Acta Crystallographica Section D最新文献

{"title":"Radiation damage in sub-Ångström resolution macromolecular crystallography: a low-dose study.","authors":"Gleb Bourenkov,Elham Paknia,Claus Flensburg,Rasmus Fogh,Peter Keller,Clemens Vonrhein,Gérard Bricogne,Ashwin Chari","doi":"10.1107/s205979832600269x","DOIUrl":"https://doi.org/10.1107/s205979832600269x","url":null,"abstract":"Sub-Ångström macromolecular crystallography allows the construction of structural models without any prior chemical knowledge and stereochemical restraints, making it possible to visualize deviations from peptide planarity, distortions of the planarity of aromatic ring systems and subtle alternate conformation networks. It also holds the promise of observing aspherical density features and other quantum-mechanical phenomena. However, the degree to which radiation damage might affect the details that can be discerned from biological structures at such resolutions has remained unknown. To address this, we report here the first study of radiation-damage effects at sub-Ångström resolution on Pyrococcus abyssi rubredoxin. Our data, collected at 100 K, indicate that the oxidized (Fe3+) state of the Fe atom is preserved to a large degree at a dose of 50 kGy, whereas at 1 MGy it is partially reduced to the Fe2+ state. Isomorphous difference maps reveal extensive conformational changes at 1 MGy that are most likely coupled to the reduction of Fe3+. At 1 MGy, hydrogen densities that are visible at 50 kGy are preserved, but are distinctly blurred by dose. These findings suggest that the `global' radiation damage in reciprocal space and the associated blurring of electron density in real space proceed through small structural changes that are much more extended than the local damage at specific sites which is normally seen at lower resolution. Thus, the `global' and `specific' damage could be viewed as two sides of the same coin. Our study highlights the unique benefits of using low-dose data-collection protocols on large crystals fully bathed in a large `top-hat' beam with a uniform fluence profile for accurate sub-Ångström structural investigations on macromolecules, as only this experimental configuration can deliver the uniform spatial distribution of dose necessary to resolve the fine details of progressive radiation damage: a capability for which we propose the term `resolution in dose'.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"260 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147663730","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":"Damage-limited resolution for X-ray and electron microscopy of organic specimens.","authors":"Ray F Egerton,Colin Nave","doi":"10.1107/s2059798326002445","DOIUrl":"https://doi.org/10.1107/s2059798326002445","url":null,"abstract":"Analytical expressions for the damage-limited resolution (DLR) are developed and applied to X-ray and electron imaging of beam-sensitive specimens, allowing for variation of the characteristic radiation dose with spatial resolution. The dependence of DLR on specimen thickness is illustrated for the common modes of X-ray and transmission electron-microscope imaging. Similarities and differences between the radiolysis damage caused by electrons and X-rays are discussed. The meaning of a `Bragg boost' in diffracted intensity is discussed.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"221 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147641781","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":"Radiation-induced peroxide rupture and its temperature-dependent repair probed by homogeneous X-ray irradiation.","authors":"Symeon Koulas,Soi Bui,Julius B Kirkegaard,Gleb Bourenkov,Roberto A Steiner","doi":"10.1107/s2059798326002688","DOIUrl":"https://doi.org/10.1107/s2059798326002688","url":null,"abstract":"Specific radiation damage frequently compromises structural analysis in macromolecular crystallography. However, it can also offer key mechanistic information. Here, we investigate the X-ray-induced radiolysis of the catalytic C5-peroxide adduct in crystals of the cofactor-independent enzyme urate oxidase. Using a top-hat X-ray beam to ensure homogeneous dose distribution, we monitored the occupancy of the peroxide species across extensive dose series at 100 K and room temperature (RT). We observe a fundamental kinetic phase transition between these thermal regimes. At RT, the peroxide decays rapidly following zero-order kinetics, consistent with a flux-limited regime where the radiolytically cleaved O2 product diffuses out of the active site to be replaced by water. Conversely, at 100 K the decay is markedly retarded and follows first-order kinetics. Bayesian kinetic modelling demonstrates that this cryoprotection arises from a recombination mechanism: the cleaved O2 molecule remains trapped in the active site with the organic species, enabling efficient recombination that competes with irreversible degradation, effectively resulting in radiation-induced in crystallo catalysis.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"67 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147663734","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":"Bayesian perspective for orientation determination in cryo-EM with application to structural heterogeneity analysis.","authors":"Sheng Xu,Amnon Balanov,Amit Singer,Tamir Bendory","doi":"10.1107/s2059798326001415","DOIUrl":"https://doi.org/10.1107/s2059798326001415","url":null,"abstract":"Accurate orientation estimation is a crucial component of 3D molecular structure reconstruction, both in single-particle cryo-electron microscopy (cryo-EM) and in the increasingly popular field of cryo-electron tomography (cryo-ET). The dominant approach, which involves searching for the orientation that maximizes cross-correlation relative to given templates, is suboptimal, particularly under low signal-to-noise conditions. In this work, we propose a Bayesian framework for more accurate and flexible orientation estimation, with the minimum mean-square error (MMSE) estimator serving as a key