Studying membrane modulation mechanisms by electron cryo-tomography

Human Immunodeficiency virus type 1 (HIV-1) is a lentivirus that infects non-dividing cells of the immune system. In non-dividing cells, nuclear import of the viral genome occurs through the nuclear pore complex (NPC), a large macromolecular assembly that forms a channel of ~40 nm across the nuclear envelope. This process requires interactions between NPC components known as nucleoporins and CA (capsid) proteins of HIV-1 post-fusion complexes. In mature HIV-1 virions, multiple copies of CA assemble into a lattice of hexamers and pentamers, forming a cone-shaped capsid core of ~60 nm in width and ~110 nm in length encasing the viral genome. Because of its large size, it was generally believed that the capsid core entirely or partially disassembled prior to its translocation through the NPC. However, nuclear entry and uncoating are rare events and challenging to characterize and therefore subject to a long-standing debate. Here, cryo-electron tomography (cryo-ET) on cryo-focused ion beam (FIB) milled T-cells was combined with subtomogram averaging (SA) to study the ultrastructure of HIV-1 capsids in the process of nuclear entry. Using a HIV-1 variant arrested at the nuclear pore due to a defect in binding of the CA lattice to the host cell protein CPSF6, snapshots of HIV-1 nuclear entry at multiple stages were captured. Reverse transcription-competent HIV-1 complexes were identified in the cytosol, docking to and within the NPC, and in the nucleoplasm of T cells. Surprisingly, in the cytosol and at the NPC, the viral complexes retained cone-shaped capsids highly resembling in size and geometry the mature capsid cores within intact virions. The density at their interior suggested that they were associated with the viral genome. The cone-shape capsids deeply enter into the NPC central channel with their narrow ends. