Journal of Structural Biology: X

Cryogenic electron microscopy (cryo-EM) has enabled rapid progress in structural biology throughout the last decade. However, cryo-EM sample preparation still represents a major bottleneck in high-resolution structure determination. Many proteins and macromolecular complexes interact with the air-water interface during sample preparation, which can cause protein denaturation, complex disassembly, and preferred particle orientation. Development of methods to alleviate these issues is an active field of investigation. However, a universally applicable solution to this problem has not been found so far. Here, we describe the simple and cost-effective usage of synthetic peptides with defined amino acid sequence to protect cryo-EM samples from complex disassembly and reduce preferred orientation. We show benefits during cryo-EM grid preparation of a large E3 ubiquitin ligase termed the anaphase-promoting complex (APC/C), and the small CDK11:cyclin L:SAP30BP complex.

Mitochondrial NADH-ubiquinone (UQ) oxidoreductase (respiratory complex I) reduces UQ within its long tunnel-like cavity, which is coupled to triggering proton translocation at the membrane domain. Although mammalian complex I adopts distinct "open" and "closed" conformations, the architecture of the UQ-accessing tunnel in the open conformation remains poorly defined owing to partial disorder of the UQ catalytic region near Fe-S cluster N2 in the tunnel. Here, we applied cryo-electron microscopy (EM) single particle analysis to determine the structures of complex I in bovine heart submitochondrial particles in the presence or absence of a mycothiazole-type inhibitor. Preceding photoaffinity labeling experiments using a synthetic photoreactive derivative ([125I]pMT) revealed that mycothiazole specifically labels the 49-kDa and ND1 subunits, both of which form a part of the UQ-accessing tunnel. Cryo-EM analysis demonstrated that bound mycothiazole reorganizes and stabilizes the UQ catalytic region in both open and closed conformations, enabling the first complete structural modeling of the entire UQ-accessing tunnel in the open conformation. Unexpectedly, in the presence of bound mycothiazole, loops of the PSST subunit forming the catalytic region in the tunnel adopted closed conformation-like features, whereas open conformation-like features were determined for the transmembrane helices of ND1 and ND6 subunits. The present study revealed that bound mycothiazole can reorganize the UQ catalytic region in the UQ-accessing tunnel and lead to a hybrid conformational state beyond the canonical open-versus-closed conformational model.

Single-particle cryo-electron microscopy (cryo-EM) plays an increasingly important role in structure-based drug discovery (SBDD) by revealing target protein-ligand interactions at the molecular level, but its application has largely focused on large- and medium-sized protein complexes. Many therapeutically important targets are smaller than 50 kDa and remain highly challenging for cryo-EM analysis because of low signal-to-noise ratios and difficulties in particle alignment. Here, we describe cryoFAST, an integrated workflow that couples ligand-compatible Fab discovery and structure-guided computational maturation with biochemical triage, fiducial-complex assembly, and single-particle cryo-EM. Through phage-display screening and/or AI-guided design, we developed high-affinity Fab binders of approximately 50 kDa that stabilize the target without disrupting ligand-target interactions and then assembled Fab-target complexes for cryo-EM analysis. Using the therapeutically relevant extracellular domain (~20 kDa) of interleukin-4 receptor (IL-4R) as a proof of concept, we determined its cryo-EM structure at 2.74 Angstrom resolution and established a framework for elucidating ligand-binding modes. This workflow provides a practical, efficient, and potentially broadly applicable route to the structural characterization of small protein targets that are otherwise challenging to study. By extending the applicability of cryo-EM to high-value but technically challenging small targets, cryoFAST may open new opportunities for structure-based drug discovery.

