Acta Crystallographica Section D最新文献

{"title":"A short story of the long road to cryo-EM in Portugal.","authors":"Célia V Romão,Pedro M Matias","doi":"10.1107/s2059798326002640","DOIUrl":"https://doi.org/10.1107/s2059798326002640","url":null,"abstract":"This letter describes the development of cryo-EM in Portugal, highlighting a strategy based on research training and the establishment of a national facility with regional support nodes. Our experience demonstrates how access to European research infrastructures can enable the creation of sustainable national platforms without investment in high-end microscopes.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147663731","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":"Model-dependent and model-independent visualization of hydrogen atoms in high-resolution cryoEM maps.","authors":"Jimin Wang","doi":"10.1107/s2059798326002512","DOIUrl":"https://doi.org/10.1107/s2059798326002512","url":null,"abstract":"In this study, two methods are described for visualizing the H atoms in cryoEM maps of macromolecules. The first involves comparing experimental charge-density (CD) maps with calculated CD maps computed using structural models from which hydrogens are excluded. After the hydrogen-free model has been refined into the experimental map, the vector-difference Fourier map one computes, i.e. the (Fobs, αobs) - (Fcalc, αcalc) residual CD map, will reveal the H atoms missing from the model. The second method is model-independent and is known as a shoulder-peak decomposition method. It relies on the difference in the one-dimensional CD profile of a non-H atom between the side of the atom that adjoins a H atom and the side that does not. Here, the utility of both methods is demonstrated using both cryoEM CD maps derived from experimental electrostatic potential (ESP) maps for mouse heavy-chain ferritin at 1.09 Å resolution and X-ray crystallographic electron-density (ED) maps reported for the human enzyme at 1.06 Å resolution. A comparison shows that hydrogen signals in cryoEM maps are about 5-10 times stronger than those of X-ray crystallographic ED maps. Both methods should be applicable to cryoEM maps with a wide range of resolutions. The model-independent method is important for nucleoprotein complexes such as the ribosome because it bypasses the necessity of modeling the contribution that unscreened atomic partial charges make to cryoEM maps, which are difficult to model properly.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147636074","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":"Alphafuser: a parsimonious approach to predicting higher-order protein complexes.","authors":"Audrey Guillotin,Stephanie Hutin,Lorelei Masselot-Joubert,Kamel Hammani,Chloe Zubieta,Max Nanao","doi":"10.1107/s2059798326003013","DOIUrl":"https://doi.org/10.1107/s2059798326003013","url":null,"abstract":"Many biological processes rely on the activity of multiple proteins acting in concert as part of higher-order oligomeric complexes. However, the majority of structural studies have routinely simplified these systems to examine only one or a few protein partners of a multi-component system. Artificial intelligence (AI) methods implemented in AlphaFold, RoseTTAFold, ESMFold and others have revolutionized our ability to not only predict the fold of individual proteins, but also to predict that of complex assemblies. A major bottleneck in exploiting AI-based protein fold prediction for multiprotein complexes, however, is identifying likely interacting partners from a given interactome. Here, we present a protein complex prediction pipeline based on AlphaFold, called Alphafuser, that combines experimental interaction data with systematic querying of possible combinations of protein partners in a computationally parsimonious manner. This versatile protein complex prediction pipeline creates a queue of all potential complexes, up to a user defined number of partners. Because a complete search of all permutations is not typically practical, we implemented a simple dead-end trimming algorithm from the dimer step onwards based on the interface probability template modeling (ipTM) score to remove low-probability subcomplexes from the queue of higher-order complexes. Experimentally structurally characterized multiprotein complexes in the Protein Data Bank, PP2A-B56γ1 holoenzyme-PME-1 complex, the human γ-secretase complex, the TFIIIC complex and the TRAPP I complex, were used to obtain a general ipTM cutoff parameter for the pipeline and to confirm that complexes without extensive direct contacts between all subunits could be identified. We applied Alphafuser to two test cases starting from yeast two-hybrid and co-immunoprecipitation/mass spectrometry (co-IP/MS) interaction data. These predictions were tested experimentally using pull-down assays, confirming direct interactions of the proteins identified computationally by the Alphafuser pipeline.