Acta Crystallographica. Section D, Structural Biology最新文献

{"title":"LowDoseWizard: rapid and standardized setup of low-dose cryo-TEM imaging in SerialEM.","authors":"Simon A Fromm, Simone Mattei","doi":"10.1107/S2059798326006327","DOIUrl":"10.1107/S2059798326006327","url":null,"abstract":"<p><p>Structure elucidation of biological macromolecules by single-particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardized setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image-shift settings for all low-dose states, and guides the user through key daily alignment procedures, including beam-shift offset calibration, objective-lens astigmatism correction and coma-free alignment. The workflow is organized into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 min. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 and 1.09 Å resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution SPA cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"990-997"},"PeriodicalIF":4.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13431637/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148597319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PETIMOT: a novel framework for inferring protein motions from sparse data using SE(3)-equivariant graph neural networks.","authors":"Valentin Lombard, Julien Nguyen Van, Sergei Grudinin, Elodie Laine","doi":"10.1107/S2059798326006054","DOIUrl":"10.1107/S2059798326006054","url":null,"abstract":"<p><p>Proteins move and deform to ensure their biological functions. Despite significant progress in protein structure prediction, approximating conformational ensembles under physiological conditions remains a fundamental open problem. This paper presents a novel perspective on the problem by directly targeting continuous compact representations of protein motions inferred from sparse experimental observations. We develop a task-specific loss function enforcing data symmetries, including scaling and permutation operations. Our method PETIMOT (Protein sEquence and sTructure-based Inference of MOTions) leverages transfer learning from pre-trained protein language models through an SE(3)-equivariant graph neural network. When trained and evaluated on the Protein Data Bank, PETIMOT shows superior performance in time and accuracy, capturing protein dynamics, particularly large/slow conformational changes, compared with state-of-the-art diffusion and flow-matching approaches, as well as traditional physics-based models. Our code and protocols are available at https://github.com/PhyloSofS-Team/PETIMOT.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"862-885"},"PeriodicalIF":4.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13431647/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148519826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An unattended image-processing pipeline for on-the-fly quality assessment and 3D exploration in cryo-EM.","authors":"Daniel Marchán Torres, Pablo Conesa, Alberto Garcia, Mikel Iceta, Ludovic Broche, Marcos Gragera, Romain Linares, Hok Sau Kwong, Francisco Javier Chichón, Olof Svensson, Rocío Arranz, Grégory Effantin, Eaazhisai Kandiah, José María Carazo, Carlos O S Sorzano","doi":"10.1107/S205979832600656X","DOIUrl":"10.1107/S205979832600656X","url":null,"abstract":"<p><p>Single-particle analysis (SPA) by cryogenic electron microscopy (cryo-EM) has become a cornerstone of structural biology; yet, the workflow from data acquisition to a 3D structure remains a time-consuming, manual and significant task. Researchers often invest days of valuable microscope time collecting massive datasets with little to no real-time feedback on sample quality or the ultimate feasibility of achieving a high-resolution reconstruction. To address this challenge, we have developed an automated, end-to-end processing workflow designed for on-the-fly analysis. Built within the Scipion framework, the pipeline integrates a cascade of automated quality-control filters, a novel consensus-based strategy for training a data-specific particle-picking model, and a parallel 2D/3D validation scheme to ensure robust processing outcomes. We validated the workflow on a diverse benchmark of 32 datasets (CryoPPP), achieving a 94% overall processing success rate and obtaining high-quality 3D reconstructions in 78% of cases. Real-world deployment at the European Synchrotron (ESRF) cryo-EM facility further validated its practical utility: the pipeline demonstrated processing speeds exceeding data acquisition, delivering preliminary 3D maps in under 3 h. Approximately 70% of user experiments converged to interpretable structures, with half achieving 3-4 Å resolution, despite the inherent complexity of facility-collected samples. By providing researchers with rapid, actionable feedback and identifying both promising and problematic datasets in real time, this workflow transforms cryo-EM data collection from a passive process into an active, data-driven experiment. It serves as a powerful diagnostic and decision-support tool, accelerating structural determination and optimizing microscope time in high-throughput cryo-EM environments.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"915-939"},"PeriodicalIF":4.