Search Results (313)

Determining RNA secondary structures is a fundamental challenge in computational biology and molecular sensing. Experimental techniques such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy can reveal RNA structures with atomic precision, but their high cost and time consuming nature limit large-scale applications. To address this issue, we introduce the Structure-Sensing Nucleotide Attention Learning framework (NTFold), a virtual sensing framework based on deep learning for accurate RNA secondary structure prediction. NTFold integrates a Nucleotide Attention Module (NAM) to explicitly model dependencies among nucleotides, thereby capturing fine-grained sequence correlations. The resulting correlation map is subsequently refined by a Structural Refinement Module (SRM), which preserves hierarchical spatial information and enforces structural consistency. Through this two stage learning paradigm, NTFold produces high-precision contact maps that enable reliable RNA secondary structure reconstruction. Extensive experiments demonstrate that NTFold outperforms existing deep learning-based predictors, highlighting its capability to learn both local and global nucleotide interactions in an sensor inspired manner. This study provides a new direction for integrating attention driven correlation modeling with structure-sensing refinement toward efficient and scalable RNA structural sensing. Full article

Lipid raft-associated gangliosides facilitate the early stages of SARS-CoV-2 entry by triggering the exposure of the receptor-binding domain (RBD) within the trimeric spike protein, which is initially sequestered. A broad range of in silico, cryoelectron microscopy and physicochemical approaches indicate that the RBD becomes accessible after a ganglioside-induced conformational rearrangement originating in the N-terminal domain (NTD) of one protomer and propagating to the neighboring RBD. We previously identified a triad of amino acids, Q134-F135-N137, as a strictly conserved element on the NTD. In the present review, we integrate a series of structural and experimental data revealing that this triad may act as a conformational transducer connected to a chain of residues that are capable of transmitting an internal conformational wave within the NTD. This wave is generated at the triad level after physical interactions with lipid raft gangliosides of the host cell membrane. It propagates inside the NTD and collides with the RBD of a neighboring protomer, triggering its unmasking. We also identify a chain of aromatic residues that are capable of controlling electron transfer through the NTD, leading us to hypothesize the existence of a dual conformational/quantum wave. In conclusion, the complete conservation of the Q134-F135-N137 triad despite six years of extensive NTD remodeling underscores its critical role in the viral life cycle. This triad represents a potential Achilles’ heel within the hyper-variable NTD, offering a stable target for therapeutic or vaccinal interventions to disrupt the conformational wave and prevent infection. These possibilities are discussed. Full article

The function of the highly conserved GTPase LepA, a homolog of elongation factor EF-G, remains unknown in translation. However, there is biochemical data that it implicates in the 30S ribosomal subunit biogenesis. Here, using cryo-electron microscopy, we characterized 30S subunits isolated from an Escherichia coli strain with a deleted lepA gene. The cryo-EM maps for ∆lepA 30S particles were divided into classes corresponding to consecutive assembly intermediates: from particles characterized by unformed helices h44/h45 of the central decoding center (CDR) and highly flexible head, through intermediates with a distorted CDR and a partial stabilization of the head, to near-mature 30S subunits with correctly docked h44 in the CDR, accessible 3′ end of 16S rRNA for translation but significant flexibility in head domain. Cryo-EM analysis of ΔlepA 30S intermediates revealed that they predominantly proceed to nearly mature functional state and exhibit suboptimal flexibility in the head domain. This finding suggests that LepA likely contributes to the final proper stabilization of the 3′ domain of the 30S subunit during ribosome assembly. Full article

