Pub Date : 2025-07-04DOI: 10.1016/j.jsb.2025.108232
Priyanka Garg , Xiangsong Feng , Swastik De , Joachim Frank
This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a microsprayer. We find a reproducible increase, rather than the expected decrease in GFP production as monitored by fluorescence, which may suggest heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios. The results have a bearing on the design and interpretation of validation experiments in time-resolved cryo-EM based on microfluidic chips.
{"title":"Passage of ribosomes through microsprayer increases functional activity – Implications for activity assays in time-resolved cryo-EM","authors":"Priyanka Garg , Xiangsong Feng , Swastik De , Joachim Frank","doi":"10.1016/j.jsb.2025.108232","DOIUrl":"10.1016/j.jsb.2025.108232","url":null,"abstract":"<div><div>This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a microsprayer. We find a reproducible <em>increase</em>, rather than the expected <em>decrease</em> in GFP production as monitored by fluorescence, which may suggest heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios. The results have a bearing on the design and interpretation of validation experiments in time-resolved cryo-EM based on microfluidic chips.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108232"},"PeriodicalIF":3.0,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144575723","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 : 2025-06-26DOI: 10.1016/j.jsb.2025.108230
Sebastian Dorawa , Katarzyna Biniek-Antosiak , Magdalena Bejger , Anna-Karina Kaczorowska , Karol Ciuchcinski , Agnieszka Godlewska , Magdalena Płotka , Gudmundur O. Hreggvidsson , Lukasz Dziewit , Tadeusz Kaczorowski , Wojciech Rypniewski
We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW = 39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37 %) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of T. thermophilus phages: φYS40 (87 %) and φTMA (88 %). The enzyme exhibits lytic activity mainly against bacteria of the genus Thermus and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (Tm = 98.3 °C). It retains ∼ 50 % of its lytic activity after 90 min of incubation at 99 °C. Crystallographic analysis, at 2.2 Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40 % of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn2+ ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of E. coli peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein’s thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.
{"title":"Crystal structure, enzymatic and thermodynamic properties of the Thermus thermophilus phage Tt72 lytic endopeptidase with unique structural signatures of thermal adaptation","authors":"Sebastian Dorawa , Katarzyna Biniek-Antosiak , Magdalena Bejger , Anna-Karina Kaczorowska , Karol Ciuchcinski , Agnieszka Godlewska , Magdalena Płotka , Gudmundur O. Hreggvidsson , Lukasz Dziewit , Tadeusz Kaczorowski , Wojciech Rypniewski","doi":"10.1016/j.jsb.2025.108230","DOIUrl":"10.1016/j.jsb.2025.108230","url":null,"abstract":"<div><div>We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic <em>Thermus thermophilus</em> MAT72 phage vB_Tt72. The protein of 346-aa (MW = 39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37 %) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of <em>T. thermophilus</em> phages: φYS40 (87 %) and φTMA (88 %). The enzyme exhibits lytic activity mainly against bacteria of the genus <em>Thermus</em> and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (T<sub>m</sub> = 98.3 °C). It retains ∼ 50 % of its lytic activity after 90 min of incubation at 99 °C. Crystallographic analysis, at 2.2 Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40 % of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn<sup>2+</sup> ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of <em>E. coli</em> peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein’s thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108230"},"PeriodicalIF":3.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522821","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 : 2025-06-23DOI: 10.1016/j.jsb.2025.108229
Mia Argyrou , Eleni Pitsillou , Andrew Hung , Assam El-Osta , Tom C. Karagiannis
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogenic agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, uses the trimeric spike protein to gain entry into the host cell. Structural studies have revealed that the spike protein is comprised of the S1 and S2 subunits. The S1 subunit of the spike protein contains the receptor-binding domain (RBD), which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor. The interaction between the RBD and ACE2 facilitates membrane fusion and host cell infection. The SARS-CoV-2 spike protein also contains a unique insertion of four amino acids that results in the 682-RRAR↓S-686 polybasic furin cleavage motif at the boundary of the S1 and S2 subunits. The furin cleavage motif contributes to the high infectivity and transmissibility of SARS-CoV-2. This review provides a comprehensive analysis of the molecular interactions of the spike protein, with a specific focus on the RBD and furin cleavage site. In addition to examining the binding characteristics with ACE2, the interactions with alternative receptors, such as neuropilin-1 (NRP1) and the nicotinic acetylcholine receptors (nAChRs) are highlighted. The ability of the spike protein to bind alternative receptors and host factors has been linked to the pathophysiology of COVID-19 and the persistence of symptoms in the post COVID-19 condition. Furthermore, we examine the impact of spike protein mutations on receptor affinity and disease severity. SARS-CoV-2 continues to evolve, with variants remaining an ongoing threat to public health. Understanding these molecular interactions is critical for the development of novel therapeutic interventions.
