Pub Date : 2025-05-20DOI: 10.1016/j.jsb.2025.108212
Xiyong Song , Jesús Baltanás-Copado , Muniyandi Selvaraj , Shrikant B. Kokate , Esa-Pekka Kumpula , Senena Corbalán-García , Juha T. Huiskonen
Fascins are crucial actin-binding proteins linked to carcinomas, such as cancer metastasis. Fascins crosslink unipolar actin filaments into linear and rigid parallel bundles, which play essential roles in the formation of filopodia, stereocilia and other membrane protrusions. However, the mechanism of how fascin bundles actin filaments has remained elusive. Here, we studied the organization of reconstituted fascin-actin bundles by cryo-electron tomography and determined the structure of the fascin–actin complex at 9 Å resolution by subtomogram averaging. Consistent with earlier findings, fascin molecules decorate adjacent actin filaments, positioned at regular intervals corresponding to the half-pitch of actin filaments. The fascin–actin complex structure allows us to verify the binding orientation of fascin between the two actin filaments. Fitting of the previously solved fascin crystal structure facilitates the analysis of the interaction surfaces. Our structural models serve as a blueprint to understand the detailed interactions between fascin and actins and provide new insights for the development of drugs targeting fascin proteins.
{"title":"The mechanism underlying fascin-mediated bundling of actin filaments unveiled by cryo-electron tomography","authors":"Xiyong Song , Jesús Baltanás-Copado , Muniyandi Selvaraj , Shrikant B. Kokate , Esa-Pekka Kumpula , Senena Corbalán-García , Juha T. Huiskonen","doi":"10.1016/j.jsb.2025.108212","DOIUrl":"10.1016/j.jsb.2025.108212","url":null,"abstract":"<div><div>Fascins are crucial actin-binding proteins linked to carcinomas, such as cancer metastasis. Fascins crosslink unipolar actin filaments into linear and rigid parallel bundles, which play essential roles in the formation of filopodia, stereocilia and other membrane protrusions. However, the mechanism of how fascin bundles actin filaments has remained elusive. Here, we studied the organization of reconstituted fascin-actin bundles by cryo-electron tomography and determined the structure of the fascin–actin complex at 9 Å resolution by subtomogram averaging. Consistent with earlier findings, fascin molecules decorate adjacent actin filaments, positioned at regular intervals corresponding to the half-pitch of actin filaments. The fascin–actin complex structure allows us to verify the binding orientation of fascin between the two actin filaments. Fitting of the previously solved fascin crystal structure facilitates the analysis of the interaction surfaces. Our structural models serve as a blueprint to understand the detailed interactions between fascin and actins and provide new insights for the development of drugs targeting fascin proteins.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108212"},"PeriodicalIF":3.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144115347","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}
Pub Date : 2025-05-16DOI: 10.1016/j.jsb.2025.108208
Nathanael Leung , Robert A. Harper , Bin Zhu , Stuart A. Bartlett , Konstantin Ignatyev , Richard M. Shelton , Gabriel Landini , Tan Sui
Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process. Though considerable efforts have been directed at studying natural and artificial carious lesions, most characterisations remain either constrained to 2D analyses or have been unable to achieve fine resolution in 3D due to limited field of view. To overcome this challenge, the present study combined X-ray diffraction (XRD) and scanning transmission X-ray microscopy (STXM) tomography techniques to analyse the mineral density, scattering intensity, and crystallite size in normal, carious, 30 % artificially demineralised, and 50 % artificially demineralised dentine. Combined XRD and STXM tomography was performed on the I18 beamline at Diamond Light Source, using a 15 keV monochromatic beam with 2 × 2 µm spotsize and scanning with translation steps of 2 µm, providing a reconstructed voxel size of 2 × 2 × 2 µm. Natural carious dentine showed a reduction in hydroxyapatite (HAp) crystallite size due to chronic demineralisation. This was unlike artificially demineralised dentine samples that underwent short, continuous demineralisation, which created a zone of fully demineralised dentine, near the sample surface, and a zone of partially demineralised dentine that had a reduced mineral density but an increased average crystallite size.