Preferred particle orientation, which results from the adsorption of proteins to the air-water interface, is a frequently encountered issue in single-particle cryo-electron microscopy (cryo-EM). Particles then present only a limited number of views, which are oversampled during data acquisition at the expense of throughput, while other views may be missing altogether. In severe cases, a high-resolution reconstruction becomes unattainable. We have recently established that ultrasonic excitation of the sample during vitrification improves the angular distribution by detaching particles from the air-water interface and reshuffling their orientations. Here, we describe an implementation of this principle for a plunge freezing apparatus, in which the ultrasonic transducer is integrated into the head of the pair of tweezers holding the sample, and show that this design is able to reduce preferred orientation. We demonstrate this for hemagglutinin, the canonical example of preferred orientation, and show that the fraction of side and tilted views increases with the excitation amplitude. This allows us to obtain a complete reconstruction of the trimer at 2.5 [A].

The visualisation of large biological samples in cryo-electron tomography requires both few-nanometre resolution and a wide, ideally >10 micrometrre, field of view. For a single image, increasing magnification trades increased resolution for a smaller field of view. Montaging, where high-magnification images are stitched together to increase field of view, addresses the trade-off but is dose-inefficient with round beams that are standard in transmission electron microscopy (TEM), since this illumination shape does not tessellate. Our workflow greatly simplifies montaging schemes for dose-sensitive specimens, allowing large montages of any shape - from individual cells or other features of interest up to whole lamellae - to be collected at high resolution without compromising electron dose efficiency. We demonstrate the workflow by acquiring montage tomograms of whole yeast (Saccharomyces cerevisiae) cells in cryo-FIB lamellae and of human neurons.

Tardigrades are minute animals renowned for environmental stress tolerance, and their remarkable biology has motivated extensive molecular studies using biochemical and biophysical approaches. Yet the native structures of endogenous macromolecular assemblies in tardigrades remain largely inaccessible, in part because only limited biological material is available. Here we show that purification-free single-particle cryo-EM, guided by long-read cDNA sequence assignment, can recover multiple endogenous assemblies directly from crude extract of a wild-derived Macrobiotus cf. shonaicus culture. We reconstructed a dimeric vitellogenin (Vg) particle at 3.10 [A] resolution, a previously uncharacterized tardigrade ferritin at 3.09 [A] resolution, and a low-resolution ribosome map. The Vg particle is a glycosylated and metal-containing assembly with structural evidence of proteolytic processing and contains lipid-like densities occupying a broad chamber whose walls include N-linked glycans. The ferritin structure revealed a divergent C-terminal helix that lines a mineral-like density observed at a position corresponding to previously described mineralization sites in ferritin-family cages. These results establish purification-free cryo-EM as a route to structural analysis of endogenous macromolecular assemblies from minute animal samples. In tardigrades, this approach provides a foundation for future state-dependent structural studies, including desiccation, rehydration and reproduction.

Structural model building and refinement need many decisions between different programs. These decisions often depend on an experienced researcher who knows how the programs work together and how to read experimental data. We used Claude Code with Claude Opus 5.5 to supervise this work for 24 cryo-EM and X-ray cases. The work finished in about 36 hours on one workstation. The agent prepared the inputs, ran established programs, read the validation results, compared candidate models and chose the next step. For 15 deposited cryo-EM models with weak starting geometry, the median MolProbity score improved from 2.62 to 1.76. For three crystal structures, the agent models came close to the published Rfree values. Chemical information such as ligands, ions and modifications must be provided by the researcher, in the same way as the sequence. With this information, the agent can place and check these components. These results show that agent supervision is acceptable when every decision can be inspected. The researcher makes the final scientific judgment. We suggest practical requirements for this kind of use. Future AI agents may help researchers obtain high quality and reliable structures of biological macromolecules.