{"title":"Insights into the pathogenic mechanisms associated with the SARS-CoV-2 spike protein","authors":"Mia Argyrou , Eleni Pitsillou , Andrew Hung , Assam El-Osta , Tom C. Karagiannis","doi":"10.1016/j.jsb.2025.108229","DOIUrl":"10.1016/j.jsb.2025.108229","url":null,"abstract":"<div><div>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogenic agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, uses the trimeric spike protein to gain entry into the host cell. Structural studies have revealed that the spike protein is comprised of the S1 and S2 subunits. The S1 subunit of the spike protein contains the receptor-binding domain (RBD), which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor. The interaction between the RBD and ACE2 facilitates membrane fusion and host cell infection. The SARS-CoV-2 spike protein also contains a unique insertion of four amino acids that results in the 682-RRAR↓S-686 polybasic furin cleavage motif at the boundary of the S1 and S2 subunits. The furin cleavage motif contributes to the high infectivity and transmissibility of SARS-CoV-2. This review provides a comprehensive analysis of the molecular interactions of the spike protein, with a specific focus on the RBD and furin cleavage site. In addition to examining the binding characteristics with ACE2, the interactions with alternative receptors, such as neuropilin-1 (NRP1) and the nicotinic acetylcholine receptors (nAChRs) are highlighted. The ability of the spike protein to bind alternative receptors and host factors has been linked to the pathophysiology of COVID-19 and the persistence of symptoms in the post COVID-19 condition. Furthermore, we examine the impact of spike protein mutations on receptor affinity and disease severity. SARS-CoV-2 continues to evolve, with variants remaining an ongoing threat to public health. Understanding these molecular interactions is critical for the development of novel therapeutic interventions.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108229"},"PeriodicalIF":3.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144481082","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 : 2025-06-22DOI: 10.1016/j.jsb.2025.108226
Roman Ye. Brodskii , Olga V. Vashchenko
Lipid membranes are uniquely complex biological structures with large and still undisclosed regulatory potential in many living processes caused by versatile changes in their structure while adsorption of various guest molecules (dopants). This work is devoted to exploring spontaneous dopant-driven formation of lipid domains in a monolipid membrane observed experimentally for dopants with bimodal adsorption. The work offers the results obtained for a wide range of different cases exploiting our proposed original simulation method and numerical model. The central idea of the approach is dopant binding ‘like the surroundings’, i.e. preferential binding.
The value range of the preferential binding extent was determined, where stable domains are formed and their size distribution becomes steady. The density of domain size distribution is power-law, i.e. the domain patterns possesses self-similarity. Outside this range, only one phase dominates if the extent is too large, whereas if it is too small, great dispersion of membrane was observed, so the membrane is physically homogeneous. Various neighboring as well as different methods of calculation of dopant binding probabilities are considered. The results obtained differed quantitatively but not qualitatively. The suggested model and the domain definition are similar to those used in percolation theory. Thus, the results can be applicated to percolation problems.
Grounding on analysis of literature data on domain patterns formed in various lipid systems, we suggested that the preferential binding mechanism is in line with the mechanism of preferential neighboring which is implicitly assumed in such systems irrespective of their specific nature.
