Journal of Structural Biology: X最新文献

{"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":"2025-05-24","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":"Crystal structure of the CCA-adding enzyme from Arabidopsis thaliana","authors":"Xiao Wang,&nbsp;Yuan-Yuan Li,&nbsp;Zi-Yan Dou,&nbsp;Jia Wang,&nbsp;Lin Liu","doi":"10.1016/j.yjsbx.2025.100127","DOIUrl":"10.1016/j.yjsbx.2025.100127","url":null,"abstract":"<div><div>The 3′-terminal CCA-end of tRNA is essential for the attachment of amino acids and correct positioning of the aminoacyl-tRNA in the ribosome. In higher plants, the CCA sequence is synthesized, maintained, and repaired by class-II CCA-adding enzymes encoded by a single nuclear gene but multi-targeted to the nucleus, cytoplasm, plastids, and mitochondria. The structure of plant class-II CCA-adding enzyme remains unsolved. Here we describe the crystal structure of CCA-adding enzyme from <em>Arabidopsis thaliana</em> (<em>At</em>CCA)<em>.</em> The overall structure of <em>At</em>CCA is similar to other class-II CCA-adding enzymes<em>,</em> but significant differences occur in the body domain. Structural comparison of body and tail domains between <em>At</em>CCA and other class-II CCA-adding enzymes unravels three specific regions of <em>At</em>CCA. Based on the modeled <em>At</em>CCA-tRNA complex, <em>At</em>CCA may have a different tRNA binding pattern. The three specific regions located in the body domain of <em>At</em>CCA also provide candidate regions for multi-targeted sorting.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100127"},"PeriodicalIF":3.5,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144168679","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":"2025-05-02","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":"2025-04-29","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":"Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation","authors":"Patrick Roth,&nbsp;Dimitrios Fotiadis","doi":"10.1016/j.yjsbx.2025.100124","DOIUrl":"10.1016/j.yjsbx.2025.100124","url":null,"abstract":"<div><div>The phosphotransferase system glucose-specific transporter IICB^Glc serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC^Glc domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB^Glc domain. Furthermore, IIC^Glc consists of a transport (TD) and a scaffold domain, with the latter being involved in dimer formation. Transport is mediated by an elevator-type mechanism within the IIC^Glc domain, where the substrate binds to the mobile TD. This domain undergoes a large-scale rigid-body movement relative to the static scaffold domain, translocating glucose across the membrane. Structures of elevator-type transporters are typically captured in either inward- or outward-facing conformations. Intermediate states remain elusive, awaiting structural determination and mechanistic interpretation. Here, we present a single-particle cryo-EM structure of purified, <em>n</em>-dodecyl-β-D-maltopyranoside-solubilized IICB^Glc from <em>Escherichia coli</em>. While the IIB^Glc protein domain is flexible remaining unresolved, the dimeric IIC^Glc transporter is found trapped in a hitherto unobserved intermediate conformational state. Specifically, the TD is located halfway between inward- and outward-facing states. Structural analysis revealed a specific <em>n</em>-dodecyl-β-D-maltopyranoside molecule bound to the glucose binding site. The sliding of the TD is potentially impeded halfway due to the bulky nature of the ligand and a shift of the thin gate, thereby stalling the transporter. In conclusion, this study presents a novel conformational state of IIC^Glc, and provides new structural and mechanistic insights into a potential stalling mechanism, paving the way for the rational design of transport inhibitors targeting this critical bacterial metabolic process.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100124"},"PeriodicalIF":3.5,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143580418","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":"Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct","authors":"Suruchi Singh ,&nbsp;Yi Liu ,&nbsp;Meghan Burke ,&nbsp;Vamseedhar Rayaprolu ,&nbsp;Stephen E. Stein ,&nbsp;S. Saif Hasan","doi":"10.1016/j.yjsbx.2025.100123","DOIUrl":"10.1016/j.yjsbx.2025.100123","url":null,"abstract":"<div><div>The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on <em>N</em>-glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100123"},"PeriodicalIF":3.5,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143437729","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":"Molecular biophysics and inhibition