Journal of Structural Biology: X最新文献

{"title":"Snake venom defensins: Defining the structural and functional characteristics of the toxin family","authors":"David Melendez-Martinez ,&nbsp;Adriana Morales-Martinez ,&nbsp;Iliana Vanessa Almanza-Campos ,&nbsp;Francisco Sierra-Valdez ,&nbsp;Miguel Borja ,&nbsp;Alejandro Carbajal-Saucedo ,&nbsp;Christopher L. Parkinson ,&nbsp;Jorge Benavides","doi":"10.1016/j.yjsbx.2025.100129","DOIUrl":"10.1016/j.yjsbx.2025.100129","url":null,"abstract":"<div><div>Snake venom defensins are a toxin family found in rattlesnake venoms (<em>Crotalus</em>) which are comprised of crotamine-like peptides and myotoxins. Their tertiary structure resembles the β-defensin family structure. Toxins from this family, such as crotamine (<em>C. durissus terrificus</em>) and myotoxin a (<em>C. viridis viridis</em>), have been described to generate paralysis through K_v 1.3 channel blockade, using three functional basic-hydrophobic dyads (Y-K, R-W, and R-W). However, the structural and functional properties of other snake venom defensins are scarcely described. For that reason, we evaluated the structural–functional characteristics of the rattlesnake venom defensins on the K_v 1.3 channel through <em>in silico</em> analysis. 38 snake venom defensins were found to be peptides from 41 to 48 residues with a highly conserved sequence. The three-dimensional structures had great similitude (RMSD, &lt;1.1 Å). Moreover, molecular dynamics simulations showed that the structures were stable (0.445 ± 0.23 nm). It was found that the snake venom defensins contain two or three basic-hydrophobic dyads, the first one is present in the N-terminal region of the defensin comprised by YK. The dyads two and three are contiguous, forming a motif in the γ-core, of which there are seven phenotypes: RWKW, RWRW, PWRR, PWKR, RWKR, RLGW, and GWRR. These dyads played a key role in the interaction of the defensins with the pore residues of the K_v1.3 channel. These results demonstrated that snake venom defensins have common structural and functional properties, interacting with the K_v 1.3 channel through the basic-hydrophobic dyads.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100129"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"pytom-match-pick: A tophat-transform constraint for automated classification in template matching","authors":"Marten L. Chaillet ,&nbsp;Sander Roet ,&nbsp;Remco C. Veltkamp ,&nbsp;Friedrich Förster","doi":"10.1016/j.yjsbx.2025.100125","DOIUrl":"10.1016/j.yjsbx.2025.100125","url":null,"abstract":"<div><div>Template matching (TM) in cryo-electron tomography (cryo-ET) enables <em>in situ</em> detection and localization of known macromolecules. However, TM faces challenges of weak signal of the macromolecules and interfering features with a high signal-to-noise ratio, which are often addressed by time-consuming, subjective manual curation of results. To improve the detection performance we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100125"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143922897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example","authors":"Penghui Deng ,&nbsp;Xiaoyue Zhang ,&nbsp;Jianqing Wen ,&nbsp;Mingce Xu ,&nbsp;Pengwei Li ,&nbsp;Hao Wang ,&nbsp;Yunchen Bi","doi":"10.1016/j.yjsbx.2025.100126","DOIUrl":"10.1016/j.yjsbx.2025.100126","url":null,"abstract":"<div><div>In cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is naturally devoid of light chains. The variable regions of IgNAR (VNARs) are solely responsible for antigen recognition, similar to VHHs (variable domain of the heavy chain of heavy-chain antibodies) in camelids. Although VNARs have attracted growing interest, generating VNARs against membrane proteins remains challenging. Furthermore, the structure of a VNAR in complex with a membrane protein has not yet been reported. This study features a membrane protein, Chlorella virus hyaluronan synthase (CvHAS), and provides a comprehensive methodological approach to generate its specific shark VNARs, addressing several major concerns and important optimizations. We showed that shark physiological urea pressure was tolerable for CvHAS, and indirect immobilization was strongly preferred over passive adsorption for membrane proteins. Together with optimizations to improve mononuclear cell (MC) viability and VNAR expression efficiency, we successfully generated S2F6, a CvHAS-specific VNAR with nM-level high affinity. The structure of the CvHAS-S2F6 complex was then determined by cryogenic electron microscopy (cryo-EM), reporting the first membrane protein and VNAR complex structure. It shows that S2F6 binds to the cytoplasmic domain of CvHAS, with a different epitope than the reported CvHAS-specific VHHs. This study provides valuable insights into developing VNARs for membrane proteins and their applications in structural biology.