{"title":"Preferential binding as a driving mechanism of lipid domains formation","authors":"Roman Ye. Brodskii , Olga V. Vashchenko","doi":"10.1016/j.jsb.2025.108226","DOIUrl":"10.1016/j.jsb.2025.108226","url":null,"abstract":"<div><div>Lipid membranes are uniquely complex biological structures with large and still undisclosed regulatory potential in many living processes caused by versatile changes in their structure while adsorption of various guest molecules (dopants). This work is devoted to exploring spontaneous dopant-driven formation of lipid domains in a monolipid membrane observed experimentally for dopants with bimodal adsorption. The work offers the results obtained for a wide range of different cases exploiting our proposed original simulation method and numerical model. The central idea of the approach is dopant binding ‘like the surroundings’, i.e. preferential binding.</div><div>The value range of the preferential binding extent was determined, where stable domains are formed and their size distribution becomes steady. The density of domain size distribution is power-law, i.e. the domain patterns possesses self-similarity. Outside this range, only one phase dominates if the extent is too large, whereas if it is too small, great dispersion of membrane was observed, so the membrane is physically homogeneous. Various neighboring as well as different methods of calculation of dopant binding probabilities are considered. The results obtained differed quantitatively but not qualitatively. The suggested model and the domain definition are similar to those used in percolation theory. Thus, the results can be applicated to percolation problems.</div><div>Grounding on analysis of literature data on domain patterns formed in various lipid systems, we suggested that the preferential binding mechanism is in line with the mechanism of preferential neighboring which is implicitly assumed in such systems irrespective of their specific nature.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108226"},"PeriodicalIF":3.0,"publicationDate":"2025-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144484878","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 : 2025-06-18DOI: 10.1016/j.jsb.2025.108227
Danielle Wiles , James Roest , Julian P. Vivan , Travis Beddoe
Cannabis sativa is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consumer preference for C. sativa products, driving scientific interest in optimising terpene expression profiles and shaping the selective breeding of terpene profiles in C. sativa cultivars. In particular, the monoterpene, terpinolene, is influential in defining the sensory and therapeutic qualities of many C. sativa strains due to its woody, citrus-like aroma. Here we report the 2.5 Å resolution crystal structure of terpinolene synthase (CsTOS) from C. sativa in its apo form. The structure exhibits the class I monoterpene synthase fold with an open active site conformation. Using site-directed mutagenesis, we identified H618 as a key residues in determining product specificity. Substituting H618 with charged residues resulted in the preferential formation of limonene over terpinolene, highlighting its critical role in stabilising the substrate intermediate. Additionally, novel mutations uncovered an extended epistatic network of residues within 5 Å of the active site, spanning the α-helical bundle of the terpene synthase fold. These interactions contribute to monoterpene formation by modulating substrate positioning and catalytic activity. These insights advance our understanding of monoterpene biosynthesis and enable the targeted engineering of terpene synthases for customised terpene production, offering significant potential for the C. sativa industry.
{"title":"The product specificities of terpinolene synthase, from cannabis sativa, reveals the plasticity of the terpene synthase active site","authors":"Danielle Wiles , James Roest , Julian P. Vivan , Travis Beddoe","doi":"10.1016/j.jsb.2025.108227","DOIUrl":"10.1016/j.jsb.2025.108227","url":null,"abstract":"<div><div><em>Cannabis sativa</em> is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consumer preference for <em>C. sativa</em> products, driving scientific interest in optimising terpene expression profiles and shaping the selective breeding of terpene profiles in <em>C. sativa</em> cultivars. In particular, the monoterpene, terpinolene, is influential in defining the sensory and therapeutic qualities of many <em>C. sativa</em> strains due to its woody, citrus-like aroma. Here we report the 2.5 Å resolution crystal structure of terpinolene synthase (CsTOS) from C. <em>sativa</em> in its apo form. The structure exhibits the class I monoterpene synthase fold with an open active site conformation. Using site-directed mutagenesis, we identified H618 as a key residues in determining product specificity. Substituting H618 with charged residues resulted in the preferential formation of limonene over terpinolene, highlighting its critical role in stabilising the substrate intermediate. Additionally, novel mutations uncovered an extended epistatic network of residues within 5 Å of the active site, spanning the α-helical bundle of the terpene synthase fold. These interactions contribute to monoterpene formation by modulating substrate positioning and catalytic activity. These insights advance our understanding of monoterpene biosynthesis and enable the targeted engineering of terpene synthases for customised terpene production, offering significant potential for the <em>C. sativa</em> industry.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108227"},"PeriodicalIF":3.0,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144321669","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}
Adenylyl cyclase 9 (AC9) regulates many physiologic functions through the production of cAMP, an important second messenger that regulates downstream effectors. The activation of AC9 is highly regulated by GPCR signaling. For example, AC9 is activated by the binding of Gαs, which, in turn, is activated by Gs-driven GPCRs. The structure of bovine AC9 (bAC9) was reported in 2019 using single-particle cryo-electron microscopy (cryo-EM). The structure of human AC9 (hAC9), however, has not been reported to date despite its potential benefit for drug development. Here, we analyzed the structures of hAC9 and hAC9 in complex with Gαs (hAC9-Gαs) using single-particle cryo-EM. The soluble domain of AC9-Gαs, the transmembrane (TM) domain of AC9-Gαs, and AC9 alone were analyzed at resolutions of 2.7 Å, 3.4 Å, and 3.2 Å, respectively. The results revealed three key aspects of the activation mechanism of hAC9 and its cAMP-generating function. First, a conformational change of the soluble domain was observed upon Gαs binding, resulting in a widely open catalytic site. Second, we analyzed the exact position of the C-terminus occluding the catalytic site in the hAC9-Gαs complex. Finally, we unexpectedly identified an elongated density suggestive of a single acyl chain in the TM domain. Consistent with recent reports on the allosteric regulation of AC by lipids, this finding suggests that the TM domain could serve as a potential drug target. These structural findings enhance our understanding of the structure and function of AC9 and other ACs and will provide a foundation for future AC-target drug discovery.
