Journal of structural biology最新文献

{"title":"Local microenvironments of capsomer variants in the PBCV-1","authors":"Wenhan Guo ,&nbsp;Esther Alarcon ,&nbsp;Jason E Sanchez ,&nbsp;Chuan Xiao ,&nbsp;Lin Li","doi":"10.1016/j.jsb.2025.108255","DOIUrl":"10.1016/j.jsb.2025.108255","url":null,"abstract":"<div><div>PBCV-1, a giant virus classified among the Nucleocytoviricota virus (NCV) whose structure has been determined to near atomic resolution. The majority capsomers forming the capsid of PBCV<strong>-</strong>1 are Type I capsomers while five other type of variants have been found in recent high resolution structure. Interestingly, some variants, such as Type V capsomers, are found at particular capsid locations whose roles are unclear. To reveal the roles of a Type V capsomer, we replaced the Type V capsomer by a Type I capsomer to compare the interaction among the two types of capsomer variant, especially the interactions between each of the Type V/Type I capsomer and its local capsid microenvironment. Our results revealed significant differences between Type V and Type I capsomers. Notably, the Type V capsomer demonstrated a stronger binding force to the surrounding capsomers than the Type I capsomer. Moreover, the identified salt bridges between Type V/I capsomers and their surrounding capsomers corroborate the results of electrostatic calculations, further highlighting the important residues involved in these interactions. Understanding these local capsid microenvironments will be essential to elucidate the mechanisms governing viral capsid assembly.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108255"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145345910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Helicon: Helical parameter determination and 3D reconstruction from one image","authors":"Daoyi Li ,&nbsp;Xiaoqi Zhang ,&nbsp;Wen Jiang","doi":"10.1016/j.jsb.2025.108256","DOIUrl":"10.1016/j.jsb.2025.108256","url":null,"abstract":"<div><div>Helical symmetry is a common structural feature of many biological macromolecules. However, determination of the helical parameters and <em>de novo</em> 3D reconstruction remain challenging. We have developed a computational method, Helicon, which poses helical reconstruction as a linear regression problem with the projection matrix parameterized by the helical twist, rise, and axial symmetry. A sparse search of the twist and rise parameters would allow determination of helical parameters and 3D reconstruction directly from one 2D class average or a raw cryo-EM image. The Helicon method has been validated with simulation tests and experimental cryo-EM images of helical tubes, non-amyloid filaments, and amyloid fibrils. Imaging stitching and L1 regularization of linear regression were shown to improve the robustness for low-twist amyloids and noisy raw cryo-EM images. Using Helicon, we could successfully determine the helical parameters and perform <em>de novo</em> reconstruction of a previously unreported, low-abundance tau amyloid structure from a publicly available dataset.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108256"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CDP-alcohol phosphotransferases: Structures and function of highly diverse sub-classes within a protein family","authors":"Alexia Gobet,&nbsp;Rasmus Kock Flygaard","doi":"10.1016/j.jsb.2025.108266","DOIUrl":"10.1016/j.jsb.2025.108266","url":null,"abstract":"<div><div>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.</div></div><div><h3>Teaser</h3><div>CDP-alcohol phosphotransferase proteins are highly diverged in structure while their overall function in phospholipid synthesis is conserved.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108266"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISP: A modular platform for cryo-EM image segmentation and processing with Conditional Random Field","authors":"Szu-Chi Chung,&nbsp;Po-Cheng Chou","doi":"10.1016/j.jsb.2025.108239","DOIUrl":"10.1016/j.jsb.2025.108239","url":null,"abstract":"<div><div>Distinguishing signal from background in cryogenic electron microscopy (cryo-EM) micrographs is a critical processing step but remains challenging owing to the inherently low signal-to-noise ratio (SNR), contaminants, variable ice thickness, and densely packed particles of heterogeneous sizes. Recent image-segmentation methods provide pixel-level precision and thus offer several advantages over traditional object-detection approaches: segmented-blob mass can be computed to suppress false-positive particles, particle centering can be improved by leveraging the full brightness profile, and irregularly shaped particles can be identified more reliably. However, low SNR makes it difficult to obtain accurate pixel-level annotations for training segmentation models, and, in the absence of systematic evaluation platforms, most segmentation pipelines still rely on ad-hoc design choices.