Journal of Structural Biology: X

Researchers using electron microscopy must often balance a trade-off between obtaining high-resolution structural information and preserving sufficient cellular context. At one end of this spectrum, single particle cryo-electron microscopy and cryo-electron tomography provide near-molecular detail but are typically limited to relatively small fields of view. At the other, volume electron microscopy approaches, such as scanning electron microscopy of resin-embedded specimens, capture large cellular volumes but generally at lower resolution. Consequently, linking nanoscale structural information to larger cellular architecture remains a significant challenge. To address this gap, we optimised a transmission electron tomography workflow for resin-embedded malaria parasites that allows us to visualise targeted regions of interest at nanometre-scale resolution while retaining several micrometres of surrounding cellular context. Here, we present our current best-practice pipeline for sample preparation, tomogram acquisition, and reconstruction. In addition, we introduce VolWeaver, a data-processing framework, that integrates high-resolution tomographic datasets into serial section volume reconstructions, enabling the visualisation and interpretation of ultrastructural features within their broader cellular environment.

Single-particle cryo-EM routinely delivers near-atomic structures; however, optimizing data collection requires a delicate balance among magnification, sampling bandwidth, and particle throughput. Although low-magnification super-resolution imaging can recover information beyond the physical Nyquist limit, its benefit varies substantially among specimens. This variability suggests that the effectiveness of super-resolution may depend on whether reconstruction is limited by detector sampling bandwidth or by the recoverable particle signal. However, the conditions that distinguish these two regimes remain poorly defined. Here, we systematically compared super-resolution and physical-pixel workflows at two magnifications using apoferritin (APO) and malate synthase G (MSG) as representative specimens with contrasting molecular size, symmetry, and image contrast. At low magnification, APO exhibited sampling-limited behavior, with super-resolution processing achieving 1.72 Angstrom compared with 2.74 Angstrom for physical-pixel processing. In contrast, MSG exhibited predominantly signal-limited behavior under the same conditions, yielding comparable resolutions of 3.05 Angstrom and 2.98 Angstrom for super-resolution and physical-pixel processing, respectively, despite the increased sampling bandwidth. These contrasting responses were further supported by particle-number saturation, per-particle motion correction, and Q-score analyses, which provided complementary evidence for the underlying sampling-limited and signal-limited regimes. Together, these results provide a practical framework for assessing whether reconstruction quality is predominantly constrained by sampling bandwidth or recoverable particle signal and offer a rational basis for balancing achievable resolution and particle throughput when selecting acquisition strategies.

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.

Cryo-electron ptychography is an emerging technique for studying radiation-sensitive biological specimens, developed from four-dimensional transmission electron microscopy (4D-STEM). Although ptychography has achieved ultrahigh resolution beyond conventional transmission electron microscopy limits for radiation-resistant samples, its application to frozen hydrated biological specimens currently remains at sub-nanometer resolution. Here we overcome this limitation by implementing electron counting with a hybrid-pixel detector, establishing key technical foundations for near-atomic-resolution cryo-ptychography. This counting approach significantly improves weak signal detection in convergent-beam electron diffraction, enabling ptychographic reconstruction at doses below 1 e-/[A]{superscript 2}. Additionally, we found beam-induced motion is effectively eliminated within single scans, suggesting conventional cryoEMs dose-fractionation approach may need reevaluation. Demonstrating high contrast under both low-dose and tilted conditions, along with achieving 3.59 [A] resolution for the [~]700 kDa T20S proteasome, we validate cryo-electron ptychography as a viable general imaging modality that could complement or surpass conventional phase-contrast cryoEM methods.