腺苷酸环化酶9 （AC9）通过产生cAMP来调节许多生理功能，cAMP是调节下游效应物的重要第二信使。AC9的激活受GPCR信号的高度调控。例如，AC9被g - αs结合激活，而g - αs又被gs驱动的gpcr激活。2019年，利用单粒子冷冻电镜（cryo-EM）报道了牛AC9 （bAC9）的结构。然而，人类AC9 （hAC9）的结构迄今尚未报道，尽管它对药物开发有潜在的益处。本文采用单粒子冷冻电镜分析了hAC9和hAC9与Gαs配合物（hAC9-Gαs）的结构。AC9- g - αs的可溶性结构域、AC9- g - αs的跨膜结构域和单独的AC9分别以2.7 Å、3.4 Å和3.2 Å的分辨率进行分析。结果揭示了hAC9的激活机制及其camp生成功能的三个关键方面。首先，在g - αs结合时观察到可溶性结构域的构象变化，导致催化位点广泛开放。其次，我们分析了hac9 - g - αs络合物中c -末端封闭催化位点的确切位置。最后，我们意外地发现了一个细长的密度，表明在TM结构域中有一个单酰基链。与最近关于脂质对AC变构调节的报道一致，这一发现表明TM结构域可能是一个潜在的药物靶点。这些结构上的发现增强了我们对AC9和其他ac的结构和功能的理解，并将为未来ac靶向药物的发现提供基础。
{"title":"Structural insights into human adenylyl cyclase 9 in complex with Gαs by cryo-EM","authors":"Risa Nomura , Shota Suzuki , Koki Nishikawa , Hiroshi Suzuki , Yoshinori Fujiyoshi","doi":"10.1016/j.jsb.2025.108223","DOIUrl":"10.1016/j.jsb.2025.108223","url":null,"abstract":"<div><div>Adenylyl cyclase 9 (AC9) regulates many physiologic functions through the production of cAMP, an important second messenger that regulates downstream effectors. The activation of AC9 is highly regulated by GPCR signaling. For example, AC9 is activated by the binding of Gαs, which, in turn, is activated by Gs-driven GPCRs. The structure of bovine AC9 (bAC9) was reported in 2019 using single-particle cryo-electron microscopy (cryo-EM). The structure of human AC9 (hAC9), however, has not been reported to date despite its potential benefit for drug development. Here, we analyzed the structures of hAC9 and hAC9 in complex with Gαs (hAC9-Gαs) using single-particle cryo-EM. The soluble domain of AC9-Gαs, the transmembrane (TM) domain of AC9-Gαs, and AC9 alone were analyzed at resolutions of 2.7 Å, 3.4 Å, and 3.2 Å, respectively. The results revealed three key aspects of the activation mechanism of hAC9 and its cAMP-generating function. First, a conformational change of the soluble domain was observed upon Gαs binding, resulting in a widely open catalytic site. Second, we analyzed the exact position of the C-terminus occluding the catalytic site in the hAC9-Gαs complex. Finally, we unexpectedly identified an elongated density suggestive of a single acyl chain in the TM domain. Consistent with recent reports on the allosteric regulation of AC by lipids, this finding suggests that the TM domain could serve as a potential drug target.<!--> <!-->These structural findings enhance our understanding of the structure and function of AC9 and other ACs and will provide a foundation for future AC-target drug discovery.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108223"},"PeriodicalIF":3.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144196126","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}
We present the Volume Segmentation Tool (VST), a deep learning software tool that implements volumetric image segmentation in volume electron microscopy image stack data from a wide range of biological sample types. VST automates the handling of data preprocessing, data augmentation, and network building, as well as the configuration for model training, while adapting to the specific dataset. We have tried to make VST more accessible by designing it to operate entirely on local hardware and have provided a browser-based interface with additional features for visualizations of the networks and augmented datasets. VST can utilise contour map prediction to support instance segmentation on top of semantic segmentation. Through examples from various resin-embedded sample derived transmission electron microscopy and scanning electron microscopy datasets, we demonstrate that VST achieves state of the art performance compared to existing approaches.
