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.
{"title":"Determinants of site-selectivity in human ileal bile acid-binding protein by NMR dynamic analysis of a functionally-impaired mutant","authors":"Tamara Teski , Gergő Horváth , Orsolya Toke","doi":"10.1016/j.jsb.2025.108202","DOIUrl":"10.1016/j.jsb.2025.108202","url":null,"abstract":"<div><div>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 <em>apo</em> 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.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108202"},"PeriodicalIF":3.0,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143873613","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}
The knowledge of three-dimensional structures of biological macromolecules is crucial for understanding the molecular mechanisms underlying disease pathology and for devising drugs targeting specific molecules. Single particle cryo-electron microscopy (Cryo-EM) has become indispensable for this purpose, particularly for large macromolecules and their complexes. However, its effectiveness has been limited in achieving near-atomic resolution for smaller macromolecules. This study presents the Cryo-EM structure of a 55 kDa pentameric AtFKBP53 nucleoplasmin domain at 2.0 Å nominal resolution. Our approach involves selecting the optimal grid for data collection and precise alignment of small particles to enhance the resolution of the final 3D reconstructed map. In this study, we systematically processed cryo-EM dataset of a small molecule to improve alignment, and this data processing strategy can be used as a guidance to process the cryo-EM data of other small molecules.
了解生物大分子的三维结构对于理解疾病病理的分子机制和设计针对特定分子的药物至关重要。单颗粒低温电子显微镜（Cryo-EM）已成为实现这一目的不可或缺的工具，尤其是在研究大型大分子及其复合物时。然而，它在实现较小大分子的近原子分辨率方面效果有限。本研究展示了标称分辨率为 2.0 Å 的 55 kDa 五聚体 AtFKBP53 核蛋白酶结构域的冷冻电镜结构。我们的方法包括选择最佳网格进行数据采集和精确排列小颗粒，以提高最终三维重建图的分辨率。在这项研究中，我们系统地处理了一个小分子的冷冻电镜数据集以改善配准，这种数据处理策略可用于指导其他小分子的冷冻电镜数据处理。
{"title":"2.0 Å cryo-EM structure of the 55 kDa nucleoplasmin domain of AtFKBP53","authors":"Nikhil Bharambe , Ketul Saharan , Dileep Vasudevan , Sandip Basak","doi":"10.1016/j.jsb.2025.108203","DOIUrl":"10.1016/j.jsb.2025.108203","url":null,"abstract":"<div><div>The knowledge of three-dimensional structures of biological macromolecules is crucial for understanding the molecular mechanisms underlying disease pathology and for devising drugs targeting specific molecules. Single particle cryo-electron microscopy (Cryo-EM) has become indispensable for this purpose, particularly for large macromolecules and their complexes. However, its effectiveness has been limited in achieving near-atomic resolution for smaller macromolecules. This study presents the Cryo-EM structure of a 55 kDa pentameric AtFKBP53 nucleoplasmin domain at 2.0 Å nominal resolution. Our approach involves selecting the optimal grid for data collection and precise alignment of small particles to enhance the resolution of the final 3D reconstructed map. In this study, we systematically processed cryo-EM dataset of a small molecule to improve alignment, and this data processing strategy can be used as a guidance to process the cryo-EM data of other small molecules.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108203"},"PeriodicalIF":3.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859033","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 : 2025-03-31DOI: 10.1016/j.jsb.2025.108200
Andry Mercedes Mavila , Jhon Antoni Vargas , Eloy Condori , Erick Giancarlo Suclupe Farro , Adriano Alves Furtado , Josué Manuel López , Silvia Lucila Gonzalez , Humberto D’Muniz Pereira , Jorge Luis Marapara , Roger Ruiz Paredes , Marianela Cobos , Juan C. Castro , Richard Charles Garratt , Diego Antonio Leonardo
Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
{"title":"Phylogenetic analysis and structural studies of heteromeric acetyl-CoA carboxylase from the oleaginous Amazonian microalgae Ankistrodesmus sp.: Insights into the BC and BCCP subunits","authors":"Andry Mercedes Mavila , Jhon Antoni Vargas , Eloy Condori , Erick Giancarlo Suclupe Farro , Adriano Alves Furtado , Josué Manuel López , Silvia Lucila Gonzalez , Humberto D’Muniz Pereira , Jorge Luis Marapara , Roger Ruiz Paredes , Marianela Cobos , Juan C. Castro , Richard Charles Garratt , Diego Antonio Leonardo","doi":"10.1016/j.jsb.2025.108200","DOIUrl":"10.1016/j.jsb.2025.108200","url":null,"abstract":"<div><div>Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (<em>Ankistrodesmus</em> sp.<em>)</em>. Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg<sup>2+</sup>-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from <em>Ankistrodesmus</em> sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in <em>Ankistrodesmus</em> sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108200"},"PeriodicalIF":3.0,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143768447","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}
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