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For cryo‐electron tomography (cryo‐ET) of beam‐sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal‐to‐noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample‐environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo‐ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low‐symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron‐beam damage to the specimen is an issue and the first images acquired will transfer more high‐resolution information than those acquired last. There is also an inherent trade‐off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n‐helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose‐symmetric tilt schemes for cryo‐ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose‐symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition. [ABSTRACT FROM AUTHOR]

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near‐atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD‐related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology. [ABSTRACT FROM AUTHOR]

Cryo‐electron tomography (cryo‐ET) enables molecular‐resolution 3D imaging of complex biological specimens such as viral particles, cellular sections and, in some cases, whole cells. This enables the structural characterization of molecules in their near‐native environments, without the need for purification or separation, thereby preserving biological information such as conformational states and spatial relationships between different molecular species. Subtomogram averaging is an image‐processing workflow that allows users to leverage cryo‐ET data to identify and localize target molecules, determine high‐resolution structures of repeating molecular species and classify different conformational states. Here, STOPGAP, an open‐source package for subtomogram averaging that is designed to provide users with fine control over each of these steps, is described. In providing detailed descriptions of the image‐processing algorithms that STOPGAP uses, this manuscript is also intended to serve as a technical resource to users as well as for further community‐driven software development. [ABSTRACT FROM AUTHOR]

Data acquisition and processing for cryo‐electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo‐ET workflow, Tomo Live, an on‐the‐fly solution that automates the alignment and reconstruction of tilt‐series data, enabling real‐time data‐quality assessment, has been developed. Through the integration of Tomo Live into the data‐acquisition workflow for cryo‐ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt‐series acquisition has completed, an unattended tilt‐series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non‐expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology. [ABSTRACT FROM AUTHOR]

The axoneme, a microtubule‐based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo‐EM and cryo‐ET have allowed a molecular‐level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo‐EM and cryo‐ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI‐guided tools that are transforming structural biology. [ABSTRACT FROM AUTHOR]

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism.

The breakthrough in cryo-electron microscopy (cryo-EM) technology has led to an increasing number of density maps of biological macromolecules. However, constructing accurate protein complex atomic structures from cryo-EM maps remains a challenge. In this study, we extend our previously developed DEMO-EM to present DEMO-EM2, an automated method for constructing protein complex models from cryo-EM maps through an iterative assembly procedure intertwining chain- and domain-level matching and fitting for predicted chain models. The method was carefully evaluated on 27 cryo-electron tomography (cryo-ET) maps and 16 single-particle EM maps, where DEMO-EM2 models achieved an average TM-score of 0.92, outperforming those of state-of-the-art methods. The results demonstrate an efficient method that enables the rapid and reliable solution of challenging cryo-EM structure modeling problems. [ABSTRACT FROM AUTHOR]

Cryoelectron microscopy (cryo-EM) methods have made meaningful contributions in a wide variety of scientific research fields. In structural biology, cryo-EM routinely elucidates molecular structure from isolated biological macromolecular complexes or in a cellular context by harnessing the high-resolution power of the electron in order to image samples in a frozen, hydrated environment. For structural chemistry, the cryo-EM method popularly known as microcrystal electron diffraction (MicroED) has facilitated atomic structure generation of peptides and small molecules from their three-dimensional crystal forms. As cryo-EM has grown from an emerging technology, it has undergone modernization to enable multimodal transmission electron microscopy (TEM) techniques becoming more routine, reproducible, and accessible to accelerate research across multiple disciplines. We review recent advances in modern cryo-EM and assess how they are contributing to the future of the field with an eye to the past. [Display omitted] In this review, de la Cruz and Eng discuss major developments in single-particle analysis, electron tomography, and microcrystal electron diffraction to show how cryo-EM has matured to become an ever-improving technology for visualizing molecular structure. [ABSTRACT FROM AUTHOR]

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET. [ABSTRACT FROM AUTHOR]

ABSTRACT Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24–66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.

