Nature Communications

Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of AsIII and antimonite (SbIII) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an inward-facing conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that AsIII resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.

Bacterial biofilms underpin chronic infection and antimicrobial resistance, notably in Pseudomonas aeruginosa. Here we deconvolute a commercial alginate lyase preparation from Flavobacterium quisquiliarum and identify a previously uncharacterised ~21 kDa porphyrin-binding protein (FqPBP). Structural, biophysical and docking analyses reveal high-affinity tetrapyrrole binding. Recombinant FqPBP independently inhibits and disperses P. aeruginosa biofilms, implicating porphyrin sequestration and iron homeostasis in biofilm control and highlighting a potential therapeutic strategy targeting iron acquisition pathways.

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures for Candida glabrata Cdr1 under active turnover conditions in the absence and presence of ATP-Mg{superscript 2}, itraconazole, and vanadate. Additional transient cryo-EM structures were unveiled by 3D variability analysis offering a detailed view of the step-by-step transitions triggered by ATP-hydrolysis. The motion cascade starts with a 4 [A] piston-like retraction of the C-helix from the {gamma}-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse squeeze-and-push motion of TMH-2 possibly drives substrate extrusion. High resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

Accurate prediction of CRISPR-Cas9 guide RNA (gRNA) editing efficiency remains limited, particularly outside human systems, where models trained on exogenous human datasets show poor generalization. We analyzed Cas9 efficiency and repair outcomes using novel endogenous editing data from four human cell types, two tomato cell types, and cells from giant river prawn and black soldier fly. While integrating publicly available predictors via ensemble frameworks improved performance, our analysis revealed hundreds of novel features affecting activity. Crucially, dominant features related to sites competition for gRNA, and local geometric properties varied across systems, highlighting the strong context dependence of Cas9 efficiency and arguing against a universal model. Interestingly, codon usage bias-based features also emerged as informative predictors, as they are proxies for chromatin accessibility. In contrast, trends in repair outcomes remained conserved. This work provides essential resources for more generalizable CRISPR guide design.

The eukaryotic COMMD family of proteins are core subunits of the Commander protein complex, with a central role in endosomal membrane trafficking and signalling. Previous crystal and cryoEM structures show that COMMD and COMMD-like proteins form homo-oligomeric and hetero-decameric assemblies with a central ring structure formed by their small C-terminal COMM domains. Their -helical N-terminal (HN) domains decorate each side of these rings, and in the eukaryotic Commander complex engage the coiled-coil proteins CCDC22 and CCDC93. Here, we have determined new crystal structures of the isolated HN domains of human Commd4, Commd9 and Commd10, and find that all three proteins form domain swapped structures with a remarkably consistent topology. This occurs via a conformational change in a hinge-loop between helices 2 and 3, leading to exchange of helices 3 to 6 between protomers. The hinge-loops of Commd9 and Commd10 possess several serine and threonine residues that can be phosphorylated, and we find that specific phospho-mimicking mutations in Commd10 can promote or inhibit domain swapping. Whether the unique COMMD HN domains play any roles beyond assembly with CCDC proteins is unclear, but this work suggests a common conformational switch exists with a potential to regulate their function.

Themeda triandra (family Andropogoneae) is a pan-continental grass occurring throughout Australia. Recognising its remarkable adaptive capacity, we assembled the nuclear genome sequence of a diploid accession from eastern Australia, finding all ten chromosomes of the 755 Mb assembly highly syntenic with those of Sorghum bicolor. Genotyping and cytometry of range-wide accessions suggested several historical radiation events, with polyploids dominating in arid regions, apart from a notable diploid lineage from the arid north-west of Australia (PAN). A detailed comparison of PAN with two diploids from temperate south-eastern Australia through whole-genome resequencing revealed extensive copy number variation and polymorphism, with changes in genes for heat-shock proteins and flowering regulation reflecting their environmental origin. Exploration of the rich genetic diversity in T. triandra with respect to environmental adaptation is expected to benefit grassland management programs and enable introgression of novel genes into Andropogoneae crops for climate resilience.