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147733949","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 reveals ArnA contamination during purification of a ciliary protein complex.","authors":"Xuguang Jiang,Masahide Kikkawa","doi":"10.1107/s2059798326002846","DOIUrl":"https://doi.org/10.1107/s2059798326002846","url":null,"abstract":"Endogenous Escherichia coli proteins can co-purify with recombinant targets and dominate cryo-EM datasets, yet often escape detection during standard biochemical quality control. In parallel to the recent report by Caliseki and coworkers [Caliseki et al. (2025), Acta Cryst. D81, 545-557], we independently identified ArnA contamination while purifying a soluble, low-yield KIF17-IFT70 complex, ultimately obtaining a 3.23 Å resolution cryo-EM structure of ArnA rather than the intended target. Our results reinforce that ArnA enrichment reflects general features of His-tag affinity purification and can become particularly problematic in cryo-EM workflows when the intended target is low in yield or conformationally heterogeneous. By comparing biochemical behavior and cryo-EM outcomes, we outline why ArnA may evade SDS-PAGE and size-exclusion chromatography, and be underappreciated in routine mass spectrometry-based quality control, yet becomes structurally dominant in cryo-EM. These findings broaden the scope of the original study and highlight the need for early cryo-EM screening and improved contaminant awareness in structural biology.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147680488","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":"Gaining insights into MmpL3: combining structural and computational approaches to unlock transport and inhibitor-binding mechanisms.","authors":"Satoshi Murakami,Domenico Marson,Eiki Yamashita,Bruno Broshka,Ui Okada,Maho Aoki,Giannamaria Annunziato,Erik Laurini,Emanuele Carosati,Marco Pieroni","doi":"10.1107/s2059798326003050","DOIUrl":"https://doi.org/10.1107/s2059798326003050","url":null,"abstract":"The rise of antimicrobial resistance in Mycobacterium tuberculosis underscores the urgent need for novel therapeutic strategies. Mycobacterial membrane protein large 3 (MmpL3) has emerged as a promising drug target, given its essential role in trehalose monomycolate transport and cell-wall biosynthesis. We determined the crystal structure of M. smegmatis MmpL3 in complex with a potent indolecarboxamide inhibitor, UPAR-1109, at 2.15 Å resolution, the highest reported to date. This structure provided unprecedented insights into the binding mode of the inhibitor, highlighting strong polar interactions and extensive hydrophobic contacts, which disrupt proton translocation. Speculative analysis about the molecular mechanism of inhibition has been proposed based on this model. Computational studies, including docking, molecular-dynamics and enhanced sampling simulations, revealed the remarkable plasticity of the MmpL3 binding site and confirmed the crystallographic orientation of UPAR-1109 as the most stable and biologically relevant binding mode. Together, these findings advance our understanding of the function and inhibition of MmpL3, providing valuable information for the rational design of next-generation antituberculosis agents, also with potential applications against nontuberculous mycobacteria.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147754829","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 and modulation of catalytically relevant states of a dye-decolourizing peroxidase using time-resolved serial femtosecond crystallography with drop-on-chip mixing and X-ray-driven reduction.","authors":"Marina Lučić,Johan Glerup,Pierre Aller,Danny Axford,Nicholas Devenish,Jaehyun Park,Anastasiia Shilova,Arturo Landeros de la Isla,Richard W Strange,Tiankun Zhou,Robin L Owen,Jonathan A R Worrall,Michael A Hough","doi":"10.1107/s2059798326001658","DOIUrl":"https://doi.org/10.1107/s2059798326001658","url":null,"abstract":"Metalloenzymes containing a heme cofactor catalyse a wide range of oxidative reactions critical to life. Understanding the structure and electronic states of the heme across the catalytic cycle is