Subtomogram Averaging Research Articles

Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes.

Proteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The coat protein complex II (COPII) mediates the anterograde transport of newly synthesized proteins from the endoplasmic reticulum, engaging cargoes with a wide range of size and biophysical properties. The native architecture of the COPII coat and how cargo might influence COPII carrier morphology remain poorly understood. Here we reconstituted COPII-coated membrane carriers using purified Saccharomyces cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated.

PilY1 regulates the dynamic architecture of the type IV pilus machine in Pseudomonas aeruginosa

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics.

In-Situ Structure and Topography of AMPA Receptor Scaffolding Complexes Visualized by CryoET.

Most synapses in the brain transmit information by the presynaptic release of vesicular glutamate, driving postsynaptic depolarization through AMPA-type glutamate receptors (AMPARs). The nanometer-scale topography of synaptic AMPARs regulates response amplitude by controlling the number of receptors activated by synaptic vesicle fusion. The mechanisms controlling AMPAR topography and their interactions with postsynaptic scaffolding proteins are unclear, as is the spatial relationship between AMPARs and synaptic vesicles. Here, we used cryo-electron tomography to map the molecular topography of AMPARs and visualize their in-situ structure. Clustered AMPARs form structured complexes with postsynaptic scaffolding proteins resolved by sub-tomogram averaging. Sub-synaptic topography mapping reveals the presence of AMPAR nanoclusters with exclusion zones beneath synaptic vesicles. Our molecular-resolution maps visualize the predominant information transfer path in the nervous system.

Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryo-electron tomography.

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

An approach for coherent periodogram averaging of tilt-series data for improved CTF estimation.

Cryo-electron microscopy (cryo-EM) has become an indispensable technique for determining three-dimensional structures of biological macromolecules. A critical aspect of achieving high-resolution cryo-EM reconstructions is accurately determining and correcting for the microscope's contrast transfer function (CTF). The CTF introduces defocus-dependent distortions during imaging; if not properly accounted for, the CTF can distort features in and limit the resolution of 3D reconstructions. For tilt-series data used in cryo-electron tomography (cryo-ET), CTF estimation becomes even more challenging due to the tilt of the specimen, which introduces a defocus gradient across the field of view, as well as the low dose and signal in individual tilt images. Here, we describe a simple algorithm to improve the accuracy of CTF estimation of tilted images by leveraging the tilt-series alignment parameters determined for tomographic reconstruction to explicitly account for the tilted specimen geometry. In brief, each tilt image is divided into patches, each of which are then stretched according to their defocus shift. These are then summed to provide a coherent power spectra at the tilt axis, which can then be used in standard CTF estimation algorithms. This uses all the data in each image to enhance the visibility of Thon rings, thereby improving high-resolution CTF estimation and subsequent enhancements in the resolution of subtomogram averages.

Structural basis for Vipp1 membrane binding: from loose coats and carpets to ring and rod assemblies.

Vesicle-inducing protein in plastids 1 (Vipp1) is critical for thylakoid membrane biogenesis and maintenance. Although Vipp1 has recently been identified as a member of the endosomal sorting complexes required for transport III superfamily, it is still unknown how Vipp1 remodels membranes. Here, we present cryo-electron microscopy structures of Synechocystis Vipp1 interacting with membranes: seven structures of helical and stacked-ring assemblies at 5-7-Å resolution engulfing membranes and three carpet structures covering lipid vesicles at ~20-Å resolution using subtomogram averaging. By analyzing ten structures of N-terminally truncated Vipp1, we show that helix α0 is essential for membrane tubulation and forms the membrane-anchoring domain of Vipp1. Lastly, using a conformation-restrained Vipp1 mutant, we reduced the structural plasticity of Vipp1 and determined two structures of Vipp1 at 3.0-Å resolution, resolving the molecular details of membrane-anchoring and intersubunit contacts of helix α0. Our data reveal membrane curvature-dependent structural transitions from carpets to rings and rods, some of which are capable of inducing and/or stabilizing high local membrane curvature triggering membrane fusion.