Time-resolved crystallography can be inaccessible for membrane proteins and other difficult-to-crystallize targets for which suitable crystals are scarce. Here we present an acquisition strategy that records consecutive diffraction images during a small crystal rotation synchronized to a single activation pulse. Matching image segments are merged across crystals to reconstruct successive time windows, enabling a single crystal ensemble to capture an extended structural trajectory. Using the Mesh&Collect workflow at a synchrotron, we obtained ten consecutive 7.5-ms time windows at 2 [A] resolution from 1,255 microcrystals of a bacteriorhodopsin triple mutant at room temperature. An additional 790 crystals extended the observation window to 500 ms. Rotation-based processing, retrospective time binning and dose control maximize information extracted from each crystal, while compatible measurements can be pooled across acquisitions. This approach makes millisecond time-resolved crystallography practical for crystal-limited targets at synchrotrons, with further development potentially extending the temporal resolution down to 1 ms.

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus (GkLip). The 2.47-[A] orthorhombic model contains a Ca2+ ion at the conserved metal-binding site, whereas the 2.30-[A] anisotropic triclinic model contains four non-equivalent protein chains in which no Ca2+ ion was modeled. GkLip retains the canonical /{beta}-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn2+-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca2+ coordination environment. Pro367 adopts a C{gamma}-endo pucker in the Ca2+-containing structure; three Ca2+-free chains also adopt the endo pucker, whereas one adopts a C{gamma}-exo pucker. The presence of both Pro367 pucker states in the Ca2+-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Atomistic simulations often struggle to capture the full conformational landscape of biomolecular complexes on experimentally relevant timescales, leaving gaps in our understanding of functionally relevant thermodynamics and kinetics. Cryo-electron microscopy (cryo-EM) provides high-resolution structural snapshots spanning multiple conformational states, but translating these static images into dynamic mechanisms remains challenging. Existing approaches for integrating cryo-EM data with molecular simulations either perturb the underlying dynamics through artificial biasing potentials or remain confined to conformations already present in the prior ensemble. We introduce cryoWEight, an automated and extensible Python framework that couples iterative weighted ensemble (WE) simulations with ensemble reweighting against cryo-EM particle images to steer simulation trajectories towards conformational states consistent with cryo-EM populations. We have found that reweighting in the image space introduces a directional bias towards the target distribution even when the prior ensemble has no overlap with the target distribution in the collective variable space. The reweighting requires only the state likelihoods that set the ensemble weights, and any observable that defines these likelihoods can serve as the target distribution, therefore generalizing beyond the application of cryo-EM. cryoWEight is validated on three proteins: chignolin, the N-terminal domain of ribosomal protein L9, and adenylate kinase. The framework converges towards the target conformational states across different sampling strategies and noise levels, providing a scalable route to integrate cryo-EM data with enhanced sampling approaches.

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

Nuclear segmentation in 3D microscopy remains challenging, particularly for low signal-to-noise ratio (SNR) data from timelapse imaging, in vivo experiments, or dense tissue samples. Yet accurate 3D nuclear segmentation is a prerequisite for quantitative single-cell analysis, making this a key bottleneck for high-throughput study of challenging datasets. We present NucLogic, a fast and robust 3D nuclear segmentation algorithm that combines the maturity of 2D segmentation with geometric stitching rules based on nuclear shape, orientation, and intensity, to reconstruct accurate 3D volumes. NucLogic reliably segments challenging datasets including long-term timelapse imaging of organoids, and large dense structures such as the zebrafish brain. Benchmarked against other state-of-the-art segmentation tools, NucLogic matches F1 scores at high SNR, with increasing outperformance as SNR decreases. Importantly, segmentation completes at 20-50% of the computation time, with greater speed gains on larger images. Beyond segmentation, NucLogic includes an intuitive graphical user interface allowing non-experts to segment, quantify, and visualize 3D and 4D data. This enables easy quantification of cell numbers and integration of a variety of both cell-intrinsic properties, such as phenotype, morphology, and local environment metrics, including density and spatial interactions. Together, NucLogic makes quantitative 3D nuclear analysis of previously unmanageable datasets accessible to a broad research community.