Tectonin Beta-Propeller Repeat containing 1 (TECPR1) is an essential regulator of a noncanonical autophagy pathway known as Sphingomyelin TECPR1 induced LC3 lipidation (STIL). TECPR1 forms an E3-like ligase complex and recognizes exposed sphingomyelin on damaged membranes. TECPR1 contains five folded domains however the structural basis for TECPR1 function has remained unresolved. Here, we report the first structure of full length TECPR1 resolved using cryo electron microscopy. TECPR1 forms an elongated hook shaped architecture that positions Dysferlin domains in a cis arrangement. Our structure uncovers an uncharacterized intramolecular interface between tectonin repeat 1 and PH domains. This interaction forms a stabilizing bridge that contributes to the orientation of the DysF domains. Molecular dynamics simulations further demonstrate that TECPR1 maintains the overall structural arrangement during membrane association. Our data provide a structural framework for how TECPR1 domain arrangement corresponds with membrane binding.

Emetic exotoxins secreted by Staphylococcus species (Staphylococcal enterotoxins, SEs) are a major cause of food poisoning cases worldwide and many are additionally classified as superantigens - able to potently activate T cells in an antigen independent manner. Fewer than half of the gene products of the known SE genes have been extensively characterized. The gene for Staphylococcal enterotoxin L (SEL) occurs in both foodborne and clinical isolates but no detailed structural characterization has yet been available. We report here the crystal structure of SEL and confirm its function as a superantigen via direct T cell activation assays. By comparison of the SEL sequence with that of its four closest homologues (SEI, SEK, SEM and SEQ), we have identified binding epitopes unique for SEL and mapped these regions onto the structure. These data provide the first high resolution view of SEL and the basis for the development of diagnostic procedures for its specific detection.

Transient receptor potential (TRP) channels are evolutionarily conserved polymodal cation channels that mediate diverse sensory functions across the animal kingdom. Although TRP channels play key roles in peripheral sensation, their expression and functional relevance in the cerebral cortex remain poorly defined. Here, we integrate long- and short-read transcriptomics, targeted qPCR and membrane-aware proteomics to quantify TRP family members in adult mouse cortex. Across transcriptomic platforms, cortical TRP expression is dominated by TRPML, TRPC, and TRPM subfamilies, with lower representation of TRPP/TRPV, whereas Trpa1 and Trpv1 lie near empirical detection thresholds. Our proteomic workflow yields reproducible protein-level evidence for a subset of cortical TRPs, including TRPV2, TRPC4, TRPM3, TRPM7 and TRPP2, consistent with transcript rank order, while TRPA1/TRPV1 do not meet replicate-level protein-group detection criteria under 1% FDR control. Together, these multi-platform measurements establish a quantitative reference for cortical TRP biology and a framework for profiling low-abundance ion channels in complex brain tissue.

Endurance exercise imposes extreme metabolic demands on the adult human brain, raising the question of how core brain function is preserved under physiological challenge. We previously showed that marathon running induces reversible reductions in myelin within specific white-matter tracts, suggesting adaptive structural change under metabolic stress. Here, we asked whether this process is functionally tolerated. Neurophysiological recordings revealed maintained conduction latencies across motor, somatosensory, visual, and auditory pathways within 48 hours after race completion, indicating intact axonal signal transmission despite reduced myelin content. Cognitive testing revealed selective and transient modulation of higher-order processing, including attenuated practice-related gains in processing speed and short-lived increases in interference, whereas visuomotor speed and executive flexibility were preserved. All cognitive measures normalized one month after the race, supporting an adaptive framework linking myelin change with preserved brain function under extreme metabolic stress.

P-glycoprotein is an efflux pump with an exceptionally broad substrate profile which drives its profound clinical impact. Despite its biological importance, it remains obscure how P-glycoprotein achieves its polyspecificity and how substrate binding and the lipid environment stimulate its activity. Structural data highlight the importance of transmembrane helices 4 and 10, which surround the binding pocket, and identify them as key players in substrate recognition. Here, we used cryo-EM to study P-glycoprotein in detergent and nanodiscs to strategically leverage environment- and substrate-dependent phenotypes. This approach allowed us to decipher unexpected and distinct roles of transmembrane helices 4 and 10, which structurally explain differences in ATPase activity. Our data highlights helix 4 as an environment sensor and helix 10 as the key player in substrate recognition constituting a dual regulation mechanism for the functional plasticity of P-glycoprotein, and visualizes the intricate interplay between a membrane protein and its environment.