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

Dinoflagellate chromosomes adopt a highly condensed and organized morphology, with periodic bands and arches observed by traditional Transmission Electron Microscopy (TEM). However, the limited two-dimensional field of view of TEM has prevented a precise characterization of the inherently three-dimensional organization of dinoflagellate chromosomes. Moreover, given the vast diversity among dinoflagellate species and the lack of a systematic comparison of their chromosomes, it remains unclear whether dinoflagellate chromosomes share common organizational features or instead exhibit significant cell- or species-specific differences. Here, we acquire three whole-nucleus 3D Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) datasets at 4 nm voxel size for each of four dinoflagellate species: Symbiodinium microadriaticum, Breviolum minutum, Fugacium kawagutii, and Crypthecodinium cohnii. We compile these data with previously published image volumes from four additional species and present an analysis of the largest collection of dinoflagellate FIB-SEM images to date. Common features observed across all eight species include the absence of physical confinement or spatial clustering of chromosomes in the nucleus. In addition, by decomposing each chromosome into a weighted sum of orthogonal shapes using Spherical Harmonics Expansion, we find a principal component encapsulating 88% of the total shape variance that is common to all species. However, our analysis also reveals differences in chromosome morphology across species. First, while many chromosomes exhibit surface ridges with left-handed helical twist, the proportion of chromosomes with such ridges varies extensively across species. Second, while chromosomes in most species are discrete and well-separated, chromosomes in F. kawagutii are interconnected in a single contiguous network. Lastly, to our knowledge, we report the first observation in eukaryotic cells of toroid-shaped DNA objects, whose numbers vary dramatically across cells and species. Overall, our results show that dinoflagellate chromosomes exhibit both shared organizational features and pronounced species-specific deviations.

Cryo-electron tomography of 100-200 nm lamellae prepared by focused ion beam milling enables structural analysis of macromolecules within cells, tissues, and small organisms, but lamella preparation remains time-consuming and limits throughput. Although automated workflows enable unsupervised fabrication, they commonly produce variable results because fixed, pre-calibrated parameters are applied without accounting for lamella-to-lamella variability. To overcome these limitations, we developed an Adaptive Milling approach that adapts milling parameters based on imaging feedback. We implemented this approach for the critical polishing stage as Adaptive Polishing, which uses machine-learning-based segmentation of scanning electron microscopy images to identify lamella features predictive of imminent collapse. These are used to periodically assess whether to continue or stop thinning during polishing. We performed a systematic comparison of the quality of lamellae prepared with Adaptive Polishing and conventional automation, and demonstrate that Adaptive Polishing produces thinner lamellae with greater consistency in thickness, and performs well for in situ cryo-electron tomography. Adaptive Polishing was tested on three different microscopes with minimal model retraining required, demonstrating transferability of the approach. We implemented Adaptive Polishing as an open-source plugin for fibsemOS together with machine-learning model training software and pre-trained models.

Microtubules are cytoskeletal filaments that are involved in intracellular transport, cell division, and motility. Despite their biological importance, determining their high-resolution structures via cryo-electron microscopy remains a significant technical challenge due to their polymorphisms and pseudo-helical assembly. Current processing workflows are complex, often requiring the integration of multiple software packages and custom scripts, which creates a steep learning curve for many research groups. To address these limitations, we introduce CsMT, a streamlined workflow implemented entirely within the CryoSPARC environment and using synthetic references. CsMT simplifies microtubule reconstruction by utilizing a novel protofilament-pair classification approach, which effectively handles the inherent pseudo-symmetry and structural heterogeneity of microtubules with minimal manual intervention. Our workflow is versatile, capable of processing both undecorated and decorated microtubules while accurately determining seams and performing high-resolution refinement. We demonstrate the efficacy of this workflow by achieving a 2.3 and 2.7 [A] resolution reconstruction of homotypic and heterotypic maps of undecorated microtubules, matching the best-resolved microtubule structures in the field. By unifying the pipeline into a single and portable workflow, CsMT enhances reproducibility and accessibility, empowering more laboratories to explore the structural biology of microtubules and associated proteins, yielding new insights into their function.

Cryogenic plasma focused ion beam-scanning electron microscopy (cryo-pFIB-SEM) enables nanoscale volume imaging of vitrified cells and tissues, but the fidelity of each newly exposed block-face depends on how ions interact with compositionally heterogeneous biological material. Using xenon, argon, oxygen, and nitrogen plasma beams across a 30-2 kV voltage range, we examined how ion species and accelerating voltage influence block-face quality and ultrastructural fidelity. Curtaining imposed a species-dependent lower limit on accelerating voltage, with xenon and argon supporting uniform milling to 10 kV and oxygen and nitrogen to 15 kV. Monte Carlo simulations predicted substantially reduced ion penetration and atomic displacement under these species-specific low-kV conditions. Experimentally, lowering milling voltage suppressed charging and topographic artifacts, with the largest effects observed at heterogeneous interfaces. Oxygen and nitrogen produced greater membrane sharpness and organelle contrast than the noble gases, and these differences influenced machine-learning-based recognition of fine organelle features in 3D datasets. Together, these findings show that cryo-pFIB-SEM image quality cannot be optimized by accelerating voltage or ion species independently. Instead, milling performance reflects a tradeoff among surface fidelity, charging, contrast, and curtaining that depends on local specimen composition. Species-specific low-kV milling therefore provides a practical framework for matching cryo-pFIB-SEM acquisition conditions to the structural and biophysical properties of the biological target.