{"title":"3D Multi-modal Imaging of demineralised dentine using combined synchrotron µ-XRD-CT and STXM-CT","authors":"Nathanael Leung , Robert A. Harper , Bin Zhu , Stuart A. Bartlett , Konstantin Ignatyev , Richard M. Shelton , Gabriel Landini , Tan Sui","doi":"10.1016/j.jsb.2025.108208","DOIUrl":"10.1016/j.jsb.2025.108208","url":null,"abstract":"<div><div>Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process. Though considerable efforts have been directed at studying natural and artificial carious lesions, most characterisations remain either constrained to 2D analyses or have been unable to achieve fine resolution in 3D due to limited field of view. To overcome this challenge, the present study combined X-ray diffraction (XRD) and scanning transmission X-ray microscopy (STXM) tomography techniques to analyse the mineral density, scattering intensity, and crystallite size in normal, carious, 30 % artificially demineralised, and 50 % artificially demineralised dentine. Combined XRD and STXM tomography was performed on the I18 beamline at Diamond Light Source, using a 15 keV monochromatic beam with 2 × 2 µm spotsize and scanning with translation steps of 2 µm, providing a reconstructed voxel size of 2 × 2 × 2 µm. Natural carious dentine showed a reduction in hydroxyapatite (HAp) crystallite size due to chronic demineralisation. This was unlike artificially demineralised dentine samples that underwent short, continuous demineralisation, which created a zone of fully demineralised dentine, near the sample surface, and a zone of partially demineralised dentine that had a reduced mineral density but an increased average crystallite size.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108208"},"PeriodicalIF":3.0,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094151","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}
Pub Date : 2025-05-14DOI: 10.1016/j.jsb.2025.108210
Camila Wendt , Fernanda C. de Medeiros , Raquel P. Gonçalves , Fabio Nudelman , Michelle Klautau , Marcos Farina , André L. Rossi
We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge Heteropia glomerosa (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, present a pattern of concentric lines spaced 400 nm apart when observed by scanning electron microscopy. We showed by electron backscattered diffraction that the whole spicule length has the same crystallographic orientation. Still, misorientation of up to 1.8° in adjacent regions (∼ 2 µm) and a continuous increase in the misalignment of up to 4.5° in regions separated by 300 µm were present. The sponge cells (mainly sclerocytes and pinacocytes) near the mineralization zone contain a high number of vesicles rich in calcium, which could be involved in the spicule biomineralization. We showed by electron and ion microscopies that the spicule growth occurs through the addition calcium carbonate granules, which form near the membrane of the sclerocyte, the cell responsible for biomineralization. The granules were deposited layer by layer on the surface of the spicule, increasing the biomineral thickness. Domains of 1–3 µm containing facets partially connected and surrounded by organic material were observed in an intermediate stage of the spicule growth. Misorientation between these domains was approximately 2°, similar to the misorientation obtained by electron backscattered diffraction, indicating that the spicule is formed by the addition of granules fusing in a predominant orientation.