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13431645/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148597327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural basis for the fast maturation of pcStar, a photoconvertible fluorescent protein. Erratum.","authors":"Shuping Zheng, Xiangrui Shi, Junjin Lin, Yiwei Yang, Yiting Xin, Xinru Bai, Huachen Zhu, Hui Chen, Jiasen Wu, Xiaowei Zheng, Ling Lin, Zhihong Huang, Sheng Yang, Fen Hu, Wei Liu","doi":"10.1107/S205979832600728X","DOIUrl":"10.1107/S205979832600728X","url":null,"abstract":"<p><p>Several corrections are made to the article by Zheng et al. [(2025), Acta Cryst. D81, 181-195].</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"1010-1011"},"PeriodicalIF":4.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148576612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dose-dependent structural and electron-density features in the lytic polysaccharide monooxygenase NcAA9D.","authors":"Samuel A Miller, William B O'Dell, Flora Meilleur","doi":"10.1107/S205979832600639X","DOIUrl":"10.1107/S205979832600639X","url":null,"abstract":"<p><p>Structural studies of copper-containing lytic polysaccharide monooxygenases (LPMOs) by X-ray crystallography are often complicated by radiation damage. In this study, we analyze a series of 36 X-ray crystal structures of NcAA9D, a Neurospora crassa AA9-family LPMO, determined from data collected at cryogenic temperature from a single crystal to investigate the progressive effects of radiation damage at the active site of this enzyme. We report new insights into the dose-dependence of active-site geometry in LPMOs and utilize the unique pre-bound dioxygen site of NcAA9D to analyze the impact of X-ray dose on the electron density of this species. It is well established that photoreduction of the LPMO active-site copper(II) leads to expulsion of its water ligands. We further characterize this displacement and the corresponding electron-density smearing, a phenomenon that can lead to the erroneous modeling of copper-bound dioxygen species. These findings suggest that radiation-dose series collected from a single crystal provide invaluable data to support unambiguous assignment of radiation-sensitive intermediates at the active site of LPMOs and other radiation-sensitive redox enzymes.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"900-914"},"PeriodicalIF":4.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13431641/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148597350","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical and structural characterization of a tail-spike protein with depolymerase activity identified in a marine podovirus.","authors":"Serena Sirigu, Thomas Roret, Pierre Yves Mocaër, Robert Larocque, Diane Jouanneau, Pierre Legrand, Anne Claire Baudoux, Mirjam Czjzek","doi":"10.1107/S2059798326005425","DOIUrl":"10.1107/S2059798326005425","url":null,"abstract":"<p><p>Marine phages are, through the infection of their bacterial hosts, key regulators of microbiome and carbon fluxes in the ocean. Despite their important role, the specific molecular mechanisms that underlie infection are so far understudied. Previously, the podovirus Cobetia marina virus 1 (Carin-1), which infects the marine γ-proteobacterium C. marina, was shown to display exopolysaccharide depolymerase activity. This activity is likely to mediate degradation of the host capsule to facilitate access to the bacterial membrane receptor, but no corresponding gene could be annotated in the genome of Carin-1 by comparative genomics. Biochemical characterization enabled assignment of this activity to Dpo31, a protein sharing less than 10% sequence identity with any characterized protein. Here, we report the structural domain organization and biochemical characterization of Dpo31, revealing an overall structure that is analogous to podovirus tail-spike proteins, allowing us to locate the depolymerase activity to the D3 domain and to identify original structural features that explain the absence of detectable similarity at the primary-sequence level.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"785-799"},"PeriodicalIF":4.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317679/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148269950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular architecture of the human citrate synthase-malate dehydrogenase 2 metabolon.","authors":"Angela J Kayll, Umanga Rupakheti, Renee St John, Gabriel Rementeria, Joseph J Provost, Christopher E Berndsen","doi":"10.1107/S2059798326005802","DOIUrl":"10.1107/S2059798326005802","url":null,"abstract":"<p><p>Metabolons are transient biomolecular complexes that enhance the efficiency of metabolic pathways through substrate channeling. These complexes are difficult to study because of their transient nature, thus limiting our understanding of how they are formed and regulated. The citric acid cycle is proposed to contain many such complexes, although few have been characterized structurally. Here, we provide direct structural evidence for the complex of human citrate synthase (hCS) and human mitochondrial malate dehydrogenase 2 (hMDH2), which is part of the larger proposed citric acid cycle metabolon. Our structural model supports previous cross-linking studies and suggests that hMDH2 can interact with each subunit of the hCS dimer, forming up to a hexameric complex. However, this complex appears to be transient, as titration of hMDH2 into hCS in activity assays does not saturate. We further show that the interaction site with hCS is nonspecific, as hCS could also stimulate oxaloacetate formation by cytosolic and plant MDH enzymes. This structural model will provide a basis for understanding the structure and regulation of the broader citric acid cycle metabolon.