Molecular architecture of the mammalian 2-oxoglutarate dehydrogenase complex

The 2-oxoglutarate dehydrogenase complex (OGDHc) orchestrates a critical reaction regulating the TCA cycle. Although the structure of each OGDHc subunit has been solved, the architecture of the intact complex and inter-subunit interactions still remain unknown. Here we report the assembly of native, intact OGDHc from Sus scrofa heart tissue using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and subtomogram averaging (STA) to discern native structures of the whole complex and each subunit. Our cryo-EM analyses revealed the E2o cubic core structure comprising eight homotrimers at 3.3-Å resolution. More importantly, the numbers, positions and orientations of each OGDHc subunit were determined by cryo-ET and the STA structures of the core were resolved at 7.9-Å with the peripheral subunits reaching nanometer resolution. Although the distribution of the peripheral subunits E1o and E3 vary among complexes, they demonstrate a certain regularity within the position and orientation. Moreover, we analyzed and validated the interactions between each subunit, and determined the flexible binding mode for E1o, E2o and E3, resulting in a proposed model of Sus scrofa OGDHc. Together, our results reveal distinctive factors driving the architecture of the intact, native OGDHc.

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography.

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.

Structure and inhibition of SARS-CoV-2 spike refolding in membranes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds the receptor angiotensin converting enzyme 2 (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas the S1 domain contains high sequence variability, the S2 domain is conserved and is a promising pan-betacoronavirus vaccine target. We applied cryo-electron tomography to capture intermediates of S2 refolding and understand inhibition by antibodies to the S2 stem-helix. Subtomogram averaging revealed ACE2 dimers cross-linking spikes before transitioning into S2 intermediates, which were captured at various stages of refolding. Pan-betacoronavirus neutralizing antibodies targeting the S2 stem-helix bound to and inhibited refolding of spike prehairpin intermediates. Combined with molecular dynamics simulations, these structures elucidate the process of SARS-CoV-2 entry and reveal how pan-betacoronavirus S2-targeting antibodies neutralize infectivity by arresting prehairpin intermediates.

Molecular architecture of coronavirus double-membrane vesicle pore complex

Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications1,2. SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs3–5. The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3–nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3–nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection.

TOMOMAN: a software package for large scale cryo-electron tomography data preprocessing, community data sharing, and collaborative computing.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are becoming the preferred methodologies for investigating subcellular and macromolecular structures in native or near-native environments. While cryo-ET is amenable to a wide range of biological problems, these problems often have data processing requirements that need to be individually optimized, precluding the notion of a one-size-fits-all processing pipeline. Cryo-ET data processing is also becoming progressively more complex due to an increasing number of packages for each processing step. Though each package has its own strengths and weaknesses, independent development and different data formats makes them difficult to interface with one another. TOMOMAN (TOMOgram MANager) is an extensible package for streamlining the interoperability of packages, enabling users to develop project-specific processing workflows. TOMOMAN does this by maintaining an internal metadata format and wrapping external packages to manage and perform preprocessing, from raw tilt-series data to reconstructed tomograms. TOMOMAN can also export this metadata between various STA packages. TOMOMAN also includes tools for archiving projects to data repositories; allowing subsequent users to download TOMOMAN projects and directly resume processing where it was previously left off. By tracking essential metadata, TOMOMAN streamlines data sharing, which improves reproducibility of published results, reduces computational costs by minimizing reprocessing, and enables distributed cryo-ET projects between multiple groups and institutions. TOMOMAN provides a way for users to test different software packages to develop processing workflows that meet the specific needs of their biological questions and to distribute their results with the broader scientific community.

Cryo-Electron Tomography and Sub-Tomogram Averaging of Respiratory Syncytial Virus Structural Proteins from Native Virions

Cryo-Electron Tomography and Sub-Tomogram Averaging of Respiratory Syncytial Virus Structural Proteins from Native Virions

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites.

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis . Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ , laying the foundation for understanding infection mechanisms.