{"title":"Calcium carbonate deposition in the spicules of the sponge Heteropia glomerosa (Porifera, Calcarea)","authors":"Camila Wendt , Fernanda C. de Medeiros , Raquel P. Gonçalves , Fabio Nudelman , Michelle Klautau , Marcos Farina , André L. Rossi","doi":"10.1016/j.jsb.2025.108210","DOIUrl":"10.1016/j.jsb.2025.108210","url":null,"abstract":"<div><div>We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge <em>Heteropia glomerosa</em> (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, present a pattern of concentric lines spaced 400 nm apart when observed by scanning electron microscopy. We showed by electron backscattered diffraction that the whole spicule length has the same crystallographic orientation. Still, misorientation of up to 1.8° in adjacent regions (∼ 2 µm) and a continuous increase in the misalignment of up to 4.5° in regions separated by 300 µm were present. The sponge cells (mainly sclerocytes and pinacocytes) near the mineralization zone contain a high number of vesicles rich in calcium, which could be involved in the spicule biomineralization. We showed by electron and ion microscopies that the spicule growth occurs through the addition calcium carbonate granules, which form near the membrane of the sclerocyte, the cell responsible for biomineralization. The granules were deposited layer by layer on the surface of the spicule, increasing the biomineral thickness. Domains of 1–3 µm containing facets partially connected and surrounded by organic material were observed in an intermediate stage of the spicule growth. Misorientation between these domains was approximately 2°, similar to the misorientation obtained by electron backscattered diffraction, indicating that the spicule is formed by the addition of granules fusing in a predominant orientation.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108210"},"PeriodicalIF":3.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144086436","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}
Cryo-electron tomography (cryo-ET) is an essential tool in structural biology, uniquely capable of visualizing three-dimensional macromolecular complexes within their native cellular environments, thereby providing profound molecular-level insights. Despite its significant promise, cryo-ET faces persistent challenges in the systematic localization, identification, segmentation, and structural recovery of three-dimensional subcellular components, necessitating the development of efficient and accurate large-scale image analysis methods. In response to these complexities, this paper introduces AITom, an open-source artificial intelligence platform tailored for cryo-ET researchers. AITom integrates a comprehensive suite of public and proprietary algorithms, supporting both traditional template-based and template-free approaches, alongside state-of-the-art deep learning methodologies for cryo-ET data analysis. By incorporating diverse computational strategies, AITom enables researchers to more effectively tackle the complexities inherent in cryo-ET, facilitating precise analysis and interpretation of complex biological structures. Furthermore, AITom provides extensive tutorials for each analysis module, offering valuable guidance to users in utilizing its comprehensive functionalities.
{"title":"AITom: AI-guided cryo-electron tomography image analyses toolkit","authors":"Xueying Zhan , Xiangrui Zeng , Mostofa Rafid Uddin, Min Xu","doi":"10.1016/j.jsb.2025.108207","DOIUrl":"10.1016/j.jsb.2025.108207","url":null,"abstract":"<div><div>Cryo-electron tomography (cryo-ET) is an essential tool in structural biology, uniquely capable of visualizing three-dimensional macromolecular complexes within their native cellular environments, thereby providing profound molecular-level insights. Despite its significant promise, cryo-ET faces persistent challenges in the systematic localization, identification, segmentation, and structural recovery of three-dimensional subcellular components, necessitating the development of efficient and accurate large-scale image analysis methods. In response to these complexities, this paper introduces <span>AITom</span>, an open-source artificial intelligence platform tailored for cryo-ET researchers. <span>AITom</span> integrates a comprehensive suite of public and proprietary algorithms, supporting both traditional template-based and template-free approaches, alongside state-of-the-art deep learning methodologies for cryo-ET data analysis. By incorporating diverse computational strategies, <span>AITom</span> enables researchers to more effectively tackle the complexities inherent in cryo-ET, facilitating precise analysis and interpretation of complex biological structures. Furthermore, <span>AITom</span> provides extensive tutorials for each analysis module, offering valuable guidance to users in utilizing its comprehensive functionalities.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108207"},"PeriodicalIF":3.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144068286","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}
Pub Date : 2025-05-12DOI: 10.1016/j.jsb.2025.108209
Songna Wu , Nianying Zhang , Qun Wan
Thermostable enzymes have significant advantages in industries, yet uncovering novel candidates with superior properties remains a scientific pursuit. This study identified rMxylcd, a glycoside hydrolase 11 family thermophilic xylanase from compost-soil metagenome, which exhibited a high specific activity of 5954 U·mg−1 at pH 5.5 and 80°C. rMxylcd was crystallized and diffracted to 1.5 Å resolution. Compared to the mesophilic xylanase Xyn II, rMxylcd exhibits a more compact architecture. Notably, B-factor analysis reveals a uniquely flexible thumb region, hinting at its critical role in the enzyme’s catalytic mechanism. rMxylcd contains two disulfide bonds in the thumb and the N-terminal regions. Breaking these disulfide bonds by mutagenesis has dramatically decreased activities and thermostability. Conversely, introducing an extra disulfide bond at the N-terminal region of its α-helix extended its half-life for more than five folds at 80°C. Our studies firmly establish that the disulfide bonds are essential for its high thermal stability and the flexibility of the thumb region is crucial for its activity. Comparing the rMxylcd crystal structure with the AlphaFold2-predicted model shows overall similarity, but the crystal structure offers higher local accuracy, especially in key functional regions. These findings not only deepen our understanding of the structure-function relationship of thermophilic xylanases but also inform a rational design of industrial enzymes.