</div><div>Here, we introduce a modular platform that automatically generates high-quality segmentation maps to serve as reference labels. The platform supports flexible combinations of segmentation architectures, feature extractors, and loss functions, and it integrates novel Conditional Random Fields (CRFs) with class-discriminative features to refine coarse predictions into fine-grained segmentations. On synthetic data, models trained with our reference labels achieve pixel-level accuracy, recall, precision, Intersection-over-Union (IoU), and <span><math><msub><mrow><mtext>F</mtext></mrow><mrow><mn>1</mn></mrow></msub></math></span> scores all exceeding 90%. We further show that the resulting segmentations can be used directly for particle picking, yielding higher-resolution 3D density maps from real experimental datasets; these reconstructions match those curated by human experts and surpass the results of existing particle-picking tools. To facilitate further research, we release our methods as the open-source package <em>CRISP</em>, available at <span><span>https://github.com/phonchi/CryoParticleSegment</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108239"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145027787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM","authors":"Aleksandra Skrajna ,&nbsp;Clara Lenger ,&nbsp;Emily Robinson ,&nbsp;Kevin Cannon ,&nbsp;Reta Sarsam ,&nbsp;Richard G. Ouellette ,&nbsp;Alberta M. Abotsi ,&nbsp;Patrick Brennwald ,&nbsp;Robert K. McGinty ,&nbsp;Joshua D. Strauss ,&nbsp;Richard W. Baker","doi":"10.1016/j.jsb.2025.108253","DOIUrl":"10.1016/j.jsb.2025.108253","url":null,"abstract":"<div><div>Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air–water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air–water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the “resolution revolution” of cryo-EM. Here, we provide a straightforward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found the lipid affinity grids concentrate particles away from the AWI in thin ice (∼30 nm). We determined three structures ranging from 2.4 to 3.0 Å resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 Å structure of a sub-100 kDa protein without symmetry, demonstrating the utility for a range of biological macromolecules. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air–water interface, and thicker ice.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108253"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145286510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"G-Quadruplex structures within the hfq gene regulate RNA–protein interactions in Acinetobacter baumannii","authors":"Aakriti Singh ,&nbsp;Mansee Patel ,&nbsp;Tarun Kumar Sharma ,&nbsp;Amit Kumar","doi":"10.1016/j.jsb.2025.108265","DOIUrl":"10.1016/j.jsb.2025.108265","url":null,"abstract":"<div><div>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 <em>Acinetobacter baumannii</em>, 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 <em>A. baumannii</em>, highlighting G4-Hfq interactions as potential antimicrobial targets and offering a scaffold for the broader exploration of RNA-based regulation in this pathogenic bacterium.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108265"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619937","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mind the corner: Fillets in cryo-FIB lamella preparation to minimise sample loss caused by stress concentration and lamella breakage","authors":"Sergey Gorelick ,&nbsp;Sailakshmi Velamoor ,&nbsp;Patrick Cleeve ,&nbsp;Sylvain Trépout ,&nbsp;Le Ying ,&nbsp;Vivek Naranbhai ,&nbsp;Georg Ramm","doi":"10.1016/j.jsb.2025.108249","DOIUrl":"10.1016/j.jsb.2025.108249","url":null,"abstract":"<div><div>Cryo-FIB milling of biological specimens is a critical and limiting step in the cryo-electron tomography workflow. Preparing electron-transparent cryo-lamellae is a serial, low-throughput process. Even with automation, a skilled operator can typically only produce 15–25 lamellae in a single cryo-FIB session. During sample handling, milling and transfer, the cryo-fixed cells as well as the supporting film layer face various mechanical forces and thermal stresses due to temperature fluctuations. Moreover, after cells are cryo-FIB milled, the resulting thin lamellae continue to endure external forces from mechanical handling and thermal stress. We propose a simple, yet highly effective modification to the standard rectangular milling pattern by implementing “fillets” or corner smoothing providing better mechanical stability. This adjustment helps to avoid sharp corners at the lamella edges, thereby reducing stress concentration. As a result, this modification decreases the likelihood of lamella breakage and improves the overall yield of ready-for-TEM lamellae by over 40 % as verified experimentally.