Adipic acid (1,6-hexanedioic acid) is a key building block for nylon-6,6, a widely used polymer in the global chemical industry. Current industrial production relies on petrochemical feedstocks and nitric acid oxidation of cyclohexane/cyclohexanol mixtures, releasing nitrous oxide, a potent greenhouse gas. Biotechnological routes offer sustainable alternatives but have been limited by low yields or reliance on multi-strain systems. Here we report a one-pot, single-strain microbial process for the efficient conversion of guaiacol - a lignin derived aromatic - into adipic acid. By integrating heterologous Rieske non-heme iron monooxygenases from Cupriavidus necator N-1 with systematic process optimisations in engineered Escherichia coli, we achieve near-quantitative conversion with 97% yield and titres of 1.5 g/L in aqueous, lab-scale reactions. This work demonstrates a novel and efficient strategy for lignin valorisation through engineered microbial synthesis, providing a new sustainable and scalable route to adipic acid.

The QacA DHA2 exporter from Staphylococcus aureus is a prototypical multidrug transporter that, like other bacterial efflux pumps, can extrude a wide range of cytotoxic compounds thus playing a crucial role in antimicrobial resistance. Here, we report crystal structures of wild-type QacA in three key conformational states: inward-open, outward-open and ethidium-bound, representing the first ligand-bound structure of a 14 transmembrane helices (TM) DHA2 transporter. In combination with computational and functional studies, these structures provide a mechanistic framework to understand drug recognition and extrusion. Structural analyses reveal remarkable adaptability within the binding pocket, including a ligand-induced deformation of TM5 that enables coordination of ethidium bromide in the outward-open state. Molecular dynamics simulations show spontaneous lipid entry into the transporter core and suggest that substrate binding from the inner membrane leaflet initiates a conformational transition to an outward-open state, stabilizing high-affinity interactions. Subsequent binding site protonation drives substrate extrusion. Together, these findings elucidate the structural dynamics and mechanistic underpinnings of QacA-mediated multidrug transport, highlighting conformational flexibility and proton-coupled electrostatic changes as key determinants of multidrug recognition and extrusion. This study provides a foundational framework for developing targeted inhibitors to combat bacterial multidrug resistance.

CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA ({Psi}DNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The {Psi}DNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.

The mechanosensitive channel of small conductance (MscS) is the founding member of the family of MscS-like channels, which share a structurally conserved core but feature additional structural elements that define their specific channel characteristics. Here, we characterize the structure and function of the Escherichia coli mechanosensitive channel of mini conductance (MscM), which features eight additional transmembrane (TM) helices and a large periplasmic domain. Our cryo-EM structures reveal that channel gating involves conformational changes in all domains of MscM. In particular, a cytoplasmic extension of TM7 couples the conformation of the TM domain to that of the cytoplasmic domain, resulting in gating of its lateral fenestrations, where ions enter the channel. Thus, different from all other MscS-like channels studied to date, channel gating in MscM is mediated by its cytoplasmic domain and not the TM domain, which senses changes in membrane tension and operates the cytoplasmic gates.

Mitochondrial crista junctions (CJs) operate as regulated gateways into the cristae microenvironment, whose protein, metabolite, and ion compositions are finely tuned for mitochondrial function. The Mic60-Mic19 complex of the mitochondrial contact site and cristae organizing system (MICOS) complex was suggested to span across CJs and act as a diffusion barrier, but little is known of how its dynamic architecture facilitates this task. To address this open question, we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic60. These and previous structural and biochemical data were harnessed in molecular dynamic (MD) simulations to develop a dynamic model of the human tetrameric Mic60-Mic19 subcomplex in the CJ environment, to validate its architecture using in organello cross-linking data and to computationally characterize its function as a diffusion barrier. Our integrative structural biology approach enables the functional investigation of flexible, multidomain protein complexes which escape conventional structural biology methods.