{"title":"Disulfide bonds enhance thermal stability and thumb region drives activity of the glycoside hydrolase 11 xylanase rMxylcd","authors":"Songna Wu , Nianying Zhang , Qun Wan","doi":"10.1016/j.jsb.2025.108209","DOIUrl":"10.1016/j.jsb.2025.108209","url":null,"abstract":"<div><div>Thermostable enzymes have significant advantages in industries, yet uncovering novel candidates with superior properties remains a scientific pursuit. This study identified rMxyl<sup>cd</sup>, a glycoside hydrolase 11 family thermophilic xylanase from compost-soil metagenome, which exhibited a high specific activity of 5954 U·mg<sup>−1</sup> at pH 5.5 and 80°C. rMxyl<sup>cd</sup> was crystallized and diffracted to 1.5 Å resolution. Compared to the mesophilic xylanase Xyn II, rMxyl<sup>cd</sup> exhibits a more compact architecture. Notably, B-factor analysis reveals a uniquely flexible thumb region, hinting at its critical role in the enzyme’s catalytic mechanism. rMxyl<sup>cd</sup> contains two disulfide bonds in the thumb and the <em>N</em>-terminal regions. Breaking these disulfide bonds by mutagenesis has dramatically decreased activities and thermostability. Conversely, introducing an extra disulfide bond at the <em>N</em>-terminal region of its α-helix extended its half-life for more than five folds at 80°C. Our studies firmly establish that the disulfide bonds are essential for its high thermal stability and the flexibility of the thumb region is crucial for its activity. Comparing the rMxyl<sup>cd</sup> crystal structure with the AlphaFold2-predicted model shows overall similarity, but the crystal structure offers higher local accuracy, especially in key functional regions. These findings not only deepen our understanding of the structure-function relationship of thermophilic xylanases but also inform a rational design of industrial enzymes.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108209"},"PeriodicalIF":3.0,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078757","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}
Pub Date : 2025-05-05DOI: 10.1016/j.jsb.2025.108201
Evgeny Hershkovitch Neiterman , Ayelet Heimowitz , Gil Ben-Artzi
Single-particle cryo-electron microscopy (cryo-EM) has significantly advanced macromolecular structure reconstruction. However, a key limitation is the conventional reliance on micrographs obtained by motion correction and averaging, which inherently loses the richness of information contained within each frame of the original movie. The future of cryo-EM reconstruction ideally involves harnessing the raw signal from every frame to unlock potentially higher quality structures. In this paper, we present a first essential step toward this paradigm shift, that is, a novel, non-parametric method for detecting tomographic projections across all movie frames, using temporal consistency. Our method is inspired by Structure-from-Motion (SfM), and independent of motion correction, CTF estimation, and initial reconstruction. Our experimental results demonstrate reduced outlier rate and accurate particle localization comparable to existing approaches throughout the entire movie sequence.