Two of the most fascinating bacterial nanomachines—the broadly disseminated rotary flagellum at the heart of cellular motility and the eukaryotic cell–puncturing injectisome essential to specific pathogenic species—utilize at their core a conserved export machinery called the type III secretion system (T3SS). The T3SS not only secretes the components that self-assemble into their extracellular appendages but also, in the case of the injectisome, subsequently directly translocates modulating effector proteins from the bacterial cell into the infected host. The injectisome is thought to have evolved from the flagellum as a minimal secretory system lacking motility, with the subsequent acquisition of additional components tailored to its specialized role in manipulating eukaryotic hosts for pathogenic advantage. Both nanomachines have long been the focus of intense interest, but advances in structural and functional understanding have taken a significant step forward since 2015, facilitated by the revolutionary advances in cryo-electron microscopy technologies. With several seminal structures of each nanomachine now captured, we review here the molecular similarities and differences that underlie their diverse functions. [ABSTRACT FROM AUTHOR]

The nucleosome is a DNA–protein assembly that is the basic unit of chromatin. A nucleosome can adopt various structures. In the canonical nucleosome structure, 145–147 bp of DNA is wrapped around a histone heterooctamer. The strong histone-DNA interactions cause the DNA to be inaccessible for nuclear processes such as transcription. Therefore, the canonical nucleosome structure has to be altered into different, non-canonical structures to increase DNA accessibility. While it is recognised that non-canonical structures do exist, these structures are not well understood. In this review, we discuss both the evidence for various non-canonical nucleosome structures in the nucleus and the factors that are believed to induce these structures. The wide range of non-canonical structures is likely to regulate the amount of accessible DNA, and thus have important nuclear functions. [ABSTRACT FROM AUTHOR]

A quantitative four‐dimensional scanning transmission electron microscopy (4D‐STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)‐milling of lamellae for (cryo‐)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub‐micrometer amorphous specimen using both direct detection and light conversion 2D‐STEM detectors in a coincident FIB‐SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam‐sensitive materials. [ABSTRACT FROM AUTHOR]

Cryogenic electron microscopy (cryo-EM) and electron tomography (cryo-ET) have become a critical tool for studying viral particles. Cryo-EM has enhanced our understanding of viral assembly and replication processes at a molecular resolution. Meanwhile, in situ cryo-ET has been used to investigate how viruses attach to and invade host cells. These advances have significantly contributed to our knowledge of viral biology. Particularly, prompt elucidations of structures of the SARS-CoV-2 spike protein and its variants have directly impacted the development of vaccines and therapeutic measures. This review discusses the progress made by cryo-EM based technologies in comprehending the severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2), the virus responsible for the devastating global COVID-19 pandemic in 2020 with focus on the SARS-CoV-2 spike protein and the mechanisms of the virus entry and replication.

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ (inside cells) resembles the canonical structure determined in vitro 25 years ago. Nucleosome structure in situ is otherwise poorly understood. Using cryo-electron tomography (cryo-ET) and 3D classification analysis of budding yeast cells, here we find that canonical nucleosomes account for less than 10% of total nucleosomes expected in situ. In a strain in which H2A-GFP is the sole source of histone H2A, class averages that resemble canonical nucleosomes both with and without GFP densities are found ex vivo (in nuclear lysates), but not in situ. These data suggest that the budding yeast intranuclear environment favors multiple non-canonical nucleosome conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average budding yeast nucleosome’s DNA is partially detached in situ.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments—the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin–myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo–electron microscopy and cryo–electron tomography in leading muscle research into a new era. [ABSTRACT FROM AUTHOR]

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompa and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions. [ABSTRACT FROM AUTHOR]

Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the in situ structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant. [ABSTRACT FROM AUTHOR]

The recent proliferation of cryo-electron tomography (cryo-ET) techniques has led to the cryo-ET resolution revolution. Meanwhile, significant efforts have been made to improve the identification of targets in the cellular context and the throughput of cryo-focused ion beam (FIB) milling. Together, these developments led to a surge of in situ discoveries on how enveloped viruses are assembled and how viruses interact with cells in infected hosts. In this article, we review the recent advances in cryo-ET, high-resolution insights into virus assembly, and the findings from inside infected eukaryotic and prokaryotic cells. [ABSTRACT FROM AUTHOR]

Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of armc4 caused complete loss of OAD, whereas mutation of calaxin caused only partial loss of OAD. Detailed structural analysis revealed that calaxin-/- OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates.