{"title":"A generalist deep-learning volume segmentation tool for volume electron microscopy of biological samples","authors":"Yuyao Huang , Nickhil Jadav , Georgia Rutter , Lech Szymanski , Mihnea Bostina , Duane P. Harland","doi":"10.1016/j.jsb.2025.108214","DOIUrl":"10.1016/j.jsb.2025.108214","url":null,"abstract":"<div><div>We present the Volume Segmentation Tool (VST), a deep learning software tool that implements volumetric image segmentation in volume electron microscopy image stack data from a wide range of biological sample types. VST automates the handling of data preprocessing, data augmentation, and network building, as well as the configuration for model training, while adapting to the specific dataset. We have tried to make VST more accessible by designing it to operate entirely on local hardware and have provided a browser-based interface with additional features for visualizations of the networks and augmented datasets. VST can utilise contour map prediction to support instance segmentation on top of semantic segmentation. Through examples from various resin-embedded sample derived transmission electron microscopy and scanning electron microscopy datasets, we demonstrate that VST achieves state of the art performance compared to existing approaches.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108214"},"PeriodicalIF":3.0,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191912","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 : 2025-05-28DOI: 10.1016/j.jsb.2025.108215
Pauline Roth , Utz H. Ermel , Deborah Moser , Gunnar Arctaedius , Maren Wehrheim , Margot P. Scheffer , Achilleas S. Frangakis
Biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectrum of applications. Recently, we developed a plugin called ArtiaX which extended the capabilities of UCSF ChimeraX to address the specific demands of cryo-electron tomography. Here, we introduce the evolution of ArtiaX, that can now generate models to facilitate particle selection, define camera recording paths, and execute particle selection routines. Diverse models can be generated and populated with putative particle positions and orientations. In addition, models can be used to drive the camera position, thereby simplifying the process of movie creation. The plugin incorporates fundamental image filtering options for the on-the-fly analysis of tomographic data and provides compatibility of particle lists with RELION-5 .star files. Collectively, this update of ArtiaX comprehensively encompasses essential tools for the analysis and visualization of electron tomograms. It retains its hallmark attributes of speed, reliability, and user-friendliness, fostering seamless human–machine interaction.
{"title":"ArtiaX: geometric models, camera paths and image processing tools","authors":"Pauline Roth , Utz H. Ermel , Deborah Moser , Gunnar Arctaedius , Maren Wehrheim , Margot P. Scheffer , Achilleas S. Frangakis","doi":"10.1016/j.jsb.2025.108215","DOIUrl":"10.1016/j.jsb.2025.108215","url":null,"abstract":"<div><div>Biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectrum of applications. Recently, we developed a plugin called ArtiaX which extended the capabilities of UCSF ChimeraX to address the specific demands of cryo-electron tomography. Here, we introduce the evolution of ArtiaX, that can now generate models to facilitate particle selection, define camera recording paths, and execute particle selection routines. Diverse models can be generated and populated with putative particle positions and orientations. In addition, models can be used to drive the camera position, thereby simplifying the process of movie creation. The plugin incorporates fundamental image filtering options for the on-the-fly analysis of tomographic data and provides compatibility of particle lists with RELION-5 .star files. Collectively, this update of ArtiaX comprehensively encompasses essential tools for the analysis and visualization of electron tomograms. It retains its hallmark attributes of speed, reliability, and user-friendliness, fostering seamless human–machine interaction.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 3","pages":"Article 108215"},"PeriodicalIF":3.0,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187251","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}
Xeg5A from Aspergillus oryzae belongs to glycoside hydrolase family 5 subfamily 4. This enzyme has been characterized as a xyloglucan-specific endo-β-1,4-glucanase (xyloglucanase) that cleaves the main chain of xyloglucan at both unbranched and xylosylated glucosyl residues in an endo-processive mode of action. X-ray crystallography revealed that Xeg5A is a tri-modular enzyme composed of a catalytic, an Ig-like, and a C-terminal CBM46-like domains. Xeg5A structures complexed with branched xyloglucan oligosaccharides at subsites –4 to +4 showed that the recognition of xyloglucan side-chain moieties is important for Xeg5A activity. The crystal structure also provided structural insights into the role of the CBM46-like domain in contributing to regiospecificity and, possibly, processivity.