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100126"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143918313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tracing the aggregation pathway of the scaffold protein DISC1: Structural implications for chronic mental illnesses","authors":"Abhishek Cukkemane ,&nbsp;Nina Becker ,&nbsp;Tatsiana Kupreichyk ,&nbsp;Henrike Heise ,&nbsp;Dieter Willbold ,&nbsp;Oliver H. Weiergräber","doi":"10.1016/j.yjsbx.2025.100128","DOIUrl":"10.1016/j.yjsbx.2025.100128","url":null,"abstract":"<div><div>Disrupted in schizophrenia 1 (DISC1) is a pleiotropic scaffold protein that is postulated to comprise large disordered regions and four distinct structured segments with a high proportion of helical or coiled-coil fold. DISC1 associates with over 300 proteins and is associated with several physiological roles ranging from mitosis to cellular differentiation. Yet, the structural features of the protein are poorly characterized. The C-terminal region (C-region, res. 691–836) forms a tetramer and can also aggregate into amyloid-like fibers, potentially linked to schizophrenia and other chronic mental illnesses. Using a combination of biophysical and structural biology applications, we investigate the structural heterogeneity of three mutants of the C-region, viz., the S713E, S704C and L807-frameshift mutants. We provide evidence for the plasticity of the C region; a thin border separates the conformational flexibility of DISC1 required for interaction with a myriad of partners from disruptive aggregation. Snapshots of aggregates and fibrils growing from a nucleus are presented, along with data supporting the role of the minimal fibrillizing element in the C-region, the β-core. This segment also houses a stretch of residues that is critical for the binding of NDEL1 proteins in the mitotic spindle complex and is absent in the non-binding splice variant DISC1Δ22aa. Physiologically, both the splice variant and the fibers represent loss-of-function states that disrupt cellular division. Our findings highlight the need to decipher the structural elements within the DISC1 C-region to comprehend its physiological role and aggregation-related anomalies, and to establish a rationale for drug development.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100128"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144137732","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein identification using Cryo-EM and artificial intelligence guides improved sample purification","authors":"Kenneth D. Carr ,&nbsp;Dane Evan D. Zambrano ,&nbsp;Connor Weidle ,&nbsp;Alex Goodson ,&nbsp;Helen E. Eisenach ,&nbsp;Harley Pyles ,&nbsp;Alexis Courbet ,&nbsp;Neil P. King ,&nbsp;Andrew J. Borst","doi":"10.1016/j.yjsbx.2025.100120","DOIUrl":"10.1016/j.yjsbx.2025.100120","url":null,"abstract":"<div><div>Protein purification is essential in protein biochemistry, structural biology, and protein design, enabling the determination of protein structures, the study of biological mechanisms, and the characterization of both natural and de novo designed proteins. However, standard purification strategies often encounter challenges, such as unintended co-purification of contaminants alongside the target protein. This issue is particularly problematic for self-assembling protein nanomaterials, where unexpected geometries may reflect novel assembly states, cross-contamination, or native proteins originating from the expression host. Here, we used an automated structure-to-sequence pipeline to first identify an unknown co-purifying protein found in several purified designed protein samples. By integrating cryo-electron microscopy (Cryo-EM), ModelAngelo’s sequence-agnostic model-building, and Protein BLAST, we identified the contaminant as dihydrolipoamide succinyltransferase (DLST). This identification was validated through comparisons with DLST structures in the Protein Data Bank, AlphaFold 3 predictions based on the DLST sequence from our E. coli expression vector, and traditional biochemical methods. The identification informed subsequent modifications to our purification protocol, which successfully excluded DLST from future preparations. To explore the potential broader utility of this approach, we benchmarked four computational methods for DLST identification across varying resolution ranges. This study demonstrates the successful application of a structure-to-sequence protein identification workflow, integrating Cryo-EM, ModelAngelo, Protein BLAST, and AlphaFold 3 predictions, to identify and ultimately help guide the<!--> <!-->removal of DLST from sample purification efforts. It highlights the potential of combining Cryo-EM with AI-driven tools for accurate protein identification and addressing purification challenges across diverse contexts in protein science.