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108249"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145119661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prediction of a structural change in the orientation of the cytoplasmic signaling unit of human Toll-like receptor 9 upon binding of agonistic and antagonistic DNA molecules","authors":"Kazuaki Hoshi","doi":"10.1016/j.jsb.2025.108252","DOIUrl":"10.1016/j.jsb.2025.108252","url":null,"abstract":"<div><div>Toll-like receptor 9 (TLR9) recognizes pathogenic DNA molecules containing unmethylated cytosine-phosphate-guanine motifs (CpG DNA) and initiates signaling cascades essential for enhancing immune responses. TLR9 is a type I transmembrane receptor comprising an N-terminal leucine-rich repeat (LRR) domain, a transmembrane domain, and a C-terminal Toll/interleukin-1 receptor (TIR) domain. Most studies have focused on the interaction between the LRR domain and its DNA ligands. However, the TIR domain is crucial for interacting with adapter proteins such as myeloid differentiation factor 88 (MyD88). The aim of this study was to predict changes in the orientation of the TIR domain in human TLR9 (hTLR9) and its complexes with agonistic or antagonistic DNA molecules using the AlphaFold server. AlphaFold predicted the overall structure of hTLR9 with high confidence scores, including part of the TIR domain. Interestingly, binding of agonistic and antagonistic DNA molecules to the N-terminal LRR domain induced a structural change in the orientation of the TIR domain compared to the unbound TLR9 structure. The TIR domain in the predicted hTLR9 model displayed a secondary structure similar to that of the previously reported human TLR1 crystal structure. The predicted model suggests that ligand binding to the N-terminal LRR domain causes a change in the orientation of the TIR domain of hTLR9, likely due to bending of the transmembrane region.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108252"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145280609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A data-processing strategy of asymmetric reconstruction for tailed phages by Cryo-electron Microscopy","authors":"Wenyuan Chen ,&nbsp;Jing Zheng ,&nbsp;Junquan Zhou ,&nbsp;Lingpeng Cheng ,&nbsp;Hongrong Liu","doi":"10.1016/j.jsb.2025.108262","DOIUrl":"10.1016/j.jsb.2025.108262","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108262"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145495681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Precise ligand-selective mechanism at the fab domain of a tau-recognizing antibody","authors":"Tomohiro Tsuchida ,&nbsp;Takahiro Tsuchiya ,&nbsp;Katsuhiko Minoura ,&nbsp;Yasuko In ,&nbsp;Katsushiro Miyamoto ,&nbsp;Taizo Taniguchi ,&nbsp;Toshimasa Ishida ,&nbsp;Koji Tomoo","doi":"10.1016/j.jsb.2025.108250","DOIUrl":"10.1016/j.jsb.2025.108250","url":null,"abstract":"<div><div>Insoluble aggregated tau protein in the form of paired helical filaments is a causative agent of the neurofibrillary tangles observed in Alzheimer’s disease (AD). The hexapeptide ²⁷⁵VQIINK²⁸⁰ located in the microtubule-binding domain of tau plays a crucial role in the abnormal aggregation process. Therefore, targeting the VQIINK sequence with a tau aggregation inhibitor may be a promising therapeutic approach for AD. A previous study demonstrated that the Fab domain of the tau antibody (Fab2r3) inhibits tau aggregation by binding to the VQIINK sequence. By determining the three-dimensional structures of the Fab2r3-VQIINK peptide complex and apo Fab2r3, we elucidated the recognition mechanism between Fab2r3 and the VQIINK peptide. However, the basis for the selectivity of Fab2r3 for VQIINK was not completely clear. Therefore, the objective of this report is to investigate the selective binding mechanism of Fab2r3 against VQIINK peptide. Through isothermal titration calorimetry, we show that Ile-4 in the VQIINK peptide is crucial for the selectivity of Fab2r3. X-ray structural analysis of three complexes of Fab2r3 with Ile-4 mutated peptides (VQIVYK, VQILNK, and VQIFNK) suggested that the rigid conformation of a hydrophobic pocket in Fab2r3 plays a vital role in ligand selectivity. These findings may explain the effectiveness of Fab2r3 as a tau aggregation inhibitor.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 4","pages":"Article 108250"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145125003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}