essential in understanding the oxidative chemistry performed on the substrate. This work demonstrates in crystallo manipulation of the heme-iron oxidation state in a B-type dye-decolourizing peroxidase from Streptomyces lividans (DtpB) using multiple, complementary, serial crystallography approaches. Fixed-target drop-on-chip serial femtosecond crystallography (SFX) together with dose-resolved serial synchrotron crystallography (SSX) allowed DtpB to be driven between multiple iron oxidation states. Drop-on-chip addition of hydrogen peroxide with fixed-target SFX is used to generate a ferryl [Fe(IV)=O] species, while the X-ray-driven approach modulates the iron oxidation state, with an apparent two-electron reduction leading to a return to a ferric state. The formation and dose response of the Fe(IV)-O state is highly variable between the chemically identical heme groups of the DtpB hexamer, highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147630262","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":"Mapping the effects of specific radiation damage and solvent radiolysis in buffers and crystals with online UV-Vis absorption spectroscopy.","authors":"Jack Stubbs,Nicolas Caramello,Matthew J Rodrigues,Sylvain Engilberge,Eric Mathieu,Samuel L Rose,Gwyndaf Evans,Antoine Royant,Ivo Tews","doi":"10.1107/s2059798326002743","DOIUrl":"https://doi.org/10.1107/s2059798326002743","url":null,"abstract":"Online UV-Vis absorption spectroscopy is often used during crystallographic data collection to characterize chromophores within proteins. This includes the study of electron-rich or redox-active enzyme intermediates and cofactors that are prone to specific radiation damage, necessitating suitable low-dose data-collection strategies. In crystallo spectroscopy enables the monitoring of X-ray-induced changes in the spectroscopic signatures of proteins and ligands; however, a comprehensive approach that also considers buffer components is required. Here, we present a mapping of X-ray-induced spectral changes in common crystallization chemicals and mixtures at cryogenic temperature, for which spectroscopic changes can be detected at doses as low as 1 kGy. A transient increase in absorption between 450 and 700 nm is frequently observed, arising from solvated electron absorption. Below 450 nm, several distinct absorption peaks were detected, for example for halides in buffers. In addition, Rayleigh scattering can lead to an increasing loss of photons from the optical path as the wavelength decreases, and thus to a significant elevation of the baseline at shorter wavelengths. In this study, we demonstrate the use of spectroscopic analyses to investigate the chromophoric enzyme intermediate I320 in vitamin B6 biosynthesis. In this case, X-ray-induced spectral changes were attributed to crystallization agents, cryoprotectants and light scattering, thereby excluding intrinsic alterations to the enzyme intermediate under low-dose conditions. Our study highlights the importance of monitoring spectral changes during diffraction data collection to ensure accurate interpretation of the electronic structure of chromophores.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147725945","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":"A cryo-EM processing pipeline for microtubules using CryoSPARC.","authors":"Daniel Zhang,Hugo Muñoz-Hernández,Pavel Filipcik,Kushal Sejwal,Yixin Xu,Sung Ryul Choi,Michel O Steinmetz,Michal Wieczorek","doi":"10.1107/s2059798326003062","DOIUrl":"https://doi.org/10.1107/s2059798326003062","url":null,"abstract":"Microtubules are cytoskeletal filaments that are typically characterized by a discontinuous helical lattice of α/β-tubulin heterodimers. Microtubules can also adopt variable lattice architectures both in vitro and in cellular contexts. Pseudo-helical averaging processing strategies have been developed to generate cryo-EM reconstructions of microtubules with and without decorating protein-binding partners, but these pipelines can be difficult to implement for the average user, especially for undecorated filaments. Here, we describe MiCSPARC, a cryo-EM processing pipeline developed around CryoSPARC [Punjani et al. (2017), Nat. Methods, 14, 290-296], which leverages automated particle picking and fast 3D refinement times in CryoSPARC to determine the structures of both decorated and undecorated microtubules. We generate reconstructions of undecorated GDP microtubules, as well as kinesin-1 motor domain-decorated GMPCPP filaments, at resolutions of up to 2.8 Å, demonstrating the robustness of the pipeline. Based on its convenient implementation and its ability to routinely generate high-resolution, seam-corrected microtubule reconstructions, MiCSPARC should provide a valuable tool for understanding microtubule dynamics, microtubule-associated proteins and microtubule-targeting agents.