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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.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

Abstract Background Cryo-electron tomography (Cryo-ET) is an imaging technique used to generate three-dimensional structures of cellular macromolecule complexes in their native environment. Due to developing cryo-electron microscopy technology, the image quality of three-dimensional reconstruction of cryo-electron tomography has greatly improved. However, cryo-ET images are characterized by low resolution, partial data loss and low signal-to-noise ratio (SNR). In order to tackle these challenges and improve resolution, a large number of subtomograms containing the same structure needs to be aligned and averaged. Existing methods for refining and aligning subtomograms are still highly time-consuming, requiring many computationally intensive processing steps (i.e. the rotations and translations of subtomograms in three-dimensional space). Results In this article, we propose a Stochastic Average Gradient (SAG) fine-grained alignment method for optimizing the sum of dissimilarity measure in real space. We introduce a Message Passing Interface (MPI) parallel programming model in order to explore further speedup. Conclusions We compare our stochastic average gradient fine-grained alignment algorithm with two baseline methods, high-precision alignment and fast alignment. Our SAG fine-grained alignment algorithm is much faster than the two baseline methods. Results on simulated data of GroEL from the Protein Data Bank (PDB ID:1KP8) showed that our parallel SAG-based fine-grained alignment method could achieve close-to-optimal rigid transformations with higher precision than both high-precision alignment and fast alignment at a low SNR (SNR=0.003) with tilt angle range ±60∘ or ±40∘. For the experimental subtomograms data structures of GroEL and GroEL/GroES complexes, our parallel SAG-based fine-grained alignment can achieve higher precision and fewer iterations to converge than the two baseline methods.

Abstract Background Cryo-electron tomography (cryo-ET) enables the 3D visualization of cellular organization in near-native state which plays important roles in the field of structural cell biology. However, due to the low signal-to-noise ratio (SNR), large volume and high content complexity within cells, it remains difficult and time-consuming to localize and identify different components in cellular cryo-ET. To automatically localize and recognize in situ cellular structures of interest captured by cryo-ET, we proposed a simple yet effective automatic image analysis approach based on Faster-RCNN. Results Our experimental results were validated using in situ cyro-ET-imaged mitochondria data. Our experimental results show that our algorithm can accurately localize and identify important cellular structures on both the 2D tilt images and the reconstructed 2D slices of cryo-ET. When ran on the mitochondria cryo-ET dataset, our algorithm achieved Average Precision >0.95. Moreover, our study demonstrated that our customized pre-processing steps can further improve the robustness of our model performance. Conclusions In this paper, we proposed an automatic Cryo-ET image analysis algorithm for localization and identification of different structure of interest in cells, which is the first Faster-RCNN based method for localizing an cellular organelle in Cryo-ET images and demonstrated the high accuracy and robustness of detection and classification tasks of intracellular mitochondria. Furthermore, our approach can be easily applied to detection tasks of other cellular structures as well.

Cryo-electron Tomography (cryo-ET) generates 3D visualization of cellular organization that allows biologists to analyze cellular structures in a near-native state with nano resolution. Recently, deep learning methods have demonstrated promising performance in classification and segmentation of macromolecule structures captured by cryo-ET, but training individual deep learning models requires large amounts of manually labeled and segmented data from previously observed classes. To perform classification and segmentation in the wild (i.e., with limited training data and with unseen classes), novel deep learning model needs to be developed to classify and segment unseen macromolecules captured by cryo-ET. In this paper, we develop a one-shot learning framework, called cryo-ET one-shot network (COS-Net), for simultaneous classification of macromolecular structure and generation of the voxel-level 3D segmentation, using only one training sample per class. Our experimental results on 22 macromolecule classes demonstrated that our COS-Net could efficiently classify macromolecular structures with small amounts of samples and produce accurate 3D segmentation at the same time.

Electron cryotomography is a rapidly evolving method for imaging macromolecules directly within the native environment of cells and tissues. Combined with sub-tomogram averaging, it allows structural and cell biologists to obtain sub-nanometre resolution structures in situ. However, low throughput in cryo-ET sample preparation and data acquisition, as well as difficulties in target localisation and sub-tomogram averaging image processing, limit its widespread usability. In this review, we discuss new advances in the field that address these throughput and technical problems. We focus on recent efforts made to resolve issues in sample thinning, improvement in data collection speed at the microscope, strategies for localisation of macromolecules using correlated light and electron microscopy and advancements made to improve resolution in sub-tomogram averaging. These advances will considerably decrease the amount of time and effort required for cryo-ET and sub-tomogram averaging, ushering in a new era of structural biology where in situ macromolecular structure determination will be routine. [ABSTRACT FROM AUTHOR]