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"740-750"},"PeriodicalIF":4.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317680/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148293140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural basis of regioselective double halogenation of the β-carboline tryptoline by the single-component halogenase AetF.","authors":"Simon Bork, Hendrik J Horstmeier, Bjarne Scharkowski, Hartmut H Niemann","doi":"10.1107/S2059798326005954","DOIUrl":"10.1107/S2059798326005954","url":null,"abstract":"<p><p>Flavin-dependent halogenases (FDHs) offer a biocatalytic route to regioselective halogenation of aromatic substrates under mild conditions. AetF, a rare self-sufficient single-component FDH, catalyzes sequential dibromination of tryptophan and has a broad substrate scope. Here, we report the crystal structure of AetF in complex with the non-native substrate tryptoline, a tricyclic β-carboline with pharmacological relevance. The structure reveals that tryptoline binds in a pose analogous to the natural AetF substrate L-tryptophan, positioning its C6 atom for initial halogenation. Enzymatic assays confirmed AetF-mediated monobromination and dibromination of tryptoline, yielding 6,8-dibromotryptoline as the final product. In accordance with the observed binding pose of the substrate tryptoline, the monobrominated initial product was almost exclusively 6-bromotryptoline. A crystal structure of AetF in complex with the minor monobrominated product 8-bromotryptoline provides insight into the effect of bromine substituents in different positions on the binding affinity and into potential pathways to the formation of 6,8-dibromotryptoline. These findings define the structural basis of site-selective halogenation of a non-native substrate by AetF and provide structural guidance for enzyme engineering.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"836-844"},"PeriodicalIF":4.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148293088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AlignPCA-2D: PCA-reduced Euclidean vector alignment for 2D classification in cryo-EM.","authors":"E Ramírez-Aportela, O L Zarrabeitia, Y C Fonseca, T Ceska, S Subramaniam, J M Carazo, C O S Sorzano","doi":"10.1107/S2059798326004572","DOIUrl":"10.1107/S2059798326004572","url":null,"abstract":"<p><p>Cryogenic electron microscopy (cryo-EM) has transformed structural biology by enabling the high-resolution reconstruction of macromolecular complexes from noisy projection images. However, the intrinsic heterogeneity and low signal-to-noise ratio of cryo-EM datasets make 2D classification a critical and computationally demanding step in the processing workflow. Here, we introduce AlignPCA-2D, a principal component analysis (PCA)-space Euclidean vector alignment method for fast, interpretable 2D classification in cryo-EM. By projecting particle images and class representations into a compressed latent PCA space, AlignPCA-2D reduces data dimensionality while preserving meaningful structural variability. The image-to-class assignment is then performed using Euclidean distance, enabling efficient and accurate classification. We benchmark AlignPCA-2D against established cryo-EM software, such as RELION and CryoSPARC, and demonstrate that it achieves competitive alignment accuracy while substantially reducing computational cost. This approach provides a lightweight alternative for large-scale 2D classification tasks, and its modular design makes it compatible with existing cryo-EM processing pipelines.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"727-739"},"PeriodicalIF":4.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317688/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148196794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unusual backfolded binding poses of BAZ2A bromodomain binders.","authors":"Andrea Dalle Vedove, Giulia Cazzanelli, Vito Giuseppe D'Agostino, Pablo Andres Vargas Rosales, Amedeo Caflisch, Graziano Lolli","doi":"10.1107/S2059798326004596","DOIUrl":"10.1107/S2059798326004596","url":null,"abstract":"<p><p>BAZ2A is a large multidomain protein overexpressed in aggressive prostate cancer, where it potentiates migration and invasion of other tissues. To counteract its metastasis-promoting role, small molecules interfering with the recognition of acetylated lysines by the BAZ2A bromodomain have been identified. However, unlike other bromodomains, BAZ2A has a shallow pocket, which significantly complicates the development of potent inhibitors. Here, we report the exploration of the acetyl-pyrrole scaffold, leading to the identification of BAZ2A-binding compounds assuming a peculiar, almost enclosed, conformation, as determined by X-ray crystallography. These molecules pose the basis for the development of potent BAZ2A macrocyclic inhibitors, as performed for other bromodomains.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"776-784"},"PeriodicalIF":4.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148147779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}