positive potentials (0 to 0.5 V vs. RHE) near open-circuit conditions. From −0.4 to 0.8 V vs. RHE, hydroxide coverage reaches 49%, accompanied by a balanced presence of Cu( i ) and Cu( ii ) oxides. These insights into Cu interfacial redox behavior under intermittent renewable energy operation—relevant to CO...

Achieving High-Yield Conversion of Janus Transition Metal Dichalcogenides on Diverse Substrates

Tue, 22 Jul 2025 00:00:00 +0000

Janus transition metal dichalcogenides (TMDCs) with intrinsic broken mirror symmetry and vertical dipole moment provide an additional degree of freedom to manipulate material symmetry down to atomic-layer thickness. However, despite advances in synthesis strategies, fundamental understanding of this atomic substitution process remains limited, which has impeded their implementation in advanced devices. Here, by using a room-temperature atomic-layer substitution (RT-ALS) strategy, we systematically investigate the critical factors facilitating the high-yield conversion of Janus TMDCs on diverse substrates. Combining Raman spectroscopy probes, X-ray photoelectron spectroscopy (XPS) measurements, and density functional theory (DFT) calculations, we demonstrate that substrates with enhanced electron doping or larger surface polarity substantially benefit the conversion of Janus TMDCs reaching a near-unity yield. Intriguingly, the strong affinity between Janus TMDCs and substrates...

Colloidal Dispersions of Sterically and Electrostatically Stabilized PbS Quantum Dots: Structure Factors, Second Virial Coefficients, and Film-Forming Properties

Tue, 22 Jul 2025 00:00:00 +0000

Electrostatically stabilized nanocrystals (NCs) and, in particular, quantum dots (QDs) hold promise for forming strongly coupled superlattices due to their compact and electronically conductive surface ligands. However, studies of the colloidal dispersion and interparticle interactions of electrostatically stabilized sub-10 nm NCs have been limited, hindering the optimization of their colloidal stability and self-assembly. In this study, we employed small-angle X-ray scattering (SAXS) experiments to investigate the interparticle interactions and arrangement of PbS QDs with thiostannate ligands (PbS-Sn₂S₆^4-) in polar solvents. The study reveals significant deviations from the ideal solution behavior in electrostatically stabilized QD dispersions. Our results demonstrate that PbS-Sn₂S₆^4- QDs exhibit long-range interactions within the solvent, in contrast to the short-range steric repulsion characteristic of PbS QDs...

Serial-femtosecond crystallography reveals how a phytochrome variant couples chromophore and protein structural changes

Mon, 21 Jul 2025 00:00:00 +0000

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway...

Soft X-Ray Tomography Has Evolved into a Powerful Tool for Revealing Cell Structures

Wed, 16 Jul 2025 00:00:00 +0000

Over the past three decades, soft X-ray tomography (SXT) has rapidly evolved from a proof-of-concept microscopy method into a high-throughput quantitative imaging modality. This advancement enables researchers to address central questions in cell biology. Despite its relatively short developmental period compared to light and electron microscopy, SXT has emerged as a powerful imaging technology. It enables measuring chemical changes in cellular organelles, analyzing three-dimensional structures of whole cells and creating digital cellular models to study cell motility. We discuss the unique nature of SXT to visualize cells without fixation or labeling, enabling quantitative analyses of organelle chemical composition. We explore SXT microscopes available worldwide, SXT segmentation software, and the diverse cell types studied using this technique. We conclude with emerging directions in SXT imaging, including a brief discussion of recent discoveries that are highly influential...

Transformation of TiN to TiNO Films via In-Situ Temperature-Dependent Oxygen Diffusion Process and Their Electrochemical Behavior

Tue, 15 Jul 2025 00:00:00 +0000

Titanium oxynitride (TiNO) thin films represent a multifaceted material system applicable in diverse fields, including energy storage, solar cells, sensors, protective coatings, and electrocatalysis. This study reports the synthesis of TiNO thin films grown at different substrate temperatures using pulsed laser deposition. A comprehensive structural investigation was conducted by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Non-Rutherford backscattering spectrometry (N-RBS), and X-ray absorption spectroscopy (XAS), which facilitated a detailed analysis that determined the phase, composition, and crystallinity of the films. Structural control was achieved via temperature-dependent oxygen in-diffusion, nitrogen out-diffusion, and the nucleation growth process related to adatom mobility. The XPS analysis indicates that the TiNO films consist of heterogeneous mixtures of TiN, TiNO, and TiO2 phases with temperature-dependent relative abundances. The correlation...