{"title":"A non-parametric approach to particle picking in all frames","authors":"Evgeny Hershkovitch Neiterman , Ayelet Heimowitz , Gil Ben-Artzi","doi":"10.1016/j.jsb.2025.108201","DOIUrl":"10.1016/j.jsb.2025.108201","url":null,"abstract":"<div><div>Single-particle cryo-electron microscopy (cryo-EM) has significantly advanced macromolecular structure reconstruction. However, a key limitation is the conventional reliance on micrographs obtained by motion correction and averaging, which inherently loses the richness of information contained within each frame of the original movie. The future of cryo-EM reconstruction ideally involves harnessing the raw signal from every frame to unlock potentially higher quality structures. In this paper, we present a first essential step toward this paradigm shift, that is, a novel, non-parametric method for detecting tomographic projections across all movie frames, using temporal consistency. Our method is inspired by Structure-from-Motion (SfM), and independent of motion correction, CTF estimation, and initial reconstruction. Our experimental results demonstrate reduced outlier rate and accurate particle localization comparable to existing approaches throughout the entire movie sequence.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108201"},"PeriodicalIF":3.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143998744","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}
Pub Date : 2025-05-03DOI: 10.1016/j.jsb.2025.108206
Isobel Jackson Hirst , Wesley Tien Chiang , Nien-Jen Hu , Charlotte A. Scarff , Rebecca F. Thompson , Michele C. Darrow , Stephen P. Muench
Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air–water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from Bacillus subtilis. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples.
{"title":"Untangling the effects of flexibility and the AWI in cryoEM sample preparation: A case study using KtrA","authors":"Isobel Jackson Hirst , Wesley Tien Chiang , Nien-Jen Hu , Charlotte A. Scarff , Rebecca F. Thompson , Michele C. Darrow , Stephen P. Muench","doi":"10.1016/j.jsb.2025.108206","DOIUrl":"10.1016/j.jsb.2025.108206","url":null,"abstract":"<div><div>Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air–water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from <em>Bacillus subtilis</em>. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108206"},"PeriodicalIF":3.0,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143941077","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}
Pub Date : 2025-04-26DOI: 10.1016/j.jsb.2025.108205
Razi Safadi, Lior Aram, Diede de Haan, Emanuel M. Avrahami, Assaf Gal
Nanopatterning of inorganic materials is a challenging task for contemporary science. It is therefore remarkable that unicellular organisms can form intricately shaped biominerals. A prominent example is the silica cell wall of diatoms, which usually forms in specialized intracellular organelles. Inside such an organelle, biological regulation proceeds via the concerted activity of various organic macromolecules and inorganic precursors. However, it was shown that a specific type of elongated silica structures, called setae, which characterizes the diatom genus Chaetoceros, form extracellularly, raising questions about the regulatory mechanisms of this silicification process. Here, we study a relatively large species, Chaetoceros rostratus, that forms long and intricate setae. We used in-cell cryo electron tomography to image the native state of seta formation. The high-resolution 3D data show that silica formation outside the cell membrane involves continuous organic sheath that covers the newly formed seta. This sheath has an elaborate structure and is positioned tens of nanometers away from the silica by structural macromolecules that might be involved in architectural regulation. Elucidating the structural components of this delicate living system will allow for new opportunities to learn about the biological strategies for controlled mineralization.
{"title":"Structural organization of the organic sheath that delineates extracellular seta silicification in diatoms","authors":"Razi Safadi, Lior Aram, Diede de Haan, Emanuel M. Avrahami, Assaf Gal","doi":"10.1016/j.jsb.2025.108205","DOIUrl":"10.1016/j.jsb.2025.108205","url":null,"abstract":"<div><div>Nanopatterning of inorganic materials is a challenging task for contemporary science. It is therefore remarkable that unicellular organisms can form intricately shaped biominerals. A prominent example is the silica cell wall of diatoms, which usually forms in specialized intracellular organelles. Inside such an organelle, biological regulation proceeds via the concerted activity of various organic macromolecules and inorganic precursors. However, it was shown that a specific type of elongated silica structures, called setae, which characterizes the diatom genus <em>Chaetoceros</em>, form extracellularly, raising questions about the regulatory mechanisms of this silicification process. Here, we study a relatively large species, <em>Chaetoceros rostratus</em>, that forms long and intricate setae. We used in-cell cryo electron tomography to image the native state of seta formation. The high-resolution 3D data show that silica formation outside the cell membrane involves continuous organic sheath that covers the newly formed seta. This sheath has an elaborate structure and is positioned tens of nanometers away from the silica by structural macromolecules that might be involved in architectural regulation. Elucidating the structural components of this delicate living system will allow for new opportunities to learn about the biological strategies for controlled mineralization.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108205"},"PeriodicalIF":3.0,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902151","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}
Recent AI applications have revolutionized the modeling of structurally unresolved protein regions, thereby complementing traditional computational methods. These state-of-the-art techniques can generate numerous candidate structures, significantly expanding the scope of structural biology. However, to effectively prioritize these models, a physics-based approach is required to assess the energy landscape. Such integration can bridge the gap between rapid model generation and precise determination of functional conformations. To address this challenge, we propose an integrated approach that combines molecular modeling with AI and HPC. Metadynamics simulations in latent space are used to explore potential energy landscapes based on initial approximations of flexible region structures derived from modeling tools such as AlphaFold, RosettaFold, Modeller, SwissModel, etc. The approach was validated by modeling folding of Trp-cage protein and conformational plasticity of ubiquitin. The predominant conformations of previously unresolved mobile regions in the active center of flavin-dependent 2-hydroxybiphenyl-3-monooxygenase (EC 1.14.13.44) were identified, while estimating the energy associated with these conformational changes.
