The dentin-enamel junction (DEJ) plays a critical role in tooth biomechanics, acting as a tough, crack-deflecting interface between the brittle enamel and the more resilient dentin. Although previous studies have describ...The dentin-enamel junction (DEJ) plays a critical role in tooth biomechanics, acting as a tough, crack-deflecting interface between the brittle enamel and the more resilient dentin. Although previous studies have described the DEJ using histology and electron microscopy techniques, the three-dimensional (3D) distribution and structural heterogeneity of scallop patterns along the DEJ remain poorly understood. Here, we combined high-resolution X-ray microcomputed tomography (µCT) with multiphoton microscopy (MPM) to investigate scallop morphology, spatial distribution, and collagen fiber organization across human teeth. Non-carious human teeth (n = 35) were scanned at 5 µm resolution, allowing 3D reconstruction of the DEJ surface. Scallop size, distribution, and root mean square (RMS) roughness were quantified across mesial, distal, buccal, and lingual faces of incisors, canines, premolars, and molars. MPM with second harmonic generation (SHG) provided complementary imaging of collagen fiber presence within scallop structures. Scallop size depended primarily on location but also on tooth type: the largest scallops (>150 µm) were concentrated on mesial and distal faces at interproximal contact areas, while molars lacked large scallops entirely. RMS roughness confirmed significant topographic heterogeneity between regions. SHG imaging showed high collagen density at scallop peaks. These findings provide the first whole-tooth 3D mapping of scallop patterns, supporting the hypothesis that scalloped DEJ structures enhance crack resistance and mechanical resilience. Further studies using higher-resolution imaging and comparative models across species may clarify the developmental and functional origins of these unique microstructures.
Mitochondrial morphology is central to cellular function, yet large-scale quantification is limited by the lack of high-resolution whole-cell imaging and efficient segmentation tools. Soft X-ray tomography (SXT) provides...Mitochondrial morphology is central to cellular function, yet large-scale quantification is limited by the lack of high-resolution whole-cell imaging and efficient segmentation tools. Soft X-ray tomography (SXT) provides native-state 3D whole-cells images, but organelle segmentation remains a bottleneck. We present MitoXRNet, a data- and parameter-efficient 3D deep learning model for mitochondria and nucleus segmentation in SXT tomograms. Using multi-axis 3D slicing, Sobel filter-based boundary enhancement, and a combined Binary-Cross-Entropy and Robust-Dice loss, MitoXRNet achieves a 73.8% Dice score on INS-1E cells with only 1.4 M parameters, outperforming existing methods. A larger 22.6 M variant generalized well to unseen data. Automated segmentation enabled quantitative analysis of mitochondrial remodeling under metabolic stimuli: glucose increased mitochondrial volume and matrix density, while GIP and GKA increased mitochondria number, reduced volume, and elevated density, indicating smaller, denser, more dynamic populations. MitoXRNet provides a scalable framework for high-throughput morphological and biophysical profiling of organelles in native-state SXT data.
AstaPs are water-soluble, photooxidative stress-inducible astaxanthin (AXT)-binding proteins found only in Scenedesmaceae microalgae, where they play a central role in survival under severe photooxidative stress. Here, w...AstaPs are water-soluble, photooxidative stress-inducible astaxanthin (AXT)-binding proteins found only in Scenedesmaceae microalgae, where they play a central role in survival under severe photooxidative stress. Here, we focused on the unique function of AstaP-pink1, which converts orange AXT into a pink form and generates a UVA absorption spectrum upon protein binding. AstaP-pink1 was expressed in genetically engineered Escherichia coli strains capable of synthesizing AXT. The host strain harboring pAC-Asta produced adonixanthin, AXT, and zeaxanthin in an approximate ratio of 5:3:2, whereas the strain carrying pMF573 predominantly produced AXT (∼90 % of total carotenoid). Co-expression of the gene encoding AstaP-pink1 in these strains resulted in moderate and selective AXT binding, accompanied by a spectral red shift and UVA absorption, thereby generating pink coloration. Crystal structure analysis of AXT-bound recombinant AstaP-pink1 (rAstaP-pink1) revealed both similarities and differences in AXT binding compared with rAstaP-orange1. Density functional theory (DFT) calculations based on the crystal structure suggested that the larger red shift than that of AstaP-orange1 and the distinct UVA absorption are derived from the conformation of AXT that is compelled by binding to AstaP-pink1. This study suggests that AXT binding by AstaP-pink1 not only facilitates the water solubilization of AXT but also generates the observed spectral properties.