A machine-learning-driven data labeling pipeline for scientific analysis in MLExchange

Fri, 11 Jul 2025 00:00:00 +0000

This study introduces a novel labeling pipeline to accelerate the labeling process of scientific data sets by using artificial intelligence (AI)-guided tagging techniques. This pipeline includes a set of interconnected web-based graphical user interfaces (GUIs), where Data Clinic and MLCoach enable the preparation of machine learning (ML) models for data reduction and classification, respectively, while Label Maker is used for label assignment. Throughout this pipeline, data can be accessed through a direct connection to a file system or through Tiled for access through Hypertext Transfer Protocol (HTTP). Our experimental results present three use cases where this labeling pipeline has been instrumental for the study of large X-ray scattering data sets in the area of pattern recognition, the remote analysis of resonant soft X-ray scattering data and the fine-tuning process of foundation models. These use cases highlight the labeling capabilities of...

RNA sculpting by the primordial Helix-clasp-Helix–Strand-Loop (HcH–SL) motif enforces chemical recognition enabling diverse KH domain functions

Wed, 9 Jul 2025 00:00:00 +0000

In all domains of life, the ancient K homology (KH) domain superfamily is central to RNA processes including splicing, transcription, posttranscriptional gene regulation, signaling, and translation. Proteins with 1 to 15 KH domains bind single-strand (ss) RNA or DNA with base sequence specificity. Here, we examine over 40 KH domain experimental structures in complex with nucleic acid (NA) and define a novel Helix-clasp-Helix-Strand-Loop (HcH-SL) NA recognition motif binding 4 to 5 nucleotides using 10 to 18 residues. HcH-SL includes and extends the Gly-X-X-Gly (GXXG) signature sequence "clasp" that brings together two helices as an ∼90° helical corner. The first helix primarily provides side chain interactions to unstack and sculpt 2 to 3 bases on the 5' end for recognition of sequence and chemistry. The clasp and second helix amino dipole recognize a central phosphodiester. Following the helical corner, a beta strand and its loop extension recognize the two 3' nucleotides, primarily...

A distinct LHCI arrangement is recruited to photosystem I in Fe-starved green algae

Tue, 1 Jul 2025 00:00:00 +0000

Iron (Fe) availability limits photosynthesis at a global scale where Fe-rich photosystem (PS) I abundance is drastically reduced in Fe-poor environments. We used single-particle cryoelectron microscopy to reveal a unique Fe starvation-dependent arrangement of light-harvesting chlorophyll (LHC) proteins where Fe starvation-induced TIDI1 is found in an additional tetramer of LHC proteins associated with PSI in Dunaliella tertiolecta and Dunaliella salina. These cosmopolitan green algae are resilient to poor Fe nutrition. TIDI1 is a distinct LHC protein that co-occurs in diverse algae with flavodoxin (an Fe-independent replacement for the Fe-containing ferredoxin). The antenna expansion in eukaryotic algae we describe here is reminiscent of the iron-starvation induced (isiA-encoding) antenna ring in cyanobacteria, which typically co-occurs with isiB, encoding flavodoxin. Our work showcases the convergent strategies that evolved after the Great Oxidation...

Ultrafast Energy Transfer and Charge Separation Pathways in the Photosystem II Supercomplex

Tue, 1 Jul 2025 00:00:00 +0000

Spectral congestion challenges spectroscopic studies of the Photosystem II (PSII) light harvesting antenna dynamics. The higher resolution of two-dimensional electronic-vibrational spectroscopy provides new insights into the PSII supercomplex dynamics.