mechanism of influenza virus A M2 viroporin by adamantane-based drugs – Challenges in designing antiviral agents","authors":"Kyriakos Georgiou ,&nbsp;Dimitrios Kolokouris ,&nbsp;Antonios Kolocouris","doi":"10.1016/j.yjsbx.2025.100122","DOIUrl":"10.1016/j.yjsbx.2025.100122","url":null,"abstract":"<div><div>The influenza A matrix 2 (AM2) protein is a prototype viroporin that conducts protons through an array of water molecules and sidechains of ionizable amino acid residues, with His37 being the most important. Amantadine is a prototype AM2 channel blocker and inhibitor of influenza A AM2 wild type (serine-31) replication. Amantadine received approval for prophylaxis against the influenza virus A in 1966. However, the characterization of the mechanism of action of amantadine targeting AM2 came 50 years after its approval as an anti-influenza A drug. We present results from experimental biophysical methods and molecular dynamics simulations for the complexes of the AM2 WT and amantadine-resistant mutant channels (V27A, L26F, S31N) in complex with adamantane-based ligands. Additionally, we describe critical experimental evidence from biochemical/functional and molecular biology experiments. Previous debates on the mechanism of drug binding and inhibition were due to the different membrane mimetic environment, the excess of the drug, and the method used<strong>,</strong> rather than the accuracy of the experiments. The collective knowledge acquired can inspire research for the development of new antivirals against influenza viruses and provide experience on the application of molecular biophysics to other viroporins.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100122"},"PeriodicalIF":3.5,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143445911","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":"Non-uniform Fourier transform based image classification in single-particle Cryo-EM","authors":"ZiJian Bai,&nbsp;Jian Huang","doi":"10.1016/j.yjsbx.2025.100121","DOIUrl":"10.1016/j.yjsbx.2025.100121","url":null,"abstract":"<div><div>In the single-particle Cryo-EM projection image classification, it is a common practice to apply the Fourier transform to the images and extract rotation-invariant features in the frequency domain. However, this process involves interpolation, which can reduce the accuracy of the results. In contrast, the non-uniform Fourier transform provides more direct and accurate computation of rotation-invariant features without the need for interpolation in the computation process. Leveraging the capabilities of the non-uniform discrete Fourier transform (NUDFT), we have developed an algorithm for the rotation-invariant classification. To highlight its potential and applicability in the field of single-particle Cryo-EM, we conducted a direct comparison with the traditional Fourier transform and other methods, demonstrating the superior performance of the NUDFT.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100121"},"PeriodicalIF":3.5,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143378637","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":"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":"2025-01-21","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":"SidF, a dual substrate N5-acetyl-N5-hydroxy-L-ornithine transacetylase involved in Aspergillus fumigatus siderophore biosynthesis","authors":"Thanalai Poonsiri ,&nbsp;Jan Stransky ,&nbsp;Nicola Demitri ,&nbsp;Hubertus Haas ,&nbsp;Michele Cianci ,&nbsp;Stefano Benini","doi":"10.1016/j.yjsbx.2024.100119","DOIUrl":"10.1016/j.yjsbx.2024.100119","url":null,"abstract":"<div><div>Siderophore-mediated iron acquisition is essential for the virulence of <em>Aspergillus fumigatus</em>, a fungus causing life-threatening aspergillosis. Drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in <em>A. fumigatus</em>. <em>A. fumigatus</em> has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants.</div><div>We present the first X-ray structure of SidF, revealing a two-domain architecture with tetrameric assembly. The N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrates structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor.</div><div>This study elucidates the structure of SidF and reveals its role in siderophore biosynthesis. We propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in <em>A. fumigatus</em> siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100119"},"PeriodicalIF":3.5,"publicationDate":"2024-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751504/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023943","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}