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100120"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143130613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis","authors":"Craig A. McElroy ,&nbsp;Elihu C. Ihms ,&nbsp;Deepak Kumar Yadav ,&nbsp;Melody L. Holmquist ,&nbsp;Vibhuti Wadhwa ,&nbsp;Vicki H. Wysocki ,&nbsp;Paul Gollnick ,&nbsp;Mark P. Foster","doi":"10.1016/j.yjsbx.2024.100103","DOIUrl":"https://doi.org/10.1016/j.yjsbx.2024.100103","url":null,"abstract":"<div><p>Cellular production of tryptophan is metabolically expensive and tightly regulated. The small <em>Bacillus subtilis</em> zinc binding Anti-TRAP protein (AT), which is the product of the <em>yczA/rtpA</em> gene, is upregulated in response to accumulating levels of uncharged tRNA^Trp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (<em>trp</em> RNA Binding Attenuation Protein), thereby preventing it from binding to the <em>trp</em> leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the <em>trp</em> operon. AT principally adopts two symmetric oligomeric states, a trimer (AT₃) featuring three-fold axial symmetry or a dodecamer (AT₁₂) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT₃, while heteronuclear ¹⁵N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT₃ and binding-inactive AT₁₂, with implications for TRAP binding and inhibition.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"10 ","pages":"Article 100103"},"PeriodicalIF":3.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590152424000084/pdfft?md5=571b0da5dc58532b76dc49e84cbcb4d5&pid=1-s2.0-S2590152424000084-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141486538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural analysis of the stable form of fibroblast growth factor 2 – FGF2-STAB","authors":"Gabin de La Bourdonnaye ,&nbsp;Martin Marek ,&nbsp;Tereza Ghazalova ,&nbsp;Jiri Damborsky ,&nbsp;Petr Pachl ,&nbsp;Jiri Brynda ,&nbsp;Veronika Stepankova ,&nbsp;Radka Chaloupkova","doi":"10.1016/j.yjsbx.2024.100112","DOIUrl":"10.1016/j.yjsbx.2024.100112","url":null,"abstract":"<div><div>Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"10 ","pages":"Article 100112"},"PeriodicalIF":3.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142531009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MicroED structure of the C11 cysteine protease clostripain","authors":"Yasmeen N. Ruma ,&nbsp;Guanhong Bu ,&nbsp;Johan Hattne ,&nbsp;Tamir Gonen","doi":"10.1016/j.yjsbx.2024.100107","DOIUrl":"https://doi.org/10.1016/j.yjsbx.2024.100107","url":null,"abstract":"<div><p>Clostripain secreted from <em>Clostridium histolyticum</em> is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of <em>C. histolyticum</em> clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"10 ","pages":"Article 100107"},"PeriodicalIF":3.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590152424000126/pdfft?md5=42cb6b31866b2698d485367c031389f5&pid=1-s2.0-S2590152424000126-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141594445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography” [J. Struct. Biol.: X 10(2024) 100113]","authors":"Chia-Chun Hsieh,&nbsp;Zi-Jing Lin,&nbsp;Lee-Jene Lai","doi":"10.1016/j.yjsbx.2024.100115","DOIUrl":"10.1016/j.yjsbx.2024.100115","url":null,"abstract":"","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"10 ","pages":"Article 100115"},"PeriodicalIF":3.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142554799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of submicron bone tissue composition in plastic-embedded samples using optical photothermal infrared (O-PTIR) spectral imaging and machine learning","authors":"Isha Dev ,&nbsp;Sofia Mehmood ,&nbsp;Nancy Pleshko ,&nbsp;Iyad Obeid ,&nbsp;William Querido","doi":"10.1016/j.yjsbx.2024.100111","DOIUrl":"10.1016/j.yjsbx.2024.100111","url":null,"abstract":"<div><div>Understanding the composition of bone tissue at the submicron level is crucial to elucidate factors contributing to bone disease and fragility. Here, we introduce a novel approach utilizing optical photothermal infrared (O-PTIR) spectroscopy and imaging coupled with machine learning analysis to assess bone tissue composition at 500 nm spatial resolution. This approach was used to evaluate thick bone samples embedded in typical poly(methyl methacrylate) (PMMA) blocks, eliminating the need for cumbersome thin sectioning. We demonstrate the utility of O-PTIR imaging to assess the distribution of bone tissue mineral and protein, as well as to explore the structure-composition relationship surrounding microporosity at a spatial resolution unattainable by conventional infrared imaging modalities. Using bone samples from wildtype (WT) mice and from a mouse model of osteogenesis imperfecta (OIM), we further showcase the application of O-PTIR spectroscopy to quantify mineral content, crystallinity, and carbonate content in spatially defined regions across the cortical bone. Notably, we show that machine learning analysis using support vector machine (SVM) was successful in identifying bone phenotypes (typical in WT, fragile in OIM) based on input of spectral data, with over 86 % of samples correctly identified when using the collagen spectral range. Our findings highlight the potential of O-PTIR spectroscopy and imaging as valuable tools for exploring bone submicron composition.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"10 ","pages":"Article 100111"},"PeriodicalIF":3.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142530909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}