The advent of direct electron detectors (DEDs) has driven a major breakthrough in cryo-electron microscopy (cryo-EM), particularly in single-particle analysis (SPA), establishing DEDs as essential tools for achieving nea...The advent of direct electron detectors (DEDs) has driven a major breakthrough in cryo-electron microscopy (cryo-EM), particularly in single-particle analysis (SPA), establishing DEDs as essential tools for achieving near-atomic resolution. In this study, we re-evaluated the performance of the TVIPS TemCam-XF416, an indirect scintillator-coupled CMOS camera (scintillator camera). Using a JEOL CRYOARM 300II, we performed SPA on two well-established benchmark specimens, β-galactosidase and apoferritin, at a 300 kV acceleration voltage. The resulting reconstructions reached resolutions of 2.6 Å and 2.1 Å, respectively. Notably, the apoferritin map clearly resolves the central holes of aromatic side chains-a level of detail previously considered exclusive to DEDs. These results were achieved by implementing the latest standard reconstruction workflows, including motion correction and contrast transfer function refinement, underscoring the critical role of computational methods in attaining high-resolution structures. While scintillator cameras inherently exhibit a lower signal-to-noise ratio than DEDs, our findings with XF416 demonstrate that, with appropriate data collection and processing, such cameras can deliver near-atomic resolution structures. This work establishes a crucial technical benchmark for the scintillator camera evaluated in this study on a high-end 300 kV cryo-EM platform, demonstrating its capability to achieve resolutions suitable for many structural biology applications and providing an updated perspective on its performance capabilities.
Studies in immortalized human mitotic cells demonstrated that MRN Complex Interacting Protein (MRNIP) plays a critical role in genome stability, replication fork protection, and the detection of DNA double-strand breaks...Studies in immortalized human mitotic cells demonstrated that MRN Complex Interacting Protein (MRNIP) plays a critical role in genome stability, replication fork protection, and the detection of DNA double-strand breaks via liquid-liquid phase separation. Our earlier work in mice identified its essential role in meiosis during spermatogenesis, namely, meiotic sex chromosome inactivation, highlighting its critical importance for male fertility. Apart from that, MRNIP is a poorly characterized protein with little to no data-based evidence of its biophysical and biochemical properties. In this study, we provide experimental evidence confirming that the N-terminal domain is indeed folded and contains a zinc-ribbon motif. We demonstrate that MRNIP binds a Zn ion at this site, which plays a structural role in stabilizing the folded domain. Together with structural similarity observed across species, these findings support the conserved nature of the N-terminal domain of MRNIP. Our experimental data confirms that the C-terminal region is disordered. Furthermore, we show that both the N- and C-terminal regions exhibit binding specificity for DNA rather than RNA, under low-salt conditions, suggesting low-affinity interactions, whereas no DNA or RNA binding was observed under physiological salt conditions. Our findings provide insight into the biophysical and biochemical properties of MRNIP and offer a foundation for advancing structural and functional studies of MRNIP.
Dental caries, one of the most prevalent non-communicable diseases worldwide, is characterised by the progressive deterioration of the structure and mechanical properties of dental hard tissues. In human teeth, dentine i...Dental caries, one of the most prevalent non-communicable diseases worldwide, is characterised by the progressive deterioration of the structure and mechanical properties of dental hard tissues. In human teeth, dentine is the most abundant mineralised tissue, forming the primary support material. To assess changes in the mechanical properties of dentine caused by dental caries and acid erosion, it is crucial to understand the relationship between organic and inorganic dentine components and their organisation into a 3D anisotropic structure at the nanoscale. Over the past 20 years, alterations in dentine structure caused by caries and artificial demineralisation have been reported using conventional microscopy techniques. However, due to the limited spatial resolution of these techniques, the 3D structural organisation including orientation and degree of alignment of mineralised collagen fibrils at the nanoscale, has not been fully explored. This study investigated alterations in the 3D structure of normal, carious and artificially demineralised dentine using SAXS tensor tomography (SASTT). This technique enabled the observation of differences in the local orientation of organic and inorganic components, as well as variations in local scattering intensity, resulting from natural caries and artificial demineralisation. In comparison to normal dentine, caries caused minor orientational differences of both components but had a major impact on the local X-ray scattering intensity. After artificial demineralisation of the dentine, most of the mineral was lost in the outer layers, resulting in a greater reduction in scattering intensity than that caused by caries. SIGNIFICANCE: The remarkable mechanical properties of human dentine arise from its complex hierarchical 3D structure. In this article, we have investigated the 3D structural alterations in dentine, caused by caries and artificial demineralisation. For this detailed investigation, SAXS tensor tomography (SASTT) has been implemented on the I22 beamline at Diamond Light Source, UK. SASTT is a technique that can probe the nanostructure of dentine, yielding orientation and degree of alignment of the mineralised collagen fibrils, while also providing a 3D reciprocal space map to investigate the detailed non-uniform scattering intensity distribution in all directions. The initial SASTT data provide insights into dentine structural alterations caused by caries and artificial demineralisation, facilitating further exploration of structure-mechanical property relationships, which may lead to improve the development of novel biomimetic materials for dental applications.