Dynamic Characterization of Photosynthetic Proteins on Thylakoid Membranes by High-Speed AFM

Tue, 1 Jul 2025 00:00:00 +0000

Dynamic Characterization of Photosynthetic Proteins on Thylakoid Membranes by High-Speed AFM

Robust error calibration for serial crystallography

Mon, 30 Jun 2025 00:00:00 +0000

Serial crystallography is an important technique with unique abilities to resolve enzymatic transition states, minimize radiation damage to sensitive metalloenzymes and perform de novo structure determination from micrometre-sized crystals. This technique requires the merging of data from thousands of crystals, making manual identification of errant crystals unfeasible. cctbx.xfel.merge uses filtering to remove problematic data. However, this process is imperfect, and data reduction must be robust to outliers. We add robustness to cctbx.xfel.merge at the step of uncertainty determination for reflection intensities. This step is a critical point for robustness because it is the first step where the data sets are considered as a whole, as opposed to individual lattices. Robustness is conferred by reformulating the error-calibration procedure to have fewer and less stringent statistical assumptions and incorporating the ability to down-weight low-quality lattices. We then apply this...

Architecture of Pseudomonas aeruginosa glutamyl-tRNA synthetase defines a subfamily of dimeric class Ib aminoacyl-tRNA synthetases

Mon, 23 Jun 2025 00:00:00 +0000

The aminoacyl-tRNA synthetases (AaRSs) are an ancient family of structurally diverse enzymes that are divided into two major classes. The functionalities of most AaRSs are inextricably linked to their oligomeric states. While GluRSs were previously classified as monomers, the current investigation reveals that the form expressed in Pseudomonas aeruginosa is a rotationally pseudosymmetrical homodimer featuring intersubunit tRNA binding sites. Both subunits display a highly bent, "pipe strap" conformation, with the anticodon binding domain directed toward the active site. The tRNA binding sites are similar in shape to those of the monomeric GluRSs, but are formed through an approximately 180-degree rotation of the anticodon binding domains and dimerization via the anticodon and D-arm binding domains. As a result, each anticodon binding domain is poised to recognize the anticodon loop of a tRNA bound to the adjacent protomer. Additionally, the anticodon binding domain has...

Too dim, too bright, and just right: Systems analysis of the Chlamydomonas diurnal program under limiting and excess light

Thu, 19 Jun 2025 00:00:00 +0000

Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to excessive. Little is known about how light intensity impacts the diurnal program in Chlamydomonas reinhardtii, or how diurnal rhythms in gene expression and metabolism shape photoprotective responses at different times of day. To address these questions, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light: genome-wide, waves of transcripts, and proteins peak at the same times in populations acclimated to stressful light intensities as in populations acclimated to moderate light. Yet, diurnal photoacclimation gives rise to hundreds of gene expression changes, even at night. Time course measurements of photosynthetic efficiency and pigments...

Photoprotection

Thu, 19 Jun 2025 00:00:00 +0000

Although light is required for photosynthesis, excess light can cause photo-oxidative damage that decreases the efficiency and rate of photosynthesis, a phenomenon known as photoinhibition. Like all photosynthetic organisms, Chlamydomonas reinhardtii cells employ a variety of photoprotective mechanisms, antioxidant defenses, and signaling pathways to prevent photoinhibition. These include regulation of photosynthetic light harvesting by nonphotochemical quenching, engagement of safety valves for eliminating excess energetic electrons, the synthesis of antioxidant small molecules and enzymes, and changes in gene expression that enable acclimation to excess light and photo-oxidative stress. Chlamydomonas has emerged as an excellent model organism to investigate fundamental mechanisms of photoprotection and their evolution in the green lineage.

Chapter 25 Photoprotection

Thu, 19 Jun 2025 00:00:00 +0000

Chapter 25 Photoprotection

Annihilation-limited long-range exciton transport in high-mobility conjugated copolymer films

Mon, 9 Jun 2025 00:00:00 +0000

A combination of ultrafast, long-range, and low-loss excitation energy transfer from the photoreceptor location to a functionally active site is essential for cost-effective polymeric semiconductors. Delocalized electronic wavefunctions along π-conjugated polymer (CP) backbone can enable efficient intrachain transport, while interchain transport is generally thought slow and lossy due to weak chain-chain interactions. In contrast to the conventional strategy of mitigating structural disorder, amorphous layers of rigid CPs, exemplified by highly planar poly(indacenodithiophene-co-benzothiadiazole) (IDT-BT) donor-accepter copolymer, exhibit trap-free transistor performance and charge-carrier mobilities similar to amorphous silicon. Here, we report long-range exciton transport in HJ-aggregated IDTBT thin-film, in which the competing exciton transport and exciton-exciton annihilation (EEA) dynamics are spectroscopically separated using a phase-cycling-based scheme and shown...