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.

Dynamics in proteins occur on a wide range of timescales and are crucial for protein function. On the fast end of that timescale, pico- to nanosecond dynamics have been extensively employed as proxies for entropy and their amplitude can be described by order parameters. Experimentally, NMR can be used to determine order parameters of the protein backbone and, via deuterium relaxation, of methyl groups, yet such experiments cannot be applied to large protein assemblies. In contrast, relaxation-violated coherence transfer experiments, that allow for the determination of side chain order parameters in highly deuterated, methyl-labeled proteins, are more sensitive. Here, we demonstrate that such experiments can be applied to very large, asymmetric protein assemblies by determining axial methyl order parameters for the 300 kDa fully asymmetric core of the eukaryotic RNA exosome complex. Ile-{delta}1[13CH3] methyl groups adopt a wide range of order parameters but highly flexible side chains are infrequent. High quality data, which we obtain for flexible regions, is required to observe subtle effects of RNA binding on order parameters. Local cryo-EM Q-scores correlate moderately with order parameters suggesting that Q-scores contain information on nanosecond motions. AF2{chi}, a recently described prediction tool for side-chain variability, provides good estimates of methyl order parameters, which are, in favorable cases, strongly correlated with experimental values. We thus demonstrate that relaxation-violated coherence transfer experiments can be employed to determine order parameters in large, asymmetric protein complexes that are difficult to capture by other methods, yet are crucial for the understanding of protein function. SignificanceNanosecond side chain dynamics contribute to the entropy of proteins and are therefore proxies for protein stability and binding. Here, we demonstrate that NMR can be employed to experimentally quantify nanosecond dynamics in large, asymmetric proteins paving the way to assess contributions of fast dynamics to the quality of static protein structures. Furthermore, we employ the experimental data to validate computational methods that provide structural insights into nanosecond dynamics.

Damage to biological specimens by the electron beam is the fundamental resolution-limiting factor in cryoelectron microscopy (cryo-EM) single particle analysis. There is, however, currently no method to accurately infer fluence-dependent changes to the specimen structure during electron irradiation. We develop a Bayesian framework to fit a sequence of atomic models to a series of cryo-EM reconstructions produced at increasing fluence. In particular, our algorithm is able to infer the ensemble average position and atomic displacement parameter of every atom in the macromolecule as a function of fluence. Application of the algorithm to cryo-EM datasets shows that the molecule expands during imaging and identifies environment-dependent variations in beam-induced damage. We use our results to propose a stochastic process model of this phenomenon. We envisage that our method will lead to a better mechanistic understanding of radiation damage to biological specimens and may contribute to efforts to mitigate its effects.

The ability of cryo-electron microscopy (cryo-EM) to interrogate the atomic structure and motion of biomolecules has motivated the development of a wide range of algorithms for heterogeneity analysis. However, evaluating and comparing these methods remains challenging because ground-truth structures are generally unknown for experimental samples. Here, we introduce the 2026 Community-Wide Assessment of Heterogeneous Reconstruction Algorithms (CAHRA), a community-wide challenge centered on three benchmark datasets that probe distinct tasks in heterogeneity analysis. These include (1) compositional heterogeneity arising from mixtures of distinct protein complexes, (2) continuous conformational variability and atomic modeling, and (3) entanglement between molecular conformation and particle pose. We describe the design, construction, and validation of these datasets, as well as their use in the recently completed CAHRA Challenge.

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.

