The solute carriers of the major facilitator superfamily (MFS) play significant roles in human health and disease. The melibiose transporter of Salmonella enterica serovar Typhimurium (MelBSt) catalyzes the stoichiometric symport of galactosides with Na+, H+ or Li+ and is a well studied prototype of MFS transporters. With a large body of data from extensive functional analyses using multiple transport and binding assays, structural characterizations of multiple states by X-ray crystallography and cryoEM single-particle analysis, and dynamic elucidations using hydrogen-deuterium exchange mass spectrometry (HDX-MS), the molecular recognition of the primary substrate galactoside and its coupling cation Na+, H+ or Li+ has been elucidated at the molecular level. Cooperative binding of both solutes is recognized as the core symport mechanism that supports the transporter's functions more effectively in a sugar-scarce environment. Structural and dynamic studies show that conformational dynamics significantly influence sugar binding but have minimal effect on Na+. Na+ acts as an allosteric activator, increasing sugar affinity by stabilizing the inner barrier and constraining conformational flexibility. All data are consistent with the previously constructed stepped-binding kinetic model for melibiose symport with Na+; in addition, MFS uniport and antiport mechanisms are discussed.
Cryo-electron microscopy (cryo-EM) micrographs are frequently contaminated by carbon edges, ice crystals, ethane bubbles and other high-contrast artifacts. These contaminants trigger abundant false positives in automated particle pickers, severely hampering downstream 3D reconstruction. Existing methods either avoid contamination implicitly (requiring dataset-specific tuning) or rely on rule-based filters that fail on complex contamination patterns. Here, we present QwenCryoMarker, a universal post-processing framework that converts the outputs of arbitrary particle pickers into clean, high-precision particle sets. Our pipeline consists of two core stages: (i) a visual large model (Qwen-Image-Edit-2511) fine-tuned via supervised learning to generate pixel-accurate binary contamination masks from raw micrographs and (ii) a lightweight contamination-aware filtering module that discards particles when the contamination proportion within their surrounding circular region exceeds a predefined threshold ratio. Contamination is defined as all micrograph regions unsuitable for reliable particle picking and subsequent 3D reconstruction, including carbon edges, ice crystals, ethane bubbles, miscellaneous debris and dense protein aggregates. The framework features plug-and-play deployment: it requires no per-dataset parameter tuning or extra retraining, and maintains compatibility with classical pickers (blob detection, template matching) as well as deep learning-based pickers (Topaz, crYOLO etc.). We validate QwenCryoMarker on five diverse CryoPPP benchmark datasets across four representative particle pickers. Quantitatively, our method consistently boosts precision with an average absolute gain of 0.009 and lifts the F1-score, while recall only drops slightly by an average of 0.008. Segmentation benchmarking shows our model reaches a mean intersection over union (IoU) of 0.629, surpassing that of the state-of-the-art MicrographCleaner (0.551) by 14.2%. We further compare against multiple segmentation baselines: U-Net (0.448), DeepLabV3+ (0.488), SAM (0.475), ASOCEM (0.195) and IceBreaker (0.433). A downstream reconstruction case study on EMPIAR-10017 verifies that particles filtered by QwenCryoMarker yield cleaner 2D class averages and higher resolution 3D density maps (3.88 versus 3.97 Å). Qualitative visualization also confirms that QwenCryoMarker stably eliminates false particles located on carbon films and ice crystals, independent of the upstream particle-picking algorithm. By encapsulating contamination suppression as a universal, model-agnostic post-processing module, QwenCryoMarker offers a practical, robust, easy-to-deploy toolkit that greatly improves particle-set quality without modifying existing cryo-EM workflows. The framework is fully open-source and can be seamlessly integrated into mainstream cryo-EM processing pipelines.
Adoptive cell therapy (ACT) with tumor-specific T cells can mediate durable cancer regression. The main target of tumor-specific T cells are neoantigens resulting from mutations in self-antigens over the course of malignant transformation. To understand T-cell recognition of cancer neoantigens at the atomic level, we studied a T-cell receptor (TCR 4414A) that recognizes a neoepitope arising from a driver mutation in the p53 oncogene (p53Y220D) presented by HLA-A2. Here, we report the structure of TCR 4414A bound to HLA-A2 and p53Y220D, as well as structures of unbound wild-type and mutant p53-HLA-A2 ligands. The structures reveal that the Y220D mutation induces a conformational change in the p53Y220D neoepitope that is detected by TCR 4414A, thereby rendering a normally cryptic self-peptide visible to T cells. The TCR minimizes interactions with the N- and C-terminal portions of p53Y220D, which are identical in mutant and wild-type peptides, and instead focuses on the Y220D driver mutation at the peptide center. In this way, TCR 4414A achieves highly specific recognition of mutant over wild-type p53, a critical parameter for avoiding off-target toxicities in ACT.