Discovery of highly potent and ALK2/ALK1 selective kinase inhibitors using DNA-encoded chemistry technology

Thu, 5 Jun 2025 00:00:00 +0000

Activin receptor type 1 (ACVR1; ALK2) and activin receptor like type 1 (ACVRL1; ALK1) are transforming growth factor beta family receptors that integrate extracellular signals of bone morphogenic proteins (BMPs) and activins into Mothers Against Decapentaplegic homolog 1/5 (SMAD1/SMAD5) signaling complexes. Several activating mutations in ALK2 are implicated in fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine gliomas, and ependymomas. The ALK2 R206H mutation is also present in a subset of endometrial tumors, melanomas, non-small lung cancers, and colorectal cancers, and ALK2 expression is elevated in pancreatic cancer. Using DNA-encoded chemistry technology, we screened 3.94 billion unique compounds from our diverse DNA-encoded chemical libraries (DECLs) against the kinase domain of ALK2. Off-DNA synthesis of DECL hits and biochemical validation revealed nanomolar potent ALK2 inhibitors. Further structure-activity relationship studies yielded center for drug...

NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism

Wed, 4 Jun 2025 00:00:00 +0000

Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4’s AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4’s β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4’s redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import....

The Role of Cu3+ in the Oxygen Evolution Activity of Copper Oxides

Tue, 3 Jun 2025 00:00:00 +0000

Cu-based oxides and hydroxides represent an important class of materials from a catalytic and corrosion perspective. In this study, we investigate the formation of bulk and surface Cu³⁺ species that are stable under water oxidation catalysis in alkaline media. So far, no direct evidence existed for the presence of hydroxides (CuOOH) or oxides, which were primarily proposed by theory. This work directly places CuOOH in the oxygen evolution reaction (OER) Pourbaix stability region with a calculated free energy of -208.68 kJ/mol, necessitating a revision of known Cu-H₂O phase diagrams. We also predict that the active sites of CuOOH for the OER are consistent with a bridge O* site between the two Cu³⁺ atoms with onset at ≥1.6 V vs the reversible hydrogen electrode (RHE), aligning with experimentally observed Cu^2+/3+ oxidation waves in cyclic voltammetry of Fe-free and Fe-spiked copper in alkaline media. Trace amounts of Fe (2 μg/mL (ppm)...

Origin of photoelectrochemical CO 2 reduction on bare Cu(In,Ga)S 2 (CIGS) thin films in aqueous media without co-catalysts

Tue, 3 Jun 2025 00:00:00 +0000

Photoelectrochemical (PEC) CO2 reduction (CO2R) on semiconductors provides a promising route to convert CO2 to fuels and chemicals. However, most semiconductors are not stable under CO2R conditions in aqueous media and require additional protection layers for long-term durability. To identify materials that would be stable and yield CO2R products in aqueous conditions, we investigated bare Cu(In,Ga)S2 (CIGS) thin films. We synthesized CIGS thin films by sulfurizing a sputtered Cu-In-Ga metal stack. The as-synthesized CIGS thin films are Cu-deficient and have a high enough bandgap (1.7 eV) suitable to perform CO2R. The bare CIGS photocathodes had faradaic yields of 14% for HCOO− and 30% for CO in 0.1 M KHCO3 electrolyte without the use of any co-catalysts under 1 sun illumination at an applied bias of −0.4 V vs. RHE and operated stably for 80 min. Operando Raman spectroscopy under CO2R conditions showed that the dominant A1 mode of CIGS was unaffected during operation. Post-mortem...