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients. [ABSTRACT FROM AUTHOR]

Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle. [Display omitted] • Integrated molecular architecture of assembled foamy virus particles • High-resolution in situ structures of Env surface glycoprotein and Gag CA capsid • Foamy virus Env is related to membrane fusogens of paramyxo- and coronaviruses • Gag CA capsid is icosahedral with features of pleomorphic orthoretroviral capsids The high-resolution in situ structural analysis of the prototype foamy virus icosahedral capsid and envelope glycoprotein provides insight into the viral infection process and evolutionary history. [ABSTRACT FROM AUTHOR]

Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes ex vivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila. [Display omitted] • Easy-to-use program to automatically fit protein domains into cryo-EM/ET maps • Testing shows DomainFit performs best in 3–8 Å resolution range • New ciliary protein discovered with DomainFit Cryoelectron microscopy enables structural determination of native macromolecular complexes, which might contain unidentified densities at a resolution that might prevent the identification. Gao and Tong et al. present DomainFit. By fitting predicted protein domains from AlphaFold2 prediction, DomainFit identifies the best-fitting domains in a given density, allowing protein identification. [ABSTRACT FROM AUTHOR]

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission. [Display omitted] • Dynamin is organized as a 2-start helix on membranes primed for fission • Dynamin-decorated lipid tubules achieve super-constriction through GTP hydrolysis • Cryo-EM captures PH domain variable loops 1 and 4 interacting with membranes • Cryo-ET shows dynamin assemblies in vivo as an assortment of helical polymers Dynamin severs endocytic vesicles. Jimah and Kundu et al. discovered that dynamin, in a post-GTP-hydrolysis state, organizes into a super-constricted 2-start helix on membranes, ready for spontaneous membrane fission. Cryo-ET of dynamin assemblies in vivo revealed that they can accommodate a range of diameters, enabling membrane constriction and fission. [ABSTRACT FROM AUTHOR]

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now b-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the bA-bB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains. [ABSTRACT FROM AUTHOR]

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Axonemal dyneins are the driving force of motile cilia, while cytoplasmic dyneins play an essential role in minus-end oriented intracellular transport. Their molecular structure is indispensable for an understanding of the molecular mechanism of ciliary beating and cargo transport. After some initial structural analysis of cytoplasmic dyneins, which are easier to manipulate with genetic engineering, using X-ray crystallography and single-particle cryo-electron microscopy, a number of atomic and pseudo-atomic structural analyses of axonemal dyneins have been published. Currently, several structures of dyneins in the post-power stroke conformation as well as a few structures in the pre-power stroke conformation are available. It will be worth systematically comparing conformations of dynein motor proteins from different sources and at different states, to understand their role in biological function. In this review, we will overview published high- and intermediate-resolution structures of cytoplasmic and axonemal dyneins, compare the high-resolution structures of their core motor domains and overall tail conformations at various nucleotide states, and discuss their force generation mechanism., (© 2024 The Author(s). Cytoskeleton published by Wiley Periodicals LLC.)

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dutta and Acharya.)