Volume electron microscopy (Volume-EM) has transformed structural cell biology by enabling nanometre-resolution imaging across cellular and tissue scales. Serial-section TEM, Serial Block-Face Scanning Electron Microscopy (SBF-SEM), Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and multi-beam SEM now routinely generate terabyte-scale volumes that capture organelles, synapses and neural circuits in three dimensions, while cryogenic Volume-EM extends this landscape by preserving vitrified, fully hydrated specimens in a near-native state. Together, these room-temperature and cryogenic modalities define a continuum of approaches that trade off volume, resolution, throughput and structural fidelity, and increasingly interface with correlative light microscopy and cryo-electron tomography. In parallel, advances in computation have turned Volume-EM into a data-intensive discipline. Multistage preprocessing pipelines for alignment, denoising, stitching and intensity normalisation feed into automated segmentation frameworks that combine convolutional neural networks, affinity-based supervoxel agglomeration, flood-filling networks and, more recently, diffusion-based generative restoration. Weakly supervised and self-supervised learning, multi-task objectives and human-AI co-training mitigate the scarcity of dense ground truth, while distributed storage and streaming inference architectures support segmentation and proofreading at the terascale and beyond. Open resources such as COSEM, MICRONS, OpenOrganelle and EMPIAR provide benchmark datasets, interoperable file formats and reference workflows that anchor method development and cross-laboratory comparison. In this review, we first outline the physical principles and imaging modes of conventional and cryogenic Volume-EM, then describe current best practices in data acquisition and preprocessing, and finally survey the emerging ecosystem of AI-driven segmentation and analysis. We highlight how cryo-Volume-EM expands the field towards native-state structural biology, and how multimodal integration with light microscopy, cryo-electron tomography (cryo-ET) and spatial omics is pushing Volume-EM from descriptive imaging towards predictive, mechanistic, cross-scale models of cell physiology, disease ultrastructure and neural circuit function. Full article

Evidence increasingly indicates that synaptic vesicle dysfunction emerges early in Parkinson’s disease (PD), preceding overt dopaminergic neuron loss rather than arising solely as a downstream consequence of neurodegeneration. α-Synuclein (αSyn), a presynaptic protein that regulates vesicle clustering, trafficking, and neurotransmitter release under physiological conditions, exhibits dose-, conformation-, and context-dependent actions that distinguish its normal regulatory roles from pathological effects observed in disease models. This narrative review synthesizes findings from a structured search of PubMed and Scopus, with emphasis on α-syn-knockout (αSynKO) and BAC transgenic (αSynBAC) mouse models, which do not recapitulate the full human PD trajectory but provide complementary insights into αSyn physiological function and dosage-sensitive vulnerability. Priority was given to studies integrating ultrastructural approaches—such as cryo-electron tomography, high-pressure freezing/freeze-substitution TEM, and super-resolution microscopy—with proteomic and lipidomic analyses. Across these methodologies, several convergent presynaptic alterations are consistently observed. In vivo and ex vivo studies associate αSyn perturbation with impaired vesicle acidification, consistent with altered expression or composition of vacuolar-type H⁺-ATPase subunits. Lipidomic analyses reveal age- and genotype-dependent remodeling of vesicle membrane lipids, particularly curvature- and charge-sensitive phospholipids, which may destabilize αSyn–membrane interactions. Complementary biochemical and cell-based studies support disruption of SNARE complex assembly and nanoscale release-site organization, while ultrastructural analyses demonstrate reduced vesicle docking, altered active zone geometry, and vesicle pool disorganization, collectively indicating compromised presynaptic efficiency. These findings support a synapse-centered framework in which presynaptic dysfunction represents an early and mechanistically relevant feature of PD. Rather than advocating αSyn elimination, emerging therapeutic concepts emphasize preservation of physiological vesicle function—through modulation of vesicle acidification, SNARE interactions, or membrane lipid homeostasis. Although such strategies remain exploratory, they identify the presynaptic terminal as a potential window for early intervention aimed at maintaining synaptic resilience and delaying functional decline in PD. Full article

One of the most important events in the pathogenesis of Parkinson’s disease and related disorders is the formation of abnormal fibrils via the aggregation of α-synuclein (α-syn) with β-sheet-rich organization. The use of Cryo-EM has uncovered different polymorphs of the fibrils, each having unique structural interfaces, which has made the design of inhibitors even more challenging. Here, a structure-guided framework incorporating AI-assisted peptide generation was set up with the objective of targeting the conserved β-sheet motifs that are present in various forms of α-syn fibrils. The ProteinMPNN, then, AlphaFold-Multimer, and PepMLM were employed to create short peptides that would interfere with the growth of the fibrils. The two selected candidates, T1 and S1, showed a significant inhibition of α-syn fibrillation, as measured by a decrease in the ThT fluorescence and the generation of either amorphous or fragmented aggregates. The inhibitory potency of the peptides was in line with the predicted interface energies. This research work illustrates that the integration of cryo-EM structural knowledge with the computational design method leads to the quick discovery of the wide-spectrum peptide inhibitors, which is a good strategy for the precision treatment of neurodegenerative diseases. Full article

The application of cryo-electron microscopy (cryo-EM) in membrane protein structural biology has catalyzed unprecedented advances in our understanding of fundamental biological processes and transformed drug discovery paradigms. This review briefly describes the biological achievements enabled using cryo-EM techniques, including single particle analysis (SPA), micro-electron diffraction (microED), and subtomogram averaging (STA), in elucidating the structures and functions of membrane proteins, ion channels, transporters, and viral glycoproteins. We highlight how these structural insights have revealed druggable sites, enabled structure-based drug design, and provided mechanistic understanding of disease processes. Key biological targets include G protein-coupled receptors (GPCRs), ion channels implicated in neurological disorders, respiratory chain complexes, viral entry machinery, and membrane transporters. The integration of cryo-EM with computational drug design has already yielded clinical candidates and approved therapeutics, marking a new era in membrane protein pharmacology. Full article

Polymyxin antibiotics are often the last line of defense against multidrug-resistant Gram-negative pathogens. A key resistance mechanism involves the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, mediated by the bifunctional enzyme ArnA. However, the evolutionary rationale and structural basis for ArnA’s domain fusion, hexameric assembly, and catalytic coordination remain mechanistically unresolved. Here, we integrate evolutionary genomics, high-resolution cryo-electron microscopy (cryo-EM), and computational protein design to provide a comprehensive mechanistic analysis of ArnA. Our evolutionary analysis reveals that the dehydrogenase (DH) and formyltransferase (TF) domains evolved independently and were selectively fused in Gammaproteobacteria, suggesting an adaptive advantage. A 2.89 Å cryo-EM structure of apo-ArnA resolves the flexible interdomain linker and reveals a DH-driven hexameric architecture essential for enzymatic activity. 3D variability analysis captures intrinsic conformational dynamics, indicating a molecular switch that may coordinate sequential catalysis and substrate channeling. Structure-based peptide inhibitors targeting the hexamerization and predicted ArnA–ArnB interaction interfaces were computationally designed, offering a novel strategy for disrupting L-Ara4N biosynthesis. These findings illuminate a previously uncharacterized structural mechanism of antimicrobial resistance and lay the groundwork for therapeutic intervention. Full article