Summary: Cryo‐electron tomography (cryo‐ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo‐tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo‐applications, provides advances in automation, imaging and the preparation of high‐pressure frozen bulk samples using cryo‐lift‐out technology. In addition, correlative cryo‐fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo‐microscopy, cryo‐FIB and cryo‐ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo‐tomography workflow, and we discuss the challenges that lie ahead. Lay Description: This article describes our recent developments for the cryo‐electron tomography (cryo‐ET) workflow. Cryo‐ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo‐ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200–300 nm thin cryo‐lamella from the cellular sample using a cryo‐focused ion beam (cryo‐FIB) microscope. Cryo‐correlative light and electron microscopy (cryo‐CLEM) is used within the workflow to guide the cryo‐lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo‐ET workflow and show new developments for cryo‐CLEM, which facilitate the connection between the cryo‐light microscope and the cryo‐FIB. Next, we present our progress in cryo‐FIB software automation to streamline cryo‐lamella preparation. In the final section we demonstrate how the cryo‐FIB can be used for 3D imaging and how bulk‐frozen cellular samples (obtained by high‐pressure freezing) can be processed using the newly developed cryo‐lift‐out technology. [ABSTRACT FROM AUTHOR]

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do. [ABSTRACT FROM AUTHOR]

Alphaviruses are spherical, enveloped RNA viruses with two-layered icosahedral architecture. The structures of many alphaviruses have been studied using cryogenic electron microscopy (cryo-EM) reconstructions, which impose icosahedral symmetry on the viral particles. Using cryogenic electron tomography (cryo-ET), we revealed a polarized symmetry defect in the icosahedral lattice of Chikungunya virus (CHIKV) in situ, similar to the late budding particles, suggesting the inherent imperfect symmetry originates from the final pinch-off of assembled virions. We further demonstrated this imperfect symmetry is also present in in vitro purified CHIKV and Mayaro virus, another arthritogenic alphavirus. We employed a subparticle-based single-particle analysis protocol to circumvent the icosahedral imperfection and boosted the resolution of the structure of the CHIKV to ∼3 Å resolution, which revealed detailed molecular interactions between glycoprotein E1-E2 heterodimers in the transmembrane region and multiple lipid-like pocket factors located in a highly conserved hydrophobic pocket. This complementary use of in situ cryo-ET and single-particle cryo-EM approaches provides a more precise structural description of near-icosahedral viruses and valuable insights to guide the development of structure-based antiviral therapies against alphaviruses., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

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., Competing Interests: The authors declare no conflict of interest.

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., 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 Asarnow, Becker, Bobe, Dubbledam, Johnston, Kopylov, Lavoie, Li, Mattingly, Mendez, Paraan, Turner, Upadhye, Walsh, Gupta and Eng.)

Background: Electron transport chain (ETC) complexes, pyruvate dehydrogenase complex (PDC), and α-ketoglutarate dehydrogenase complex (KGDC) are important elements in mitochondrial metabolism. The localization of the aforementioned protein complexes differs since oxidative phosphorylation complexes are membrane proteins, while dehydrogenase complexes (DCs) are contained in the mitochondrial matrix. Our previous cryo-electron tomography (cryo-ET) studies showed the existence of a full oxidative phosphorylation system supercomplex consisting of ETC complexes and ATP synthases (Nesterov et al., 2021). Literature data also shows the binding of fatty acid oxidation enzymes to ETC complex I (Wang et al., 2010). Although it has long been shown that PDCs can bind to complex I (Sumegi et al., 1984) in vitro, this has not been visualized directly in mitochondria and the binding mechanisms are still unknown. Methods: The mitochondria were isolated from Wistar rat heart ventricles according to a standard procedure (Nesterov et al., 2021). The dense mitochondrial suspension was diluted to ~0.3mg/ml in a respiration medium. Phosphorylation was started 10 minutes prior to vitrification. Experimental data was obtained by cryo-ET using Titan Krios and processed with IMOD and RELION. Results: The tomograms show that the significant part of DCs is localized near the inner membrane of partially destroyed mitochondria in an array-like fashion. Sole PDCs and KGDCs can be identified on the images and their position appears to be close to ETC complex I. Subtomogram averaging of close to the membrane DCs showed that there is no specific density between them, suggesting that they are not linked with identical proteins or that this link may be soft. Significant damage to the mitochondrial membrane leads to the formation of membrane-unbound DCs fraction. It suggests that coupling of DCs with ETC complexes can be controlled in vivo by the topology of the inner mitochondrial membrane and the volume of the mitochondrial matrix. Conclusion: The obtained results show a possibility of unprecedentedly large multienzyme complex formation, including almost all main mitochondrial metabolic systems. Although cryo-ET of partially destroyed mitochondria showed close localization of PDC and KGDC to complex I, further studies are required in intact mitochondria. The mechanism of their binding also remains an open question.