{"title":"Structural insights into substrate recognition of tri-modular xyloglucanase from Aspergillus oryzae","authors":"Yusuke Nakamichi , Naoki Shimada , Masahiro Watanabe , Tatsuya Fujii , Katsuro Yaoi , Tomohiko Matsuzawa","doi":"10.1016/j.jsb.2025.108213","DOIUrl":"10.1016/j.jsb.2025.108213","url":null,"abstract":"<div><div>Xeg5A from <em>Aspergillus oryzae</em> belongs to glycoside hydrolase family 5 subfamily 4. This enzyme has been characterized as a xyloglucan-specific <em>endo</em>-β-1,4-glucanase (xyloglucanase) that cleaves the main chain of xyloglucan at both unbranched and xylosylated glucosyl residues in an <em>endo</em>-processive mode of action. X-ray crystallography revealed that Xeg5A is a tri-modular enzyme composed of a catalytic, an Ig-like, and a C-terminal CBM46-like domains. Xeg5A structures complexed with branched xyloglucan oligosaccharides at subsites –4 to +4 showed that the recognition of xyloglucan side-chain moieties is important for Xeg5A activity. The crystal structure also provided structural insights into the role of the CBM46-like domain in contributing to regiospecificity and, possibly, processivity.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108213"},"PeriodicalIF":3.0,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142858","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 : 2025-05-20DOI: 10.1016/j.jsb.2025.108211
Md Samsuddin Ansari , Muhammad Zohib , Meera Kumari , Vikash Yadav , Ravi Kant Pal , Sarita Tripathi , Anupam Jain , Bichitra Kumar Biswal , Ashish Arora
The members of the PadR family of transcriptional regulators are important for cell survival in toxic environments and play an important role in detoxification, pathogenicity, and multi-drug resistance. Rv0047c of Mycobacterium tuberculosis H37Rv is annotated as a PadR family protein. We have characterized the stability and structure of Rv0047c. Rv0047c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 55.3 °C. The crystal structure of Rv0047c was determined at a resolution of 3.15 Å. The structure indicates the biological unit to be a dimer with each monomer having a characteristic N-terminal winged-helix-turn-helix DNA binding domain and a C-terminal dimerization domain. The N-terminal domain is composed of four helices, α1, α2, α3, and α4 and two beta strands β1 and β2. The C-terminal dimerization domain (CTD) consists two long helices α6 and α7. The two domains are connected by helix α5. A short helical turn (helix αa, residue 89–92), leads to compaction of the α4-α5 loop. Rv0047c exhibits specificity in binding to an upstream region having an inverted repeat sequence. This binding is dependent upon Y18 and Y40 residue of Rv0047c, which are highly conserved among the PadR family. Overall, our results suggest a transcription regulatory role for Rv0047c, similar to other PadR family proteins.
{"title":"Structural and biophysical characterization of PadR family protein Rv0047c of Mycobacterium tuberculosis H37Rv","authors":"Md Samsuddin Ansari , Muhammad Zohib , Meera Kumari , Vikash Yadav , Ravi Kant Pal , Sarita Tripathi , Anupam Jain , Bichitra Kumar Biswal , Ashish Arora","doi":"10.1016/j.jsb.2025.108211","DOIUrl":"10.1016/j.jsb.2025.108211","url":null,"abstract":"<div><div>The members of the PadR family of transcriptional regulators are important for cell survival in toxic environments and play an important role in detoxification, pathogenicity, and multi-drug resistance. Rv0047c of <em>Mycobacterium tuberculosis</em> H37Rv is annotated as a PadR family protein. We have characterized the stability and structure of Rv0047c. Rv0047c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 55.3 °C. The crystal structure of Rv0047c was determined at a resolution of 3.15 Å. The structure indicates the biological unit to be a dimer with each monomer having a characteristic N-terminal winged-helix-turn-helix DNA binding domain and a C-terminal dimerization domain. The N-terminal domain is composed of four helices, α1, α2, α3, and α4 and two beta strands β1 and β2. The C-terminal dimerization domain (CTD) consists two long helices α6 and α7. The two domains are connected by helix α5. A short helical turn (helix αa, residue 89–92), leads to compaction of the α4-α5 loop. Rv0047c exhibits specificity in binding to an upstream region having an inverted repeat sequence. This binding is dependent upon Y18 and Y40 residue of Rv0047c, which are highly conserved among the PadR family. Overall, our results suggest a transcription regulatory role for Rv0047c, similar to other PadR family proteins.</div></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"217 2","pages":"Article 108211"},"PeriodicalIF":3.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144127989","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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