Ischemic stroke remains one of the most catastrophic diseases in neurology, in which, due to a disturbance in the cerebral blood flow, the brain is acutely deprived of its oxygen and glucose oligomer, which in turn rapidly leads to energetic collapse and progressive cellular death. There is now increasing evidence that this type of stroke is not simply a type of ‘oxidative stress’ but rather a programmable loss-of-redox homeostasis, within which electron flow and the balance of oxidants/reductants are cumulatively displaced at the level of the single molecule and at the level of the cellular area. The advances being made in cryo-electron microscopy, lipidomics, and spatial omics are coupled with the introduction of a redox code produced by the interaction of the couples NADH/NAD⁺, NADPH/NADP⁺, GSH/GSSG, BH₄/BH₂, and NO/SNO, which determine the end results of the fates of the neurons, glia, endothelium, and pericytes. Within the mitochondria, pathophysiological events, including reverse electron transport, succinate overflow, and permeability transition, are found to be the first events after reperfusion, while signals intercommunicating via ER–mitochondria contact, peroxisomes, and nanotunnels control injury propagation. At the level of the tissue, events such as the constriction of the pericytes, the degradation of the glycocalyx, and the formation of neutrophil extracellular traps underlie microvascular failure (at least), despite the effective recanalization of the vessels. Systemic influences such as microbiome products, oxidized lipids, and free mitochondrial DNA in cells determine the redox imbalance, but this generally occurs outside the brain. We aim to synthesize how the progressive stages of ischemic injury evolve from the cessation of flow to the collapse of the cell structure. Within seconds of injury, there is reverse electron transport (RET) through mitochondrial complex I, with bursts of superoxide (O₂•⁻) and hydrogen peroxide (H₂O₂) being produced, which depletes the stores of superoxide dismutase, catalase, and glutathione peroxidase. Accumulated succinate and iron-induced lipid peroxidation trigger ferroptosis, while xanthine oxidase and NOX2/NOX4, as well as uncoupled eNOS/nNOS, lead to oxidative and nitrosative stress. These cascades compromise the function of neuronal mitochondria, the glial antioxidant capacity, and endothelial–pericyte integrity, leading to the degradation of the glycocalyx with microvascular constriction. Stroke, therefore, represents a continuum of redox disequilibrium, a coordinated biochemical failure linking the mitochondrial metabolism with membrane integrity and vascular homeostasis. Full article

Alzheimer’s disease is driven by multiple molecular drivers, including the pathological behavior of two intrinsically disordered proteins, amyloid-β (Aβ) and tau, whose aggregation is regulated by sequence-encoded ensembles and liquid–liquid phase separation (LLPS). This review integrates recent advances in biophysics, structural biology, and computational modeling to provide a multiscale perspective on how sequence determinants, post-translational modifications, and protein dynamics regulate the conformational landscapes of Aβ and tau. We discuss sequence-to-ensemble principles, from charge patterning and aromatic binders to familial mutations that reprogram structural ensembles and modulate LLPS. Structural studies, including NMR, SAXS, cryo-EM, and cryo-electron tomography, trace transitions from disordered monomers to fibrils and tissue-level structures. We highlight experimental challenges in LLPS assays, emerging standards for reproducibility, e.g., LLPSDB, PhaSePro, and FUS benchmarks, and computational strategies to refine and condensate modeling. Finally, we explore the therapeutic implications, including condensate-aware medicinal chemistry, ensemble-driven docking, and novel insights from clinical trials of anti-Aβ antibodies. Together, these perspectives underscore a paradigm shift toward environment- and ensemble-aware therapeutic design for Alzheimer’s and related protein condensation disorders. Full article

Chitin is an essential polysaccharide of the fungal cell wall, critical for structural integrity, cell division and, in pathogenic fungi, virulence. As chitin is absent in both plant and mammalian systems, chitin synthases are considered attractive targets for the specific control of fungal pathogens. Yet despite decades of research, structural information on chitin synthases was lacking and inhibitors have failed to gain approval in the clinic. Current inhibitors are also ineffective against major agricultural pathogens such as Aspergillus and Fusarium species, largely due to the presence of multiple chitin synthase isoforms in filamentous fungi and the cell wall compensatory response induced under stress. However, recent cryo-electron microscopy structures of Class I chitin synthases from yeasts Saccharomyces cerevisiae and Candida albicans and an oomycete chitin synthase have provided unprecedented insights into the structural and mechanistic properties of these large, transmembrane proteins. These studies revealed conserved, domain-swapped homodimer architectures, distinct substrate binding and catalytic pockets, and sophisticated intrinsic regulatory mechanisms. With these breakthroughs, this review summarises our current understanding of fungal chitin biosynthesis, the challenges that remain to fully biochemically characterise these enzymes, and considers how the new structural insights may guide the development of broad-spectrum antifungals. Full article