In molluscs, chitin synthases are essential for biologically controlled biomineralization, with some variants possessing a myosin motor domain that may link polymer synthesis to the cytoskeleton. Experimentally, we established a reliable workflow for expressing ArCS1_E22TM in Dictyostelium discoideum and developed effective purification methods to reconstitute ArCS1_E22TM in nanodiscs using MSPs and specific lipid composition. MSP1D1deltaH5 proved optimal for nanodisc formation, yielding homogeneous, monodisperse discs (~8.2 nm). Lipids were refined to POPC:POPE:POPG (3:1:1) with 20% cholesterol, improving nanodisc quality and uniformity as observed by negative-stain EM. The full-length ArCS1 and its subdomains were modeled using AlphaFold3; the myosin motor, glycosyltransferase, and transmembrane regions are well-defined internally but loosely constrained relative to one another, suggesting flexible linking and conformational coupling. Modelling with Mg2+ and oleic acid as ligands and comparative analyses with bacterial cellulose synthase and yeast chitin synthase 1 provided insights into substrate binding and a potential mechanism for chitin polymerization and translocation. This research establishes a standard procedure for comprehensive structural analyses of recombinant molluscan chitin synthase in near-native or biomimetic membranes. This sets the stage for high-resolution cryo-electron microscopy to determine the first experimentally resolved structure of a molluscan chitin synthase and to provide insight into the enzyme's architecture and the regulatory mechanisms of biomineralization.

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].

Visualizing macromolecules within their native cellular context by cryo-electron tomography (cryo-ET) is fundamentally limited by the trade-off between field of view and resolution: Capturing high-resolution information about biomolecules requires high magnification, which restricts the field of view and obscures the cellular context in which those biomolecules function. Collecting montage data by tiling the electron beam over the region of interest offers one solution, although traditional round electron beams cause excessive radiation damage across overlapping regions. We previously made electron beams square in shape, enabling montage collection with minimal overlap and thereby reducing excessive exposure and loss of high-resolution information. Here, we create a pipeline for collecting and processing montage cryo-ET data with square electron beams. We show that square beam montages retain high-resolution information by reconstructing virus-like particles to 3.5 [A] resolution using sub-tomogram averaging, and apply the workflow to imaging a glial cell and malaria parasite lamellae over fields of view up to 65 m2. We also provide a comprehensive protocol to make square beams accessible to the community.

Bacterial flagellar filaments are often modified by glycans, but the structural basis and physiological significance of flagellin glycosylation remain poorly understood in many species. Vibrio alginolyticus produces lateral flagella for surface-associated motility in viscous environments, with LafA forming its filament as flagellin. A maf homolog encoding a putative flagellin glycosylation factor is located immediately downstream of lafA, suggesting that the lateral filament is glycosylated. To investigate this possibility, we purified the lateral flagellar filament from V. alginolyticus and determined the structure at 2.37 [A] resolution by electron cryomicroscopy. Upon model building of LafA in the map, we identified additional densities connected to five serine residues, possibly corresponding to O-linked pseudaminic acid modifications. Mass spectrometric analyses identified these modifications as pseudaminic acid attached to Ser148, Ser173, Ser183, Ser189, and Ser197. Deletion of maf abolished lateral flagella formation and motility, and these defects were restored by complementation with maf. These results demonstrate that the Vibrio lateral flagellar filament is extensively modified by pseudaminic acid and that Maf is required for filament formation. Our findings provide the first structural insight into the glycosylation of Vibrio lateral flagellar filament and establish a framework for understanding the role of flagellin glycosylation in surface-associated motility.

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.

Here, we present MC-Bayes, a Python-based script for processing cryo-electron microscopy EER movies on one or more GPUs using MotionCor3 in a user-friendly manner. Further, it generates the .star files necessary for RELION to perform Bayesian polishing (a.k.a.: reference-based motion correction) with EER movies. Until now, Bayesian polishing of EER data was only possible if the CPU-based "RELIONCor" implementation of MotionCor2 was used, which is sub-optimal on GPU-heavy cryo-EM processing systems. This wrapper was created for those facilities and/or users who may have (many) powerful GPUs, but for whatever reason have few CPU cores or less system RAM. Leveraging MotionCor3, MC-Bayes allows motion correction of EER data 2 or more times faster (depending on system) than the RELION CPU implementation, except in circumstances where dozens or hundreds of CPU cores with high quantities of system RAM can be utilised.