The Guest Editors introduce the special issue based on talks at the CCP4 Study Weekend 2024. The virtual issue is available at https://journals.iucr.org/special_issues/2026/CCP42024/.
The determination of protein-ligand complex structures by X-ray crystallography is a cornerstone of modern structure-guided drug discovery. However, the process is complex and fraught with potential pitfalls at every stage, from data collection to final model deposition. The presence of flawed or misinterpreted ligand models in the Protein Data Bank (PDB) can misdirect scientific efforts that rely on them as the basis for new hypotheses and experiments. This article outlines a practical approach for ligand validation during structure determination. We discuss the application of validation tools, such as those in Coot, MolProbity, Mogul and Buster-report, to avoid errors. By re-examining several deposited PDB entries, we illustrate key pitfalls, including (i) modelling a ligand into absent or ambiguous electron density, (ii) incorrect chemical definitions (e.g. chirality, tautomers), (iii) poor fit of parts of a ligand to the electron density and (iv) data/model mismatches during deposition. We emphasize the importance of a `null-hypothesis' approach and continuous critical assessment throughout the modelling process to improve the reliability of deposited structures.
We report here the structure of Pyrococcus abyssi rubredoxin determined at a resolution of 0.43 Å. This, to the best of our knowledge, represents the highest resolution protein structure yet determined. Experimentally, the determination of this structure has been made possible by a series of technical innovations and streamlined procedures that are described herein. The structure model refined with spherical scattering factors, the so-called independent atom model (IAM), reveals many positive difference densities that can be attributed to electrons at the midpoint of chemical bonds. To validate the interpretation that these do indeed represent bonding electrons, we have connected the DiSCaMB transferable aspherical atom model (TAAM) library to BUSTER. TAAM refinements resolve all positive density features in midpoints of chemical bonds. Extrapolating the findings and methodology reported here, we anticipate that it should now be possible to routinely acquire accurate X-ray diffraction data for quantum crystallography of biological macromolecules. This is particularly relevant to the study of enzyme mechanisms, which are well documented to entail quantum-mechanical phenomena.
There has been a rapid expansion in new approaches to sample preparation for single-particle cryoEM. One drive has been around speeding up the process both for the application of time-resolved studies and in outrunning some of the negative effects which may come from the sample interacting with the air-water interface, such as degradation and preferred orientation. Here, we set out to significantly improve the speed of grid preparation by removing the plunging stage and directly spraying onto pre-cooled grids which are situated in the cryogen, allowing us to produce grids where two samples are mixed and frozen on the grid in less than 1 ms. We have previously shown a preliminary proof of principle with this new approach and here we provide a detailed description of the approach taken and the methodology behind it. This approach allows us to move beyond the millisecond timescale to open up more systems to time-resolved EM and potentially improve samples.
Metal-dependent formate dehydrogenases (Fdhs) are a promising research target in efforts to mitigate climate change by developing active, efficient, selective and safe industrial catalysts for CO 2 reduction. Here, we report the room-temperature (RT) serial synchrotron X-ray crystallography (SSX) structure of Nitratidesulfovibrio vulgaris formate dehydrogenase AB and compare this structure with previously reported cryogenic structures. The comparison reveals structural differences likely arising from the absence of cryoprotectants, differences in data-collection temperature and the much lower radiation dose accumulated by each crystal during the SSX experiments. The RT-SSX structure provides the first step towards time-resolved serial crystallography experiments targeting possible catalytic intermediates in order to fully understand the catalytic mechanism of metal-dependent Fdhs.