The blue light photoreceptor cum transcription factors, aureochromes (Aureos), are present exclusively in photosynthetic stramenopiles. Co-existence of Light-Oxygen-Voltage (LOV) and basic leucine zipper (bZIP) is unique...The blue light photoreceptor cum transcription factors, aureochromes (Aureos), are present exclusively in photosynthetic stramenopiles. Co-existence of Light-Oxygen-Voltage (LOV) and basic leucine zipper (bZIP) is unique to Aureos - therefore ideal to study light-dependent DNA binding/transcriptional regulation. Further, Aureos' inverse effector-sensor topology, resembling several sensory eukaryotic transcription factors, makes them prototypical optogenetic scaffolds. In absence of 3D data, this study aims for a thorough investigation of the bZIP domains from Aureos and others, and their interaction with substrate DNA using tools from sequence/structural bioinformatics, network theory, molecular dynamics simulation and in vitro experiments. An in-depth comparison of 173 Aureo/plant/opisthokont bZIPs reveals Aureos' uniqueness and evolutionary significance in DNA binding specificity as well as dimer stability. An all-atom network analysis on representative bZIP-DNA co-crystal structures, especially the measurement of eigenvector centrality, further adds importance to hydrophobic interactions in the zipper region to stabilize bZIP dimer and facilitate DNA binding in Aureos and other bZIPs. The most notable finding is the unique presence of histidine at the basic region of Aureos unlike other bZIPs. Histidine not just promotes blue light independent substrate DNA-binding affinity but also serves as a potential switch point in Aureo/bZIP evolution.
Studies of protein structure and stability have traditionally focused on individual domains, treating them as autonomous units, even though most proteins consist of multiple domains. This raises the question to what exte...Studies of protein structure and stability have traditionally focused on individual domains, treating them as autonomous units, even though most proteins consist of multiple domains. This raises the question to what extent can multidomain proteins be considered as sums of their individual domains, and how neighboring domains influence one another. Chitinase Chi60 from the psychrophilic bacterium Moritella marina consists of four domains linked in sequence: a catalytic domain, two consecutive Ig-like domains, and a chitin-binding module. The modular architecture of this enzyme provides an opportunity to examine the structure and stability of a protein from which domains are systematically excised. A series of deletion mutants of the chitinase was designed and constructed, and their structures and thermal melting profiles were analyzed. The different domains exhibit distinct melting temperatures. The catalytic domain shows a complex melting profile. Each domain can fold and maintain its structural integrity when isolated, including the two tandem Ig-like domains that share sequence similarity. Although the interfaces between domains in this modular protein are small, it is still possible to detect the influence neighboring domains exert on one another. Some artificial combinations of domains are unstable and prone to degradation. This long, flexible molecule may be stabilized through dimerization when not engaged with the chitin substrate, with two of its domains participating in the interaction.