The COVID-19 pandemic, driven by SARS-CoV-2, continues to challenge global health due to emerging variants and the potential risk posed by related sarbecoviruses. Neutralizing antibodies targeting the spike (S) glycoprotein, particularly the receptor-binding domain (RBD), play a crucial role in viral neutralization and vaccine design. Although broadly neutralizing anti-RBD antibodies have been identified, the nature of cross-reactive humoral responses induced by natural infection with ancestral SARS-CoV-2 strains remains incompletely understood. Here, we isolated 105 S-specific monoclonal antibodies (mAbs) from individuals recovered from prototype SARS-CoV-2 infection. Of these, 30 mAbs cross-recognized SARS-CoV-1, including 25 RBD-directed mAbs, of which 12 displayed cross-neutralizing activity against both viruses. Among them, mAb 12C2 potently neutralized SARS-CoV-1 and multiple SARS-CoV-2 variants, likely through mechanisms that include inhibition of membrane fusion and potential destabilization of the S trimer. Cryo-electron microscopy revealed that 12C2 engages the outer face of the RBD, overlapping with the epitope recognized by the broadly neutralizing antibody S309 derived from SARS-CoV-1 convalescent. Collectively, these findings demonstrate that ancestral SARS-CoV-2 infection can elicit robust cross-neutralizing antibody responses and provide valuable insights for the design of broadly protective antibodies and vaccines. Full article

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers. Full article

Current gene therapy treatments utilizing adeno-associated virus (AAV) vectors are based on capsids of primate origin. However, pre-existing neutralizing anti-AAV antibodies, that are present in a significant portion of the population, can lead to vector inactivation and reduced therapeutic efficacy. Advances in DNA sequencing have facilitated the discovery of many AAVs from non-primate species, including isolates from pigs, which exhibit up to 50% capsid protein sequence divergence, compared to primate AAV serotypes. In this study, AAVs isolated from porcine tissues (AAVpo.1 and AAVpo.6) were selected for structural characterization due to their low capsid protein VP1 sequence identity compared to each other and to AAV9. The AAV vectors were produced via the standard triple transfection system in HEK293 cells using AAV2 rep to package AAV2-ITR vector genomes and were purified by iodixanol density gradient ultracentrifugation. The capsid structures of AAVpo.1 and AAVpo.6 were determined using cryo-electron microscopy and then compared to each other in addition to the AAV5 and AAV9 structures. Given that porcine AAVpo.6 has been reported to infect human cells and the ability to cross the blood–brain barrier, the functional characterization was focused on the identification of a potential glycan receptor utilized by the porcine capsids. Additionally, the porcine AAV capsid reactivity to human derived anti-AAV antibodies was assessed to evaluate the potential for these capsids to be used as alternative vectors for gene therapy, particularly for patients with pre-existing immunity to primate-derived AAV serotypes. Full article

Neurodegenerative diseases such as Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD), and α-synucleinopathies—including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)—are characterized by the accumulation of misfolded protein aggregates. Advances in positron emission tomography (PET) imaging have enabled in vivo visualization of these pathologies, particularly tau and α-synuclein fibrils, facilitating early diagnosis and differential classification. Tau PET tracers such as ¹⁸F-florzolotau have demonstrated robust imaging of both AD-type and 4-repeat tauopathies, including atypical parkinsonian syndromes in FTLD such as progressive supranuclear palsy and corticobasal degeneration. Cryo-electron microscopy has elucidated the molecular interactions underlying tracer binding, highlighting hydrophobic grooves in cross-βstructures as binding components commonly present in multiple tau fibril types. For α-synucleinopathies, new tracers with a modified cross-β-binding scaffold, including ¹⁸F-SPAL-T-06 and ¹⁸F-C05-05, have shown promise in detecting MSA-related pathology and, more recently, midbrain pathology in PD and DLB. However, sensitive detection of pathologies in early PD/DLB stages remains a challenge. The integration of high-resolution PET technologies and structurally optimized ligands may enable earlier and more accurate detection of protein aggregates, supporting both clinical decision-making and the development of targeted disease-modifying therapies. Full article