example. Through simulations, we demonstrate that the MMSE estimator consistently outperforms the cross-correlation-based method, especially in challenging low signal-to-noise scenarios, and we provide a theoretical framework that supports these improvements. When incorporated into iterative refinement algorithms in the 3D reconstruction pipeline, the MMSE estimator markedly improves reconstruction accuracy, reduces model bias and enhances robustness to the `Einstein from Noise' artifact. Crucially, we demonstrate that orientation-estimation accuracy has a decisive effect on downstream structural heterogeneity analysis. In particular, integrating the MMSE-based pose estimator into frameworks for continuous heterogeneity recovery yields accuracy improvements approaching those obtained with ground-truth poses, establishing MMSE-based pose estimation as a key enabler of high-fidelity conformational landscape reconstruction. These findings indicate that the proposed Bayesian framework could substantially advance cryo-EM and cryo-ET by enhancing the accuracy, robustness and reliability of 3D molecular structure reconstruction, thereby facilitating deeper insights into complex biological systems.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147461637","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":"The human TIMP-1 unbound structure provides a platform for fragment screening.","authors":"Ahmed Shemy,Jana Van Broeckhoven,Niels Hellings,Arnout Voet","doi":"10.1107/s2059798326001749","DOIUrl":"https://doi.org/10.1107/s2059798326001749","url":null,"abstract":"Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a critical regulator of extracellular matrix remodelling and an important mediator of remyelination in demyelinating disorders such as multiple sclerosis. In addition, TIMP-1 has emerged as a promising therapeutic target in cancer due to its interaction with CD63, which promotes tumorigenic signalling and carcinogenesis. Although several structures of TIMP-1 bound to matrix metalloproteinases have been reported, no unbound structure with all druggable sites available has previously been reported. Here, we present the first unbound crystal structure of human TIMP-1, resolved at 1.95 Å resolution. Comparison with the MMP-bound complex reveals localized conformational changes and altered intramolecular hydrogen bonding in the unbound structure, indicating increased structural plasticity in the absence of the protease. Crystals were obtained in multiple conditions, but only two diffracted to high resolution. Although optimization and seeding did not significantly improve the morphology, the additive screen enhanced both the morphology and reproducibility and provided intrinsic cryoprotection. The resulting crystal form proved compatible with soaking-based screening campaigns, providing a robust structural basis for the discovery of TIMP-1 ligands with clinical potential.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"10 8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147461638","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":"Automated workflows for strategy computation and data collection at synchrotron beamlines.","authors":"Rasmus H Fogh,Peter Keller,Claus Flensburg,Clemens Vonrhein,Wlodek Paciorek,Leigh Carter,Gérard Bricogne","doi":"10.1107/s2059798326001920","DOIUrl":"https://doi.org/10.1107/s2059798326001920","url":null,"abstract":"The Global Phasing workflow derives and executes experimental strategies for macromolecular crystallography, optimized in real time for the crystal symmetry and mounting orientation and the expected experimental resolution. By acquiring sweeps at multiple orientations, the workflow averages out the differences in Lorentz intensity enhancement and multiplicity to produce equally weighted merged reflections for processing. Values for various classes of parameters can be read from configuration files, submitted diffraction plans or user interaction, with optimal transmission settings derived from the beam flux density and the intended experimental resolution. The program can be executed in user-driven or unattended mode. Strategies are available for native or phasing experiments in either basic or advanced versions. The workflow has been shown to improve resolution, the number of unique reflections measured and average I/σ compared with a single-sweep experiment on equivalent crystals with the same total radiation dose. The workflow is a standalone program that could be interfaced to any beamline-control system, and is currently available through its integration with MXCuBE on a number of beamlines.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147483663","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 structure of a glycoside hydrolase family 30, subfamily 12 endo-1,4-β-xylanase.","authors":"Franz J St John,Casey Crooks,Michael Endres,Lily Pakdaman,Lisa Koch,Loreen Bynum,Nathaniel Kuch,Andrzej Joachimiak,Kemin Tan","doi":"10.1107/s2059798326002160","DOIUrl":"https://doi.org/10.1107/s2059798326002160","url":null,"abstract":"We have determined the X-ray crystallographic protein structure of endo-1,4-β-xylanase (EX) A from Anaerobacterium chartisolvens (AchXyn30A), a homologue of the recent biochemically characterized glycoside hydrolase family 30, subfamily 12 (GH30_12) EX from Acetivibrio clariflavus (AcXyn30B). The N-terminal GH30 catalytic domains (CDs) of these two enzymes share approximately 63% amino-acid sequence identity and the full-length proteins each consist of the GH30_12 CD, a family 6 carbohydrate-binding module and a C-terminal dockerin domain. In this report, we offer additional support for the recent subfamily classification of these EXs and provide detailed X-ray crystallographic protein structure analysis of AchXyn30A, the first protein structure from this newly defined GH30 subfamily. We also provide comparative structural analysis using a generated AcXyn30B homology model as well as other GH30 subfamily enzymes. Additionally, we examine potential xylan-chain interactions informed by the protein structure. These characterized EXs further illustrate the diversity of xylan-degrading enzymes which have evolved within glycoside hydrolase family 30.