Background: Currently, different approaches of active and passive targeted drug delivery are being developed. One of the most promising methods of targeted drug delivery is the use of capsules. For instance, colloidosomes—capsules consisting of the shell formed by colloidal particles at the interface of the emulsion—can be used for targeted delivery of antitumor drugs or any other drugs in liquid form. Here we present results of cryo-EM study of submicrocapsules with the soybean oil core and with the shell consisting of SiO2 nanoparticles and detonation nanodiamonds (DNDs) stabilized with chitosan and alginate. Methods: Сryo-electron tomography (Cryo-ET) was used to identify the morphological features of the submicrocapsules. Preliminary screening of samples and cryo-ET data collection were performed using Titan Krios cryo-EM (ThermoFisher Scientific, US) equipped with Falcon 2 direct electron detector. The restoration of the tomographic series was carried out using IMOD software. Eman2 was used for segmentation and UCSF Chimera was used for visualization of the 3D model. Submicron capsules were obtained by stabilizing oil droplets with a mixture of SiO2 nanoparticles and DNDs. To form a stable shell, an additional layer of silica particles and polyelectrolyte layers of alginate/chitosan were applied to the droplets of the dispersed phase of the emulsion by physical adsorption. Results: Cryo-EM data showed the presence of submicrocapsules with a diameter in the range of 200-900 nm. Although a significant fraction of submicrocapsules was found to be partially destroyed, results of cryo-ET study of intact capsules demonstrated that silicon dioxide nanoparticles form a net, while DNDs form clusters. Conclusion: Here we demonstrate the results of the study of submicron capsules with a shell of silica nanoparticles and DNDs. It was found that a uniform distribution of DNDs is not a prerequisite for the creation of submicron capsules that contradicts the theoretical model.

Background: Antibiotic resistance becomes one of the main problems of modern medicine; therefore, the development of new antibacterial compounds is absolutely necessary. The ribosome is the target for a lot of different antibiotics; there are several main binding sites on the ribosome – decoding center, peptidyl-transferase center, and ribosome exit tunnel. Modification or mutation of nucleotides in these sites could make cells resistant to structurally different antibiotics. Methods: pDualrep2 reporter system was used for detection of the protein synthesis inhibitors in cultural broths of new soil bacteria. By means of a cell-free translation system, the inhibitory activity and mechanism of action of Auraplanin were estimated. CryoEM data collection was performed on a Titan Krios operated at 300 kV, equipped with a Falcon II direct electron detector. Results: In this work, we have found a new inhibitor of protein synthesis, which binds in a completely new binding site. This compound is produced by Actinoplanes sp. VKM Ac-2862 and by Cryo-EM study of its complex with E.coli ribosome, it was shown, that it binds close to 560 loop of 30S ribosomal subunit. The new compound is a derivative of tetramic acid and we called it Auraplanin, because of bright orange color of the producer strain. Structural data are in good agreement with genetic results – resistant mutations were located close determined binding site. Substitutions C564G, G558U, and G566A significantly increase minimal inhibitory concentration, all these mutations were not detected previously. We also observed resistant mutation in ribosomal protein S4, this mutation was previously identified as error-prone. Interestingly, ribosomal ambiguity mutations, G299A and G347U, also increased resistance to Auraplanin. Conclusion: On the basis of the genetic, structural and biochemical studies we hypothesized that Auraplanin acts prevent the transfer from an open to a closed conformation of 30S subunit, in contrast to streptomycin, which promotes the formation of a closed state.

Background: Cryo-electron tomography (cryo-ET) is currently the only technique that can be used for quaternary structure determination of polyribosomal complexes under near physiological conditions. Here we describe, in more detail, the cryo-ET pipeline from a sample preparation for data processing from the poster talk “Cryo-ET structural analysis of polyribosomes from HeLa cells” by Zhanna A. Afonina. Methods: The investigations were carried out in a Titan Krios 60-300 TEM/STEM (FEI, USA), equipped with Falcon II DED (FEI, USA) and Cs-image corrector (CEOS, Germany). Data acquisition were done automatically using FEI Tomography software. All data processing stages including sub-tomogram averaging after tomographic reconstruction using IMOD were carried out using computing resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute”. Results: Cryo-ET with sub-tomogram averaging was allowed us to determine spatial structure of polyribosomes with 17.5Å resolution. The relative orientations of ribosomes in polyribosomes and the mRNA pathways in each individual polyribosome were determined. Conclusion: Cryo-ET in combination with sub-tomographic averaging is the most promising technique in cryo-electron microscopy. This approach in comparison of single particle analysis imposes additional requirements both at the stages of data acquisition and data processing. On the example of this work we demonstrated all the stages of the Cryo-ET pipeline. Taking into account the experimental equipment used, bottlenecks and possible solutions were shown.