Cryo-electron tomography (Cryo-ET) is an emerging technology for three-dimensional (3D) visualization of macromolecular structures in the near-native state. To recover structures of macromolecules, millions of diverse macromolecules captured in tomograms should be accurately classified into structurally homogeneous subsets. Although existing supervised deep learning–based methods have improved classification accuracy, such trained models have limited ability to classify novel macromolecules that are unseen in the training stage. To adapt the trained model to the macromolecule classification of a novel class, massive labeled macromolecules of the novel class are needed. However, data labeling is very time-consuming and labor-intensive. In this work, we propose a novel few-shot learning method for the classification of novel macromolecules (named FSCC). A two-stage training strategy is designed in FSCC to enhance the generalization ability of the model to novel macromolecules. First, FSCC uses contrastive learning to pre-train the model on a sufficient number of labeled macromolecules. Second, FSCC uses distribution calibration to re-train the classifier, enabling the model to classify macromolecules of novel classes (unseen class in the pre-training). Distribution calibration transfers learned knowledge in the pre-training stage to novel macromolecules with limited labeled macromolecules of novel class. Experiments were performed on both synthetic and real datasets. On the synthetic datasets, compared with the state-of-the-art (SOTA) method based on supervised deep learning, FSCC achieves competitive performance. To achieve such performance, FSCC only needs five labeled macromolecules per novel class. However, the SOTA method needs 1100 ∼ 1500 labeled macromolecules per novel class. On the real datasets, FSCC improves the accuracy by 5% ∼ 16% when compared to the baseline model. These demonstrate good generalization ability of contrastive learning and calibration distribution to classify novel macromolecules with very few labeled macromolecules.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Microvilli are actin-bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause gastrointestinal disorders; however, mechanisms controlling microvilli formation and organization remain unresolved. Here, we study microvilli by vitrifying the Caenorhabditis elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them with cryo-focused ion-beam milling, followed by cryo-electron tomography and subtomogram averaging. We find that many radial nanometer bristles referred to as nanobristles project from the lateral surface of nematode and mouse microvilli. The C. elegans nanobristles are 37.5 nm long and 4.5 nm wide. Nanobristle formation requires a protocadherin family protein, CDH-8, in C. elegans. The loss of nanobristles in cdh-8 mutants slows down animal growth and ectopically increases the number of Y-shaped microvilli, the putative intermediate structures if microvilli split from tips. Our results reveal a potential role of nanobristles in separating microvilli and suggest that microvilli division may help generate nascent microvilli with uniformity. [ABSTRACT FROM AUTHOR]

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo‐tomography (cryo‐ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS‐ring. In high‐torque motors of Beta‐, Gamma‐, and Epsilon‐proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS‐ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine. Synopsis: The assembly of bacterial flagellar motors is an "inside‐out" process, starting from the inner membrane. Cryo‐ET reveals novel structures that surround the cytoplasmic C‐ring of high‐torque motors that intriguingly, are only associated with certain assembly stages and are not present in the fully assembled motor. Transient cytoplasmic rings surround the motors of various Beta‐, Gamma‐, and Epsilon‐proteobacteria during assembly.These rings appear to surround the FliG‐ring of the assembling motors.In Helicobacter pylori, these rings are dependent on components of the flagellar type III secretion system. [ABSTRACT FROM AUTHOR]

[Display omitted] • We present a method that allows the cryo-EM/ET (electron cryo-microscopy/tomography) imaging of rod-shaped bacteria orthogonal to their longest axis, which is practically impossible using standard vitrification methods on cryo-EM grids. • We demonstrate straightforward manufacture of the honeycomb discs we introduce. • We present the first ever complete view of an FtsZ ring in E. coli using the method in 1). • We demonstrate that the honeycomb discs are superior in terms of mechanical and imaging performance, because the cells are surrounded by conducting gold metal. • We show that molecular resolutions can be obtained after subtomogram averaging from the FIB-milled honeycomb lamellae (ribosomes at 6.7 Å resolution). • We discuss how the honeycomb discs may have other important applications, for example to overcome preferred orientations of single particles in cryo-EM. Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations. [ABSTRACT FROM AUTHOR]

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond. [Display omitted] • Capsid assembly of BTV in host cells visualized by cryo-FIB-based cellular cryo-ET • High-resolution structures of BTV assembly intermediates by single-particle cryo-EM • ATPase VP6 pentamer bound underneath the VP3 shell of the pre-subcore for RNA threading • Duality model for BTV RNA packaging with features of both dsDNA and ssRNA viruses Integrated structural analyses captured a total of eleven assembly states of bluetongue virus (BTV), revealing insights into the mysterious RNA packaging and capsid assembly of dsRNA viruses. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (Cryo-ET) has been regarded as a revolution in structural biology and can reveal molecular sociology. Its unprecedented quality enables it to visualize cellular organelles and macromolecular complexes at nanometer resolution with native conformations. Motivated by developments in nanotechnology and machine learning, establishing machine learning approaches such as classification, detection and averaging for Cryo-ET image analysis has inspired broad interest. Yet, deep learning-based methods for biomedical imaging typically require large labeled datasets for good results, which can be a great challenge due to the expense of obtaining and labeling training data. To deal with this problem, we propose a generative model to simulate Cryo-ET images efficiently and reliably: CryoETGAN. This cycle-consistent and Wasserstein generative adversarial network (GAN) is able to generate images with an appearance similar to the original experimental data. Quantitative and visual grading results on generated images are provided to show that the results of our proposed method achieve better performance compared to the previous state-of-the-art simulation methods. Moreover, CryoETGAN is stable to train and capable of generating plausibly diverse image samples. [ABSTRACT FROM AUTHOR]