Anaerobic carbon transformation in freshwater sediments drives substantial methane emissions globally, yet the microbial taxa linking complex carbon degradation to methane production remain poorly characterized. Here, we combined metagenomics with the first metatranscriptomic dataset from the anoxic sediments of meromictic Lake Cadagno (Swiss Alps) to identify the active microbial clades, metabolic pathways, and extrachromosomal elements (ecDNA) across a depth gradient within the upper 56 cm of sediment. We recovered 802 species-level metagenome-assembled genomes (MAGs) spanning 66 phyla and identified a Bacteroidota clade (VadinHA17) as one of the most abundant and transcriptionally active populations in the sediment. This clade encodes and transcribes a broad range of diverse glycoside hydrolases (GH), indicating a central role in complex carbohydrate degradation. Transcriptional profiles suggest that this clade ferments organic substrates to acetate and hydrogen, which are key substrates for methanogenesis. In line with this, the acetoclastic methanogen Methanothrix and hydrogenotrophic Methanoregula were among the most abundant and transcriptionally active archaea in the same depth layers. Beyond microbial genomes, we detected 86,905 viral OTUs (vOTUs) and 2,136 plasmid OTUs (pOTUs), with free viruses and plasmids accounting for 5-10% and 0.2% of all sequencing reads, respectively. Notably, plasmids and viruses associated with Bacteroidota VadinHA17 encode and transcribe GHs that could augment host carbohydrate-degrading capacity. Together, these findings reveal new details on how methane production in anoxic lake sediments emerges from a network spanning primary fermentation, methanogenesis and ecDNA-mediated metabolisms.

Shiga toxin-converting bacteriophages play a critical role in the emergence and virulence of pathogenic Escherichia coli strains. Despite their significance, detailed structural information on these phages remains scarce. Here we present a high-resolution cryo-electron microscopy and proteomic analysis of the phi24B bacteriophage, revealing an icosahedral capsid with T=9 symmetry, decorated by a processed esterase protein (gp84) and stabilized by cementing proteins. The tail assembly comprises a dodecameric portal, two rings of adapter proteins sharing a common fold, a hexameric nozzle, six lateral tail fibers, and a flexible central needle fiber. The binding sites of the fibers are described. Comparative analysis indicates conservation of the tail structure with related podoviruses but very different peripheral features.

Quantifying muscle health at scale has been limited by the difficulty of segmenting individual muscles on MRI. We developed an automated 3D deep-learning framework that segments 20 bilateral hip and thigh muscles from Dixon MRI, enabling muscle level quantification of volume and relative fat fraction (rFF). Applied to 10,840 baseline and 2,766 longitudinal UK Biobank scans, this framework supports population-scale phenotyping across demographic, metabolic and treatment exposures. Segmentation accuracy was robust, and increased with muscle size. Men had greater muscle volumes, whereas women showed consistently higher rFF. Fat infiltration was highest in postural and pelvic-stabilising muscles and lowest in the quadriceps, revealing pronounced anatomical heterogeneity. Over two years, most muscles showed small but consistent volume declines, with losses more uniform in men and more heterogeneous in women; rFF increased more prominently in women, suggesting early compositional deterioration. In T2D, men showed widespread volume loss and elevated rFF, whereas women showed minimal volume loss and heterogeneous fat changes, revealing sex-specific disease signatures. Automated muscle-specific MRI phenotyping resolves structural and compositional changes obscured by compartment-level measures and provides a scalable platform for population-level studies of musculoskeletal ageing, metabolic disease, and therapeutic response.

Quinoa (Chenopodium quinoa) is a nutrient-rich pseudocereal with diverse specialized metabolites, yet the genetic basis of this metabolic diversity is poorly understood. Here we integrate whole-genome sequencing and multi-tissue metabolic profiling of 603 quinoa accessions. We detected 4,688 metabolic features and identified over 1,000 metabolites in seeds, leaves, and roots. Using multi-tissue genome-wide association, we mapped the genetic architecture of quinoa metabolome by identifying 584 quantitative trait loci (QTL) and prioritized 219 candidate genes across 58 major QTL governing saponin, betalain, and flavonoid biosynthesis. Moreover, we constructed a drought-responsive multi-omics regulatory network and uncovered additional key genes involved in quinoa stress signalling and metabolic pathways. Finally, we cloned and functional validated the roles of CYP76AD1 in betalamate accumulation, UGT91C1 in flavonoid glycosylation, and CYP72A154 and soyasapogenol B glucuronide galactosyltransferase (SGT) in saponin biosynthesis. This multi-omic framework provides a high-resolution map of the quinoa metabolome and a foundation for breeding nutrient-rich and stress-resilient quinoa cultivars.