Background: Chromatin higher order structure has been for decades a matter of debate, mainly due to its mesoscale nature, which precluded its accurate analysis by optical microscopy, while electron microscopy studies rendered controversial results reflecting highly dynamic and heterogeneous DNA folding prone to sample preparation artifacts. On the other hand, lack of adequate tools for nondestructive yet selective staining of DNA as a whole, and specific chromosomal loci in particular, added another layer of uncertainty to the fuzzy picture of how DNA is organized in the cell nucleus. Methods: Here we propose an approach for specific labeling of endogenous chromosomal loci compatible with the direct visualization of DNA folding motifs at near native conditions by cryo-electron tomography. The approach is based on use of tagged DNA-binding proteins for mapping chromosomal loci, with subsequent introduction of Nanogold-conjugated ligands into the cell nuclei by microinjection.. Results: Our preliminary data demonstrated feasibility of above-mentioned approach with a set of DNA-binding tools (LacO-lac-rep system, TALEs, dCas) tagged with GFP, 6His and subsequent detection with anti-GFP antibodies, GBP, or Ni-NTA, conjugated with 1.4-nm gold. The passage to the cryo-ET would require further development of robust 3D-CLEM tools for location of target loci and lamella preparation with cryo-FIB. Conclusion: A combination of nondestructive in vivo labeling of endogenous chromosomal loci with correlative cryo-FIB – cryo-ET would open new perspectives to our efforts to understand how genome is organized and functions in living cells.

The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.

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., Competing Interests: JH, PC, SN, SH, KB, SM, RL, WA, KZ No competing interests declared, JC, FL, JC Reviewing editor, eLife, (© 2023, Hwang, Chai, Nawaz et al.)

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., 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 © 2023 Bodakuntla, Kuhn, Biertümpfel and Mizuno.)

Historically, structural biology has been largely centered on in vitro approaches as the dominant technique to obtain indispensable high-resolution data. In situ structural biology is now poised to contribute with high-precision observations in a near-physiological context. Mass spectrometry, electron tomography, and fluorescence microscopy are opening up new opportunities for structural analysis, including the study of the protein machinery in living cells. The complementarity between studies is increasingly used to reveal biologically significant observations. Here we compare two complementary studies addressing the mechanisms of vesicle tethering with in vitro and in situ approaches. Cryoelectron microscopy and live-cell imaging assisted by anchoring platforms team up to explore elusive mechanisms of exocytosis, showing directions of future research. Irastorza-Azcarate et al. summarize recent cryo-EM and live-cell imaging studies on the exocyst, a protein complex involved in tethering secretory vesicles to the plasma membrane in exocytosis. The comparative analysis demonstrates the power of integrating in vitro and in situ approaches to unravel the structure and mechanism of macromolecular complexes. [ABSTRACT FROM AUTHOR]

Background Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms—3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ , we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. Findings Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiae collected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. Conclusions Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students. [ABSTRACT FROM AUTHOR]

• MBIR can significantly boost contrast of tomograms of cryo specimens. • MBIR can significantly reduce missing wedge artifacts and retain sharp features. • MBIR can partially restore the missing information in the unsampled angular region. • MBIR can improve the resolution of subtomogram averages. Cryo-Electron Tomography (cryo-ET) has become an essential technique in revealing cellular and macromolecular assembly structures in their native states. However, due to radiation damage and the limited tilt range, cryo-ET suffers from low contrast and missing wedge artifacts, which limits the tomograms to low resolution and hinders further biological interpretation. In this study, we applied the Model-Based Iterative Reconstruction (MBIR) method to obtain tomographic 3D reconstructions of experimental cryo-ET datasets and demonstrated the advantages of MBIR in contrast improvement, missing wedge artifacts reduction, missing information restoration, and subtomogram averaging compared with other reconstruction approaches. Considering the outstanding reconstruction quality, MBIR has a great potential in the determination of high resolution biological structures with cryo-ET. [ABSTRACT FROM AUTHOR]