Serial-sectioning electron microscopy allows the exploration of the 3D ultrastructure of cells, tissues or whole organisms. Sample preparation is a critical step for this technique, which determines the quality of downstream imaging and analysis. Here we optimize a high-pressure freezing sample-preparation workflow for larvae of the marine annelid Platynereis dumerilii. We tested several fixation and staining methods and screened a large number of specimens by synchrotron X-ray imaging to evaluate specimen integrity and contrast. We further evaluated selected blocks by array tomography SEM to select the best performing protocols. Our protocols and robust workflow could be adapted to similar marine planktonic organisms including ciliated invertebrate larvae.

Glycosyltransferases have broad diversity in their acceptor substrates, yet the molecular and evolutionary basis of this diversity remains poorly understood. NodZ is an -1,6-fucosyltransferase that fucosylates Nod factors, lipochitooligosaccharide molecules involved in the establishment of rhizobium-legume symbioses. While NodZ is the only known bacterial member of the GT23 family of -1,6-fucosyltransferases, the determinants of its acceptor selectivity and the evolutionary origins thereof are unclear. Here, a phylogenetic analysis of 332 NodZ-like proteins revealed a distinct rhizobial clade and significantly greater structural diversity in the N-terminus than in the C-terminus. Kinetic analysis of a Mesorhizobium japonicum NodZ demonstrated selectivity for longer chitooligosaccharide acceptors (comprised of four to five GlcNAc units), while alanine scanning revealed W139 as a primary determinant of this chain length preference. We propose that W139 forms an aromatic platform that engages distal GlcNAc units present only in longer acceptors, coupling productive binding to acceptor chain length. We then used the NodZ phylogeny to reconstruct ancestral NodZ homolog sequences, which indicated that W139 emerged at the base of the rhizobial clade, and thus may represent a key evolutionary step towards canonical NodZ function. Additionally, the most recent resurrected ancestor was capable of fucosylating chitooligosaccharides with lower efficiency than the extant NodZ, suggesting that efficiency towards chitooligosaccharides was improved during the evolution of this family. Results described here demonstrate that evolutionary diversification of the N-terminal region can remodel acceptor specificity while preserving the conserved catalytic architecture of GT23 fucosyltransferases, providing a framework for understanding and engineering fucosyltransferase specificity.

Nitrate is an important oxyanion in bacterial physiology, with major roles as a nitrogen source for assimilation and as an alternative electron acceptor in anaerobic respiration. Accordingly, diverse nitrate-responsive regulatory systems sense nitrate availability and control the expression of genes involved in nitrate transport and metabolism. In the NreABC system, the soluble GAF-like domain protein NreA functions as a cellular nitrate sensor. To date, our molecular understanding of nitrate recognition by NreA has been derived primarily from studies of the Staphylococcus carnosus protein, with less known about homologues from other species. Here, we identified 736 NreA homologues through bioinformatic analysis of the GAF-like domain superfamily and selected five staphylococcal representatives for thermodynamic characterization by isothermal titration calorimetry. Despite conservation of the nitrate-binding pocket, these homologues resolved into high- and low-affinity groups with distinct thermodynamic profiles. Comparative structural modeling and chimeragenesis of a low-affinity homologue revealed that the C-terminal region is a non-coordinating determinant of nitrate-binding affinity. Together, these findings show that a conserved nitrate-binding pocket preserves recognition, while distal sequence variation shapes thermodynamics and tunes the nitrate concentration range over which NreA sensors can function.