Electron cryo‐microscopy (cryo‐EM) has lately emerged as a powerful method in structural biology and cell biology. While cryo‐EM single‐particle analysis (SPA) is now routinely delivering structures of purified proteins and protein complexes at near‐atomic resolution, the use of electron cryo‐tomography (cryo‐ET), together with subtomogram averaging, is allowing visualization of macromolecular complexes in their native cellular environment, at unprecedented resolution. The unique ability of cryo‐EM to provide information at many spatial resolution scales from ångströms to microns makes it an invaluable tool that bridges the classic "resolution‐gap" between structural biology and cell biology domains. Like in many other fields of biology, in recent years, cryo‐EM has revolutionized our understanding of pathogen biology, host–pathogen interaction and has made significant strides toward structure‐based drug discovery. In a very recent example, during the ongoing coronavirus disease (COVID‐19) pandemic, the structure of the stabilized severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein was deciphered by SPA. This led to the development of multiple vaccines. Alongside, cryo‐ET provided key insights into the structure of the native virion, mechanism of its entry, replication, and budding; demonstrating the unrivaled power of cryo‐EM in investigating pathogen biology, host–pathogen interaction, and drug discovery. In this review, we showcase a few examples of how different imaging modalities within cryo‐EM have enabled the study of microbiology and host–pathogen interaction. [ABSTRACT FROM AUTHOR]

How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1-C2B-MUN-C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to "primed" ready-release vesicles in which SNAREpins are clamped and ready to release (state 3). [ABSTRACT FROM AUTHOR]

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three‐dimensional reconstruction, we show that there is extensive helical ordering of the envelope‐associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub‐tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M‐dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M‐layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N‐RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly. Synopsis: CryoEM and tomography analysis of the human pathogen respiratory syncytial virus shows the formation of filamentous virions that exhibit helical ordering of the envelope associated matrix protein layer, which in turn coordinates helical ordering and clustering of viral glycoproteins. 16 Å resolution sub‐tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope.Respiratory syncytial virus filamentous virions have a helically ordered matrix layer that is formed by a lattice of M dimers.Viral glycoproteins embedded in the envelope also show helical ordering and frequently cluster in pairs, these properties are coordinated by the underlying matrix layer.In addition to packing multiple copies of the viral genome in the form of helical ribonucleoprotein complexes or nucleocapsids, virions also package an abundance of ring‐shaped assemblies likely formed of the viral nucleocapsid protein (N) and RNA. [ABSTRACT FROM AUTHOR]

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes. Klykov et al. review how MS-based structural methods benefit cryo-EM single-particle analysis and cryo-ET label-free visual proteomics pipelines. Recent reports are highlighted wherein combinations of data outputs from MS and cryo-EM/ET methods were utilized. Integration of both methods is discussed on the data output and hardware levels. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environment at molecular resolution. Most cells and all tissues, however, are too thick to be imaged directly by transmission electron microscopy (TEM). Overcoming this limitation requires the production of thin biological sections called lamellae. The procedure to obtain lamellae of cells, either seeded or grown directly on electron microscopy grids, requires cryo-focused ion beam (cryo-FIB) milling to thin the samples. This method faces an additional challenge when dealing with tissues and multicellular organisms, as these samples must be high-pressure frozen, which embeds the sample in a thick layer of ice. Nonetheless, lamellae can still be prepared from such samples by extracting a small volume and transferring it to a receiver grid for lamella preparation, a process called lift-out. Here, we describe the available workflows to produce lamellae by lift-out at cryogenic conditions and recent developments in gas injection system (GIS)-free approaches to the lift-out transfer. These advances expand the applications of cryo-ET, enabling the investigation of tissues and whole organisms in situ at molecular resolution. [ABSTRACT FROM AUTHOR]

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET. [ABSTRACT FROM AUTHOR]

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.