{"title":"Characterization of conformational flexibility in protein structures by applying artificial intelligence to molecular modeling","authors":"Kirill Kopylov , Evgeny Kirilin , Vladimir Voevodin , Vytas Švedas","doi":"10.1016/j.jsb.2025.108204","DOIUrl":"10.1016/j.jsb.2025.108204","url":null,"abstract":"<div><div>Recent AI applications have revolutionized the modeling of structurally unresolved protein regions, thereby complementing traditional computational methods. These state-of-the-art techniques can generate numerous candidate structures, significantly expanding the scope of structural biology. However, to effectively prioritize these models, a physics-based approach is required to assess the energy landscape. Such integration can bridge the gap between rapid model generation and precise determination of functional conformations. To address this challenge, we propose an integrated approach that combines molecular modeling with AI and HPC. Metadynamics simulations in latent space are used to explore potential energy landscapes based on initial approximations of flexible region structures derived from modeling tools such as AlphaFold, RosettaFold, Modeller, SwissModel, etc. The approach was validated by modeling folding of Trp-cage protein and conformational plasticity of ubiquitin. The predominant conformations of previously unresolved mobile regions in the active center of flavin-dependent 2-hydroxybiphenyl-3-monooxygenase (EC 1.14.13.44) were identified, while estimating the energy associated with these conformational changes.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108204"},"PeriodicalIF":3.0,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888019","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}
Pub Date : 2025-04-21DOI: 10.1016/j.jsb.2025.108202
Tamara Teski , Gergő Horváth , Orsolya Toke
Human ileal bile acid-binding protein (hI-BABP), a member of the family of intracellular lipid-binding proteins, has a key role in the enterohepatic circulation of bile salts. The two internal binding sites of hI-BABP exhibit positive cooperativity accompanied by a site preference of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in the human body. Previous study of Q51A hI-BABP in its apo state, a mutant with lost site-selectivity, suggests that disruption of the hydrogen-bonding network in the vicinity of the C/D-turn has long-range dynamic effects. To improve our understanding of the determinants of site-selectivity in hI-BABP, a comparative NMR chemical shift and spin relaxation analysis of homo- and heterotypic bile salt complexes of wild-type and Q51A hI-BABP was carried out. The wild-type GCDA-complex shows a striking similarity with the thermodynamically most stable hI-BABP:GCDA:GCA complex in terms of both structure and dynamic behaviour, suggesting that the bound GCDA at site 1 has a decisive role in conveying key stabilizing interactions in the physiologically most abundant heterotypic complex. Destabilization of hI-BABP-GCDA by the functionally impairing mutation Q51A is indicated by both the increase of ms-timescale motions in key segments of the protein as well as by increased ps-ns local fluctuations superimposed on slow motions. Our study suggests that binding interactions in hI-BABP might be modulated by altering the dynamic behaviour of specific segments in the protein with implications for targeting the intracellular trafficking of bile salts and bile salt-induced stimulation of nuclear receptors.
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