Bacterioferritin from Brucella melitensis ( Bm Bfr) is a protein that plays essential roles in oxidative stress management and metal homeostasis in the bacterium. It presents itself as a 24-mer forming a spherical nanocage, allowing further applications such as drug delivery and nanoparticle synthesis. Studying the oligomerization process is thus important for a deeper understanding of the protein. In this work, two forms of the protein were studied: with the heme cofactor (holo Bm Bfr) and without the cofactor (apo Bm Bfr). Analyses were performed using size-exclusion chromatography, thermal shift analysis and structure determination of apo Bm Bfr by X-ray crystallography. This revealed that oligomerization is not possible with the apo form, since the heme is essential for dimer formation, which is the first oligomerization step. Further oligomerization can occur at a pH above 5.0, where interface interactions are favorable, to form a highly stable 24-mer state. Those findings allowed us to suggest an oligomerization mechanism for Bm Bfr.
Several corrections are made to the article by Zheng et al. [(2025), Acta Cryst. D81, 181-195].
Endo-β-1,6-galactanases hydrolyze β-1,6-linked galactosyl linkages in galactans, yielding β-1,6-linked galacto-oligosaccharides, predominantly galactobiose. Here, we report to our knowledge the first crystal structure of an endo-β-1,6-galactanase, together with its β-1,6-galactobiose-bound complex, revealing the structural basis for substrate recognition by this enzyme. Endo-β-1,6-galactanase from Streptomyces avermitilis ( Sa 16Gal30A) is a member of glycoside hydrolase family 30 (GH30) subfamily 5. Sa 16Gal30A consists of two structural domains: a catalytic (β/α) 8 -barrel domain and an appended β-sandwich domain. The β-1,6-galactobiose complex structure revealed two β-1,6-galactobiose molecules bound within the catalytic domain: one at the catalytic site and another at the distinct surface site distal to the catalytic center. This structure represents the first reported structure of a GH30 subfamily 5 enzyme and provides structural insights into the molecular basis of β-1,6-galactan recognition within the catalytic cleft. Sa 16Gal30A possesses three extended regions, loops 2, 4 and 8, in the catalytic domain compared with enzymes from other GH30 subfamilies, and these loops appear to modulate substrate specificity towards β-1,6-galactan by shaping the architecture of the catalytic cleft. In addition, a secondary β-1,6-galactan-binding site was identified at a distal location, which may function as a distal subsite, thereby facilitating the efficient hydrolysis of long β-1,6-galactan chains.
Simultaneous inhibition of oncogenic signaling and metabolic pathways represents a promising approach for cancer therapy. KPT-9274, a clinical stage compound, has been reported as a dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT), but its structural basis has remained undefined. Here, we present high-resolution crystal structures of PAK4 and NAMPT in complex with KPT-7523, an analog of KPT-9274, determined at 2.20 and 1.45 Å resolution, respectively. In PAK4, the 2-aminopyridine moiety of KPT-7523 enables dual binding, occupying the adenine-binding site for ATP and simultaneously engaging the substrate-binding cleft in the C-lobe, thereby interfering with both catalytic and regulatory functions. In NAMPT, the same scaffold inserts into the NAD+ active site in an extended conformation that preserves critical interactions. Biophysical assays revealed distinct affinities across the two targets. These findings highlight the 2-aminopyridine moiety as a versatile pharmacophore that is adaptable to structurally unrelated proteins and provide a framework for designing next-generation dual inhibitors in cancer therapy.