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147753068","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":"Structures of variants of Escherichia coli flavodiiron-type nitric oxide reductase reveal changes in the di-iron site.","authors":"Patrícia T Borges,Filipe Folgosa,Maria C Martins,Guillaume Gotthard,Peter van der Linden,Philippe Carpentier,Miguel Teixeira,Carlos Frazão,Célia V Romão","doi":"10.1107/s2059798326002214","DOIUrl":"https://doi.org/10.1107/s2059798326002214","url":null,"abstract":"Flavodiiron proteins (FDPs) are NO and/or O2 reductases which contain a di-iron catalytic center. Interestingly, they exhibit different selectivities towards each one of these substrates, despite having the same ligands of the iron ions. Escherichia coli FDP is a selective NO reductase that protects this bacterium against nitric oxide by catalyzing two-electron reduction to the nontoxic N2O. Previously, based on kinetic studies, we explored the possible role of two amino acids located in the di-iron second coordination sphere, Lys53 and Tyr271, in modulation of the substrate selectivity of Entamoeba histolytica FDP, a selective O2 reductase. In this work, we replaced the structurally equivalent residues in E. coli FDP, Asp52 and Ser262, by those present in the O2-selective FDP and determined their crystal structures in both oxidized and reduced states. Furthermore, the molecular-substrate tunnels were experimentally identified using krypton pressurization of the crystals. The data obtained corroborated previous molecular-dynamics calculations on this FDP. The side chains of residues in both positions 52 and 262 of E. coli FDP variants and wild type are in the vicinity of the shorter intramolecular tunnel, which is suggested to be the exit route for the reaction products N2O and H2O. The E. coli FDP S262Y variant shows photoreduction of the di-iron center and partial loss of electron density in some of its coordinating ligands after X-ray exposure, and these effects are consistent with increased radiation sensitivity. The kinetic properties of the variants towards NO and O2 were not significantly different from the wild type, contrary to what was observed previously for E. histolytica FDP.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147630260","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":"Electrons and X-rays for diffraction and imaging.","authors":"Colin Nave,Pedro Nunes,Alistair Siebert","doi":"10.1107/s2059798326002056","DOIUrl":"https://doi.org/10.1107/s2059798326002056","url":null,"abstract":"A comparison is provided between the use of electrons and X-rays for collecting diffraction data from small protein crystals and imaging data from cells and tissues. The paper contains a review element written to enable an understanding of the relevant properties of electrons by those (including one of the authors) more used to X-ray imaging and diffraction. Radiation-damage mechanisms, sample thickness-dependent dose efficiency and energy-dependent scattering cross sections are discussed, together with contrast mechanisms in electron and X-ray imaging. Crossover points are calculated for diffraction from crystals where electrons and X-rays could yield equivalent data quality in the presence of radiation damage. Increasing the electron energy from 300 to 1000 keV results in a ∼43% rise in the electron/X-ray crossover point. However, the maximum information coefficient (useful signal/absorbed dose) for electrons alone occurs for a 250 nm crystal examined at around 800 keV. The impact of inelastic scattering on electron imaging and diffraction is examined, including its role in coherence loss, Bragg spot broadening and background elevation. The possibilities are investigated for locating regions of interest using X-rays for subsequent higher resolution imaging using electrons. For locating a 30 nm diameter protein or virus, the required X-ray dose would be much less than the tolerable dose for electron imaging at 0.5 or 0.25 nm. Overall, these findings are relevant for imaging at different length scales while minimizing dose-induced structural damage.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147630261","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}