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147502239","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":"The crystal structure of human transport and Golgi organization 2 homolog (TANGO2) suggests a cysteine N-terminal nucleophile (Ntn) hydrolase.","authors":"Dayong Zhou,Lirong Chen,John Rose,Bi Cheng Wang","doi":"10.1107/s2059798326001968","DOIUrl":"https://doi.org/10.1107/s2059798326001968","url":null,"abstract":"Recently, there has been growing interest in the function and physiological importance of human TANGO2 (transport and Golgi organization 2 homolog), particularly whether it acts as a heme-trafficking protein. To address this question, we experimentally determined the three-dimensional structure of TANGO2. Our crystallographic analysis indicates that interactions between heme and TANGO2 are nonspecific. Structural comparison of the TANGO2 crystal structure with known cysteine Ntn-hydrolases allowed us to identify a putative active site, catalytic residues and a substrate-binding cavity that correspond to residues that are mutated in pathogenic TANGO2 variants. Based on these features, we propose that TANGO2 may utilize fatty-acid derivatives as substrates, suggesting a potential role in lipid metabolism. Mutations in the human TANGO2 gene cause TANGO2 deficiency disorder, a multisystem, life-threatening disease with onset in early childhood. Together, our results provide new insights into the molecular function of TANGO2 and help to resolve the ongoing debate regarding whether it functions as a heme-trafficking protein.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"18 1","pages":"383-396"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147585433","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":"Molecular analysis of 3D domain swapping in the acylphosphatase from Escherichia coli.","authors":"Sergio Martínez-Rodríguez,Jose A Gavira,M Carmen Salinas-Garcia,Montserrat Andujar-Sánchez,Ana Camara-Artigas","doi":"10.1107/s2059798326001774","DOIUrl":"https://doi.org/10.1107/s2059798326001774","url":null,"abstract":"Three-dimensional domain swapping is a mechanism by which proteins form oligomers. At present, the molecular basis that dictates whether some proteins fold in their monomeric form or as intertwined oligomers is poorly understood. Previously, we have described the first intertwined dimer of an acylphosphatase (AcP) from crystals belonging to the orthorhombic space group C222. In this work, we present the first crystallographic structure of monomeric AcP from Escherichia coli (EcoAcP) and compare it with the intertwined structure of the orthorhombic polymorph and a new intertwined dimer structure obtained from crystals belonging to the hexagonal space group P6122. The monomeric form contains two molecules in the asymmetric unit, each exhibiting some differences. One of the molecules shows a sodium cation that introduces conformational changes in loop L4 (connecting α2 and β4). This loop is located adjacent to the active site, which is formed by a cleft between loops L1 (connecting β1 and α1) and L3 (connecting β2 and β3), in which phosphate anions have been modelled. Besides, in the monomeric form, the active-site Arg20 forms a salt bridge with the carboxyl-terminal group. This interaction is absent in the intertwined dimer, where the interchange of the terminal β-strand β5 is facilitated by loop L5 (connecting β4 and β5) that serves as a hinge loop. The first residue of this loop, Pro79, has been modelled in a cis conformation in the intertwined structures, whereas it is in a trans conformation in the monomeric form. The low rate of cis-trans proline isomerization would favour the formation of the domain-swapped structure under appropriate conditions. Comparative analysis of the monomer and intertwined dimer structures would facilitate understanding of the molecular basis of oligomer formation.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147483666","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":"Decision-making in serial crystallography: a simple test to quickly determine whether sufficient data have been collected.","authors":"David von Stetten,Arwen R Pearson","doi":"10.1107/s2059798326001324","DOIUrl":"https://doi.org/10.1107/s2059798326001324","url":null,"abstract":"In standard rotational data collection for macromolecular crystallography, data are normally collected from a single crystal, and the resulting data processing delivers metrics for data completeness and signal to noise that are well established. However, in serial crystallography it can be difficult to assess quickly whether enough data have been recorded to deliver a well scaled and complete dataset with sufficient signal to noise to address the scientific question being asked. Completeness alone is not an appropriate metric, as a nominally complete dataset can be obtained with a much smaller number of images, and thus multiplicity, than is needed to produce a final dataset with well estimated merged intensity values. Insufficient data result in alarmingly reasonable processing statistics and plausible electron-density maps that contain almost no experimental signal, instead being dominated by the phases from the phasing model. We have therefore established a simple electron-density-based test to determine whether enough data have been collected, and implemented this in the autoprocessing pipeline at the T-REXX endstation on beamline P14 at PETRA III. Importantly, the results of this perturbed model real-space difference density (PMRDD) test help to guide decisions as to whether more data should be collected or whether the experimenter can move onto a new time-point or sample.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"12 1","pages":"227-236"},"PeriodicalIF":0.0,"publicationDate":"2026-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147329602","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}