Cryo-electron tomography (cryo-ET) enables 3D visualization of biological structures in their native state, but high-fidelity tomogram reconstruction remains challenging due to low signal-to-noise ratios and limited angu...Cryo-electron tomography (cryo-ET) enables 3D visualization of biological structures in their native state, but high-fidelity tomogram reconstruction remains challenging due to low signal-to-noise ratios and limited angular sampling. In this work, we present CryoETGS, a differentiable learning framework that reconstructs tomograms through adaptive 3D Gaussian representations of biological structures for cryo-ET. This representation enables efficient and interpretable reconstruction through a hardware-accelerated differentiable rendering pipeline aligned with the cryo-ET imaging geometry. CryoETGS incorporates hierarchical initialization, adaptive densification, and a tilt-weighted optimization strategy to enhance convergence and reconstruction fidelity. The framework further supports real-time projection synthesis and bidirectional conversion between voxel-based and Gaussian representations. Extensive experiments on both simulated and experimental datasets demonstrate that CryoETGS achieves state-of-the-art reconstruction performance, effectively mitigates missing wedge artifacts, and exhibits high computational efficiency. Source code is publicly available at https://github.com/JachyLikeCoding/ETGS.
Endothelial monocyte activating polypeptide II (EMAP II) is the C-terminal domain of its precursor, AIMP1/p43 polypeptide, a multifunctional protein with diverse functional cytokine activities and tRNA-binding abilities....Endothelial monocyte activating polypeptide II (EMAP II) is the C-terminal domain of its precursor, AIMP1/p43 polypeptide, a multifunctional protein with diverse functional cytokine activities and tRNA-binding abilities. Several X-ray crystallographic structures of EMAP II are available in the PDB database. However, its NMR-based structure, presented in this work indicates partition of its structure into two domains characterized by substantial differences in structural dynamics. Based on N relaxation experiments, hydrogen-deuterium exchange (HDX) data and molecular dynamics simulations we conclude that observed differences in dynamics may enable dual mechanisms of tRNA binding and cytokine function of EMAP II. Although in general in-solution EMAP II structure studied here is highly similar to its in-crystal X-ray structure, the N-terminal segment responsible for cytokine activity exhibited reduced deuterium exchange rate and demonstrated higher accessibility to the solvent compared to X-ray structures and the AlphaFold2 model. Such differences may be important for understanding cytokine function of EMAP II. The tRNA-binding motif characterized by different orientation of the side chain of tryptophan 128, which can play a significant role in the regulation of tRNA binding.
Phosphodiesterase-5 (PDE5A) is a critical therapeutic target for treating male erectile dysfunction (ED). PDE5A inhibitors like sildenafil are clinically effective, but exhibit side effects due to non-specific inhibition...Phosphodiesterase-5 (PDE5A) is a critical therapeutic target for treating male erectile dysfunction (ED). PDE5A inhibitors like sildenafil are clinically effective, but exhibit side effects due to non-specific inhibition of related PDE isoforms. This necessitates the discovery of safer and specific PDE5A inhibitors. The prepared folium of Epimedium sagittatum (PFES) is a primary sovereign drug in traditional Chinese medicine (TCM) formulations for ED. As part of ongoing research into its pharmacodynamic basis, this study isolated and identified forty-four flavonoids and organic acids from PFES. The inhibitory activities of these compounds against PDE5A were systematically evaluated, and three compounds (1, 5, and 6) are demonstrated to have significant PDE5A inhibition activities. Notably, the compound 6 (baohuoside I), was previously confirmed the activitywith PDE5A, and its co-crystal structure with PDE5A was also reported. To elucidate the molecular mechanisms underlying the inhibition of PDE5A by compound 1 and 5, we solved the crystal structures of the catalytic domain of PDE5A in complex with compound 1 and 5. Further structural analyses revealed distinct binding modes adopted by 1 and 5 for occupying the PDE5A active site, highlighting their specific interactions with PDE5A compared to each other and to other established inhibitors like sildenafil. In summary, these findings underscore the potential of PFES-derived natural compounds as specific PDE5A inhibitors, and provide crucial insights for the rational development of novel ED therapeutics with potentially improved specificity and reduced side effects based on TCM constituents.
Selenoproteins, defined by the incorporation of the 21st amino acid - selenocysteine (Sec) - orchestrate essential redox, endocrine, and metabolic pathways in humans, yet high‑resolution structures exist for only a minor...Selenoproteins, defined by the incorporation of the 21st amino acid - selenocysteine (Sec) - orchestrate essential redox, endocrine, and metabolic pathways in humans, yet high‑resolution structures exist for only a minority of the 25 family members. Leveraging the AlphaFold 3 (AF3), we generated full‑length atomic models for all human selenoproteins together with in‑silico Sec-to-Cys variants. AF3 achieved high confidence for 22 proteins and sub‑Å agreement with the one experimentally solved glutathione peroxidase 4 (GPX4). Global comparison of native and mutant models revealed that Sec-to-Cys substitution preserves overall fold in nineteen proteins but locally disrupts or re‑wires intramolecular selenenyl‑sulfide linkages in six cases. Structure‑based clustering uncovered a conserved "Se‑thioredoxin‑like" core in fifteen selenoproteins. AF3 additionally predicted potential GPX4 homodimeric assemblies, consistent with the dimeric forms observed in native gels from brain tissue and cell lines. Together, these AF3 models constitute the comprehensive structural atlas of the human selenoproteome, elucidate the fold‑specific positioning of Sec. The dataset provides a foundation for mechanistic dissection, evolutionary analyses, and rational drug design targeting selenium‑dependent redox biology.