With the continuous growth of the Protein Data Bank archive, the Worldwide Protein Data Bank (wwPDB) partnership anticipates that the entire complement of possible four-character PDB accession codes (for example 1ABC) will be exhausted by 2028. wwPDB is, therefore, revising the PDB accession code (PDB ID) to 12 characters by extending its length and prepending `pdb_' (for example pdb_1000axyz) in lower case. This change will enable the robust detection of references to PDB entries in published literature. On or about July 21st 2027, the PDB will convert to releasing entries with extended PDB IDs only, which will not be compatible with the legacy PDB format. A beta version of the PDB Archive (PDB Beta Archive) is now available to help communities adapt to and embrace the extended PDB IDs and PDBx/mmCIF format during a transition phase. All files in the current PDB archive are reorganized in the Beta Archive with extended PDB IDs (including file naming and directories) on an entry-level basis, mirroring the data organization of the PDB Versioned Archive. wwPDB encourages scientific journals, PDB community members and users to transition to the PDBx/mmCIF format and adopt the new PDB ID format as early as possible. The PDB Beta Archive will replace the current public archive, and 12-character PDB IDs will be solely assigned to all newly deposited PDB entries.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes classified into the auxiliary activity (AA) families of the CAZy database. They oxidatively cleave glycosidic bonds in recalcitrant polysaccharides, playing a key role in biomass degradation and contributing to the virulence of some pathogens. The redox state of the active-site copper and its coordination geometry are central to LPMO catalysis, yet the subtle structural consequences of redox and substrate-binding transitions remain insufficiently resolved. In previous work, a comparative X-ray crystallographic analysis of a model AA9 LPMO (LsAA9A) was conducted under four distinct conditions at 100 K: Cu(II), Cu(I) and the corresponding saccharide-bound states, with the Cu(I) state generated by X-ray photoreduction. In this study, LsAA9A crystals were chemically reduced with or without saccharide substrate prior to low-dose X-ray data collection to minimize radiation damage. Copper-coordination distances and angles were determined precisely through triplicate structure determinations (each from an independent crystal) for each condition, revealing small but reproducible geometry differences across key states in the LPMO catalytic pathway. In order to identify the most significant differences, statistical evaluation using one-way analysis of variance (ANOVA), followed by Tukey-Kramer post hoc tests and pairwise t-tests, was carried out. Within the assumptions made, statistically significant differences in the coordinated Cu-His1 Nδ1 and Cu-Tyr Oη distances, and in the coordination angles θ2, θ3 and θT, are observed across the four states and are discussed in terms of the mechanism and in relation to our previous study. To complement cryogenic data, multi-crystal data sets at increasing X-ray dose were collected at room temperature to track photoreduction of the copper site, with the specific aim of detecting the transition of Cu(II) to fully reduced Cu(I). This could not finally be achieved due to the onset of global radiation damage; however, a subset of the reduction-linked geometric changes were detectable, indicating that a partial transition may have occurred.
When a macromolecular crystal lattice targeted for study is closely related to a previously studied crystal lattice, phasing the target crystal by difference (fast-)Fourier transform (DFFT) methods is preferable to performing molecular replacement. The application Scotty, within the Phasertng codebase, is software for the identification of coincident lattices and downstream processing. All crystallographic PDB entries are organized into a scikit-learn `BallTree' index under the Niggli cell distance metric `NCDist'. Nearest neighbours under the metric are progressed to test structure-factor intensity correlation. The structure from the lattice with the highest correlation with the target is used to phase the target lattice, and the coordinates are taken forward to coordinate refinement using a wide convergence radius protocol. The method can identify lattice coincidences accounting for very significant non-isomorphism. The nearest-neighbour search is space-group agnostic, so that coincident lattices are identified even when the nominal space groups are different, the symmetry of one lattice being described as a subgroup of the other or a higher metric symmetry.
The application Scotty, implemented within the Phasertng codebase, was used to perform a PDB-wide analysis of lattice coincidences. Using a broad definition of lattice coincidence, the number of distinct lattice clusters is approximately half the total number of crystallographic PDB entries. In over one thousand lattice clusters entries are reported in different space groups, consistent with pseudo-symmetric variation within a common lattice framework. Space-group frequencies computed at the lattice-coincidence level update those obtained by entry-based counting, and more accurately reflect priors for novel crystal forms. Combining lattice clustering with sequence identity and deposited oligomeric annotations reveals multiple cases of inconsistent biological assembly assignments among structures sharing near-identical lattices, suggesting an opportunity to improve annotations. The survey also identifies a range of protein systems forming extended in cellulo paracrystalline arrays, including storage, sequestration, toxin and membrane-associated proteins, an understudied area of structural biology. Overall, the results demonstrate that lattice-level analysis provides a valuable perspective on macromolecular self-association.
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.
Monte Carlo neutron ray-tracing simulations of time-of-flight (TOF)-Laue neutron macromolecular crystal diffraction (n-MX) using the McStas software package were performed for the upcoming NMX Macromolecular Diffractometer at the European Spallation Source. Splitting neutron rays that arrive at the crystal leads to dramatic improvements in event formation with minimal computational overhead. The simulated event-probability data were sampled using a new single-pass weighted reservoir-sampling method, and processed like real n-MX data using DIALS. The effects of air and beamstop scatter on simulated data were investigated.