Background: Cryo-electron tomography (cryo-ET) enables the 3D visualization of cellular organization in near-native state which plays important roles in the field of structural cell biology. However, due to the low signal-to-noise ratio (SNR), large volume and high content complexity within cells, it remains difficult and time-consuming to localize and identify different components in cellular cryo-ET. To automatically localize and recognize in situ cellular structures of interest captured by cryo-ET, we proposed a simple yet effective automatic image analysis approach based on Faster-RCNN. Results: Our experimental results were validated using in situ cyro-ET-imaged mitochondria data. Our experimental results show that our algorithm can accurately localize and identify important cellular structures on both the 2D tilt images and the reconstructed 2D slices of cryo-ET. When ran on the mitochondria cryo-ET dataset, our algorithm achieved Average Precision >0.95. Moreover, our study demonstrated that our customized pre-processing steps can further improve the robustness of our model performance. Conclusions: In this paper, we proposed an automatic Cryo-ET image analysis algorithm for localization and identification of different structure of interest in cells, which is the first Faster-RCNN based method for localizing an cellular organelle in Cryo-ET images and demonstrated the high accuracy and robustness of detection and classification tasks of intracellular mitochondria. Furthermore, our approach can be easily applied to detection tasks of other cellular structures as well. [ABSTRACT FROM AUTHOR]

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting superresolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryoprotectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ~135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo- CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system. [ABSTRACT FROM AUTHOR]

[Display omitted] • Applying SRRF nanoscopy under cryo condition. • Cryo-scanning transmission Electron tomography (CSTET) allows imaging of whole organelles and their interaction in 850 nm areas of intact and near-native cells. • Combining dual-axis CSTET with deconvolution results in visually isotropic resolution also in the axial direction. Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell. [ABSTRACT FROM AUTHOR]

Modern electron tomography has progressed to higher resolution at lower doses by leveraging compressed sensing (CS) methods that minimize total variation (TV). However, these sparsity-emphasized reconstruction algorithms introduce tunable parameters that greatly influence the reconstruction quality. Here, Pareto front analysis shows that high-quality tomograms are reproducibly achieved when TV minimization is heavily weighted. However, in excess, CS tomography creates overly smoothed three-dimensional (3D) reconstructions. Adding momentum to the gradient descent during reconstruction reduces the risk of over-smoothing and better ensures that CS is well behaved. For simulated data, the tedious process of tomography parameter selection is efficiently solved using Bayesian optimization with Gaussian processes. In combination, Bayesian optimization with momentum-based CS greatly reduces the required compute time-an 80% reduction was observed for the 3D reconstruction of SrTiO3 nanocubes. Automated parameter selection is necessary for large-scale tomographic simulations that enable the 3D characterization of a wider range of inorganic and biological materials., Competing Interests: Conflict of Interest The authors declare that they have no competing interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 ., Competing Interests: ZT, SC, AN, JC, JS, LG No competing interests declared, (© 2023, Tan, Cai, Noble et al.)

Electron cryotomography (cryo-ET) is an imaging technique uniquely suited to the study of bacterial ultrastructure and cell biology. Recent years have seen a surge in structural and cell biology research on bacteria using cryo-ET. This research has driven major technical developments in the field, with applications emerging to address a wide range of biological questions. In this review, we explore the diversity of cryo-ET approaches used for structural and cellular microbiology, with a focus on in situ localization and structure determination of macromolecules. The first section describes strategies employed to locate target macromolecules within large cellular volumes. Next, we explore methods to study thick specimens by sample thinning. Finally, we review examples of macromolecular structure determination in a cellular context using cryo-ET. The examples outlined serve as powerful demonstrations of how the cellular location, structure, and function of any bacterial macromolecule of interest can be investigated using cryo-ET. [ABSTRACT FROM AUTHOR]

The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell's icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding. [ABSTRACT FROM AUTHOR]

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryoelectron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder, a site that may act as an elongation center. Subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a β sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes. [Display omitted] • Gas vesicles (GVs) are formed by ∼3-nm corrugated protein shells • Corrugation reverses at the cylinder midpoint, which may act as an elongation center • The protein shell is primarily formed by the conserved major structural protein GvpA • GvpC provides extra reinforcement by forming a helical spiral around the GV cylinder Dutka et al. used cryo-ET supported by biochemical data and computational modeling to reveal the conserved structure of Anabaena flos-aquae gas vesicles. The resulting model gives insights into the distinctive mechanical properties of gas vesicles and their assembly. [ABSTRACT FROM AUTHOR]