Semenov O, Nazarenko V, Yudenko A
… +11 more, Kovalev K, Goncharov I, Natarov I, Mikhailov A, Kuznetsova E, Nikolaev A, Yang Y, Sluchanko NN, Borshchevskiy V, Remeeva A, Gushchin I
Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their cou...Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A'α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins.
Correlating super-resolution fluorescence light microscopy with cryo-electron tomography (SRcryoCLEM) is a feasible way of targeting specific proteins of interest for high-resolution cryo-electron tomography (cryoET) ima...Correlating super-resolution fluorescence light microscopy with cryo-electron tomography (SRcryoCLEM) is a feasible way of targeting specific proteins of interest for high-resolution cryo-electron tomography (cryoET) imaging within cells. Among different approaches for performing super-resolution fluorescence microscopy on cryogenically preserved samples, cryo-single molecule localization microscopy (cryoSMLM) offers one of the highest imaging resolutions. Thus far, applications of cryoSMLM in SRcryoCLEM have been limited to targeting a single protein structure at a time, as the available palette of cryo-compatible reversibly photoswitchable fluorescent proteins, required for cryoSMLM imaging, is severely limited. Here, we present rsTagRFP and rsEGFP2 as a compatible pair of red and green fluorescent labels that enables dual-colour cryoSMLM, and thus dual-target SRcryoCLEM, in mammalian cells. We demonstrate the simultaneous targeting and identification of two separate structures, MAP2-decorated microtubules and vimentin intermediate filaments, with 30 nm accuracy and within the same cell.
Membranes are essential components of cells and their compartments. They are composed of asymmetric phospholipid bilayers that separate different environments ensuring the physiological functioning of cells. Most phospho...Membranes are essential components of cells and their compartments. They are composed of asymmetric phospholipid bilayers that separate different environments ensuring the physiological functioning of cells. Most phospholipids are synthesized in the endoplasmic reticulum and transported to the target membrane via various routes. Phosphatidic acid is the starting point for all lipid synthesis pathways, following either the Kennedy pathway for phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine or the CDP-DAG pathway for cardiolipin, phosphatidylglycerol and phosphatidylinositol. Many of the enzymes responsible for these synthesis pathways belong to the cytidine diphosphate alcohol phosphotransferase (CDP-AP) family for which a detailed structural and functional understanding is missing. In this review, we focus on the CDP-AP protein family which is divided in two classes, defined by different structures and mechanisms. The CDP-AP members are membrane proteins, and their mode of catalysis follows a bi-bi or ping-pong mechanism. Recent studies on different CDP-AP family members are bringing new molecular insights on these essential proteins. TEASER: CDP-alcohol phosphotransferase proteins are highly diverged in structure while their overall function in phospholipid synthesis is conserved.
G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory significance in bacterial gene expression. While extensively studied in eukaryotes, the roles of G4s especially two-tetrad (2G) G4s...G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory significance in bacterial gene expression. While extensively studied in eukaryotes, the roles of G4s especially two-tetrad (2G) G4s in prokaryotic systems remain greatly underexplored. In this study, we identified and characterized multiple 2G G4-forming motifs within the hfq gene of Acinetobacter baumannii, a clinically significant and highly resilient pathogen. The RNA chaperone Hfq protein plays a central role in post-transcriptional gene regulation in this organism. Using a combination of in silico prediction and biophysical techniques (NMR, CD spectroscopy, EMSA, fluorescence titration, and ITC), we determined the folding and topology of these motifs into stable G4 structures, particularly in RNA. These G4s showed high-affinity binding with BRACO-19, a known G4 ligand, and preferential interaction with full-length Hfq protein compared to its C-terminally truncated variant, underscoring the role of the glycine-rich C-terminal domain in RNA recognition. Furthermore, BRACO-19-mediated stabilization of these G4 structures resulted in significant downregulation of hfq transcript variants, especially in the glycine-rich region. Collectively, this work uncovers a novel regulatory axis involving G-quadruplexes and Hfq protein in A. baumannii, highlighting G4-Hfq interactions as potential antimicrobial targets and offering a scaffold for the broader exploration of RNA-based regulation in this pathogenic bacterium.