P-097 Studying epididymal sperm using cryo-electron microscopy

Abstract Study question What are the structural changes in epididymal sperm leading to gain of motility? Summary answer We resolved the contents of the cytoplasmic droplet in immature sperm. We further found that the outer dynein arms are in a pre-power stroke conformation. What is known already Cells exiting the testis are morphologically complete but incapable of fertilization. They first need to mature in the epididymis, where sperm gain progressive motility. Intriguingly, during this epididymal maturation the cytoplasmic droplet, a cytoplasmic remnant from spermiogenesis, migrates distally from the connecting piece to the principal piece. At the heart of the sperm’s flagellum is the axoneme, the molecular motor of the cell. It is a microtubule-based structure that houses protein complexes with different functions. For instance, it has radial spokes for signaling and rigidity. It also houses dyneins, motor proteins attached to neighboring doublets that ultimately generate flagellar beating. Study design, size, duration Bovine testes were acquired from a slaughterhouse, and sperm were isolated from different regions of the epididymis. After a washing step, these cells were directly used for imaging. Participants/materials, setting, methods Cells were prepared for imaging by rapid freezing on cryo-Electron Microscopy (cryo-EM) grids. Frozen cells were either imaged directly or first thinned by Focused Ion Beam (FIB) – milling. We applied the image analysis approaches subtomogram averaging and single particle analysis. Main results and the role of chance We resolved the structure of the 96-nm repeat of the axonemal doublet from immature sperm and showed a complete axoneme, containing radial spokes and different sets of dynein arms. Focusing our refinement on a specific set of dyneins, the outer dynein arms, revealed that they are in the pre-power stroke state. In this primed state the dyneins have ATP bound and are active. Interestingly, these cells display no movement. This could indicate that a different blocking mechanism is present in the axoneme. Furthermore, we used 3D cryo-EM to elucidate the contents of the cytoplasmic droplet. We showed that it contains many vesicles, some display a shape that is reminiscent of an autophagosome. We also find vesicles displaying a double membrane with a highly repetitive density connecting in between. This suggests that sperm cells bring along auxiliary proteins and molecules in the cytoplasmic droplet that may be required for proper maturation. Altogether we show that cryo-EM can be used to study immature sperm cells in detail, with a focus on structures important in the gain of movement. Limitations, reasons for caution Cryo-EM imaging requires frozen cells and uses electrons as imaging source, therefore we lack information on cell state (e.g. viability/motility) or protein identity. By complementing our visual data with other techniques such as CASA and mass spectrometry, we aim to get a full picture. Wider implications of the findings Elucidating protein conformation or the structural mechanism behind sperm activation could open doors into developing new therapeutics for diseases on one hand. On the other hand, understanding how sperm motility arises (and how to potentially put it to a halt) could also lay ground for developing male contraception. Trial registration number not applicable

Cryo-electron tomography pipeline for plasma membranes.

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with sub-tomogram averaging and employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. The pipeline provides fast, efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of cells.

Exploring the molecular composition of the multipass translocon in its native membrane environment.

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.

Tilt-series-based joint CTF estimation for cryo-electron tomography

Contrast transfer function (CTF) estimation is a necessary step in the cryo-electron tomography (cryoET) workflow and essential for high-resolution in situ structural determination. However, the low signal-to-noise ratio and continuous defocus variation in micrographs of cryoET tilt series make accurate CTF estimation challenging. Here, we report a tilt-series-based joint CTF estimation method implemented in the new software CTFMeasure. The joint estimation method combines all Thon-ring signals in a tilt series to improve the estimation accuracy. By using an objective function involving the CTF parameters and geometric parameters of a cryoET tilt series, CTFMeasure can estimate the CTF parameters of each micrograph and the absolute tilt angle offset of the lamellar sample relative to the sample stage plane, which is usually the glancing angle used during focused ion beam (FIB) milling. Tests on both synthetic and experimental data, as well as subtomogram averaging, demonstrated the accurate CTF estimation of cryoET tilt series by CTFMeasure.

Cryo-electron tomography reveals the packaging pattern of RuBisCOs in Synechococcus β-carboxysome

Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO2-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact β-carboxysomes from Synechococcus elongatus PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the β-carboxysome. These results provide new insights into the biogenesis of β-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.