Probing Electrode Transformation under Dynamic Operation for Alkaline Water Electrolysis

Mon, 19 May 2025 00:00:00 +0000

Abstract Alkaline water electrolyzers (AWEs) play a pivotal role in the realm of large‐scale hydrogen production. However, AWEs face significant challenges in electrode degradation particularly under dynamic operating conditions, induced by reverse current phenomenon during frequent startup/shutdown. Herein, this study aims to rationalize the degradation mechanisms of AWEs under these conditions. A three‐electrode membrane electrode assembly (MEA) setup is first utilized to decouple polarization behaviors of anode and cathode in AWEs. Following a proposed accelerated stress testing protocol, the setup allows for tracking individual electrode performance transformations during frequent reverse current operation. Integrating operando cell studies with in situ and post‐mortem characterizations, it is showed that continuous formation of highly active species, nickel (oxy)hydroxides, improves the anode performance for oxygen evolution reaction. On the contrary, irreversible oxidation...

Expanding the Landscape of Dual Action Antifolate Antibacterials through 2,4-Diamino-1,6-dihydro-1,3,5-triazines

Mon, 12 May 2025 00:00:00 +0000

Antibiotics that operate via multiple mechanisms of action are a promising strategy to combat growing resistance. Previous studies have shown that dual action antifolates formed from a pyrroloquinazolinediamine core can inhibit the growth of bacterial pathogens without developing resistance. In this work, we expand the scope of dual action antifolates by repurposing the 2,4-diamino-1,6-dihydro-1,3,5-triazine (DADHT) cycloguanil scaffold to a variety of derivatives designed to inhibit dihydrofolate reductase (DHFR) and disrupt bacterial membranes. Dual mechanism DADHTs have activity against a variety of target pathogens, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa, among other ESKAPEE organisms. Through X-ray crystallography, we confirmed engagement of the Escherichia coli DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in...

NUB1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome

Mon, 12 May 2025 00:00:00 +0000

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor NUB1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system with human components and revealed that NUB1 uses the intrinsic instability of FAT10 to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin-independent and p97-independent manner. Using hydrogen–deuterium exchange, structural modeling and site-directed mutagenesis, we identified the formation of an intricate complex with FAT10 that activates NUB1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic NUB1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These findings identified...

Role of tau versus TDP‐43 pathology on medial temporal lobe atrophy in aging and Alzheimer's disease

Wed, 7 May 2025 00:00:00 +0000

Hippocampal atrophy on magnetic resonance imaging is an important biomarker in Alzheimer's disease (AD). While hippocampal atrophy was thought to result from tau tangles in AD, different neuropathologies can lead to hippocampal atrophy, especially TAR DNA-binding protein 43 (TDP-43) pathology. In this narrative review, we evaluate existing studies on the relative contribution of tau and TDP-43 pathology to medial temporal lobe (MTL) atrophy. We report a clear association of both tau and TDP-43 neuropathology with MTL atrophy, even after correcting for other neuropathologies. Next, we discuss a potential synergism between tau and TDP-43 and the relative timing of the effects of both neuropathologies. Finally, avenues for future research will be discussed. A better understanding of the interplay between tau and TDP-43 neuropathologies and their effect on atrophy will help with the development of more specific biomarkers for limbic-predominant age-related TDP-43 encephalopathy and...

The impact of kidney function on Alzheimer’s disease blood biomarkers: implications for predicting amyloid-β positivity

Wed, 7 May 2025 00:00:00 +0000

BackgroundImpaired kidney function has a potential confounding effect on blood biomarker levels, including biomarkers for Alzheimer’s disease (AD). Given the imminent use of certain blood biomarkers in the routine diagnostic work-up of patients with suspected AD, knowledge on the potential impact of comorbidities on the utility of blood biomarkers is important. We aimed to evaluate the association between kidney function, assessed through estimated glomerular filtration rate (eGFR) calculated from plasma creatinine and AD blood biomarkers, as well as their influence over predicting Aβ-positivity.MethodsWe included 242 participants from the Translational Biomarkers in Aging and Dementia (TRIAD) cohort, comprising cognitively unimpaired individuals (CU; n = 124), mild cognitive impairment (MCI; n = 58), AD dementia (n = 34), and non-AD dementia (n = 26) patients all characterized by [18F] AZD-4694. Plasma samples were analyzed for Aβ42, Aβ40, glial fibrillary acidic protein (GFAP),...

Photosynthesis | Photosystem II: Water Oxidation, Overview ☆