The RNA binding motif 15 protein (RBM15) binds both RNA and proteins to regulate a wide repertoire of processes in the cell, including the positing of N6 methyladenosine marks on RNA, the silencing of genes on the inacti...The RNA binding motif 15 protein (RBM15) binds both RNA and proteins to regulate a wide repertoire of processes in the cell, including the positing of N6 methyladenosine marks on RNA, the silencing of genes on the inactive X-chromosome, and even hematopoiesis. Although its C-terminal SPOC domain has been found to facilitate protein-protein interactions, the structural mechanism that underlies how its three N-terminal RNA recognition motifs (RRMs) interact with RNA remains to be elucidated. In this crowdsourced study, we bioinformatically assessed publicly available, genome-wide RNA 2D structural probing and RNA binding protein (RBP) cross-linking and immunoprecipitation (CLIP) data to identify RNAs that bind with RBM15. Binding assays reveal that the RRMs work in concert to bind stem-loop structured RNA motifs with nanomolar binding affinity. Structural modeling and nuclear magnetic resonance (NMR) spectroscopy analysis suggest that RRMs 2 and 3 are coaxially stacked to form a heterodimer; they create a sandwich-like motif around structured RNA. Altogether, this work provides insight into the structural mechanism by which RBM15 interacts with RNAs to govern biological function.
Cryogenic electron tomography is an important technique that enables the three-dimensional visualisation of microscopic samples. In cryogenic electron tomography, a series of two-dimensional projection images is acquired...Cryogenic electron tomography is an important technique that enables the three-dimensional visualisation of microscopic samples. In cryogenic electron tomography, a series of two-dimensional projection images is acquired from different tilt angles of the sample and computationally reconstructed into a tomogram. The tilt range of the specimen stage is typically limited to a certain angular range. Beyond this range, the sample may become too thick for electrons to penetrate, and mechanical components such as the support grid or holder may obstruct the beam, resulting in a loss of image quality. This angular limitation leads to missing information in the reconstructed tomograms, known as the missing wedge problem. Moreover, the use of low-dose electron imaging and other experimental constraints introduces considerable noise, thereby reducing the signal-to-noise ratio of the reconstructed tomogram. In order to solve the problems of missing wedges and low signal-to-noise ratio of tomograms, the Fillnet tomogram restoration framework was designed in this study. The training pair generation module and the FFT_Unet model are specially designed in this framework to improve the accurate acquisition of three-dimensional features in tomograms. Different loss functions are also designed to improve the model's attention to the special features of the samples.
The structure of the tailed phage is composed of an icosahedral (or elongated icosahedral) head and a spiral symmetrical tail, which are connected by a portal located at a unique vertex of the icosahedron. A series of im...The structure of the tailed phage is composed of an icosahedral (or elongated icosahedral) head and a spiral symmetrical tail, which are connected by a portal located at a unique vertex of the icosahedron. A series of image-processing methods and tools have been developed to address the asymmetric structures of phages. However, the structural determination in small proteins within the head and flexible proteins of tailed phages remains a significant impediment, further hindering our deep understanding of the structural biology field. In this study, we developed a data-processing strategy for tailed phage and demonstrated its efficacy with three cryo-EM datasets, including podophage T7, siphophage T1, and myophage Mu. The proposed strategy combines conventional icosahedral reconstruction with local refinement and reconstruction and consists of four key modules: icosahedral reconstruction, selection of the unique vertex of the icosahedron, local asymmetric reconstruction and refinement, and local defocus refinement. The strategy has been successfully applied to determine the asymmetric structure of a range of tailed phages, with a particular focus on resolving the small proteins (core proteins and scaffolding proteins) within the head and flexible proteins on the tail. In addition, the local defocus refinement of our strategy approaches the approximate resolution limit of the icosahedral capsid. The proposed strategy is a viable scheme for determining the asymmetric structures of tailed phages, especially in podophages.