TIR (Toll/interleukin-1 receptor) domains are central components of innate immunity and cell-death pathways across diverse forms of life. In bacteria, TIR domain-containing proteins participate in diverse antiphage defence systems, several of which rely on NADase activity carried out by TIR domains. This nucleosidase activity is often regulated by additional domains that modulate activation and specificity in response to infection. AbTir, a TIR domain-containing protein from Acinetobacter baumannii, contains an N-terminal CC domain that negatively regulates its NADase activity, but the structural basis of this regulation remains unknown. Here, we used X-ray crystallography to investigate the molecular basis of CC domain-mediated regulation in AbTir. We determined the crystal structure of full-length AbTir in complex with a nanobody, revealing a symmetric AbTir TIR dimer in which only one of the two symmetry-related CC helices is accommodated at the dimer surface, breaking the overall symmetry of the full-length protein. Structural comparisons with other bacterial TIR domains suggest that such TIR dimers likely represent an inactive state, in which the NAD+-binding site has not yet formed. Phylogenetic analysis places AbTir within a family of bacterial TIR proteins associated with defence-related genomic loci, although phage-spot assays performed in the surrogate host Escherichia coli did not detect measurable antiphage activity. Together, these findings reveal an unusual structural mechanism of CC domain-mediated inhibition of a bacterial TIR NADase and expand our understanding of the mechanistic diversity of bacterial TIR proteins.

The clonogenic assay is the foundational standard for evaluating cellular radiosensitivity, yet its manual quantification is limited by labor-intensive processing, high inter-observer variability, and a reductive binary scoring system. Here, we present and validate a deep learning-based segmentation pipeline for the automated, high-throughput quantification of mammalian cell survival across diverse radiation qualities (photons, protons, and carbon ions). By benchmarking against expert consensus using Bland-Altman analysis, we demonstrate that this AI framework consistently operates within natural human error margins. Crucially, transitioning to automated whole-well image-based analysis enables the extraction of continuous multidimensional data, including colony area, density, and cellularity. This phenotypic profiling successfully identified and quantified high-cellularity colonies formed by radioresistant BxPC-3 cells under particle irradiation. Coupled with rapid image acquisition platforms, this methodology significantly reduces processing bottlenecks while standardizing the derivation of relative biological effectiveness (RBE). By advancing beyond binary survival metrics to continuous morphological profiling, this framework provides a vital new analytical dimension for investigating structural mechanisms of radioresistance.

While cryogenic electron microscopy (cryo-EM) has transformed structural biology, sample preparation remains a major determinant of success. Specifically, the air-water interface (AWI) can damage complexes, impose preferred orientation, and reduce particle density. Here, we introduce cryoSHIELDs, a chemically diverse library of peptide additives designed to passivate the AWI and improve cryo-EM grid preparation. Across 24 protein systems spanning GPCRs, ABC and SLC transporters, soluble and Fab-containing complexes, and enzymes, cryoSHIELDs increased the success rate of structure determination. Individual cryoSHIELDs improved particle density, angular sampling, complex stability, and, for routine targets, map quality and resolution. This toolkit enabled the structural characterization of important targets, including the ABCC6 transporter, revealing unique TMD0-mediated stabilization, and DAMGO-bound -opioid receptor-Gi complex using substantially lower protein concentration. CryoSHIELDs provide a simple, tunable, and broadly applicable platform for more reliable cryo-EM in discovery biology and structure-based drug development.

Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.

Glycine transporter 1 (GlyT1) is a clinically investigated target whose conformational plasticity enables ligands to engage distinct extracellular and intracellular binding sites. However, methods to directly quantify intracellular binding remain limited and largely rely on radioligand-based approaches. Here, we report a NanoBRET-based binding assay to quantify intracellular GlyT1 target engagement in living cells using a fluorescent tracer derived from the GlyT1 inhibitor Cmpd1. With a structure-based approach followed by pharmacological characterization we identified a suitable meta-substituted exit vector for linker and fluorophore attachment while maintaining GlyT1 binding. A series of fluorescent probes was synthesized and evaluated, leading to the development of GlyT1-probe which displayed high-affinity binding and enabled robust NanoBRET measurements. Unlike the functional uptake assay, the NanoBRET assay distinguished intracellular from extracellular GlyT1 inhibitors and resolved structure-activity relationships at the intracellular binding site. This assay provides a quantitative approach to interrogate conformation-selective GlyT1 target engagement in living cells, facilitating the characterization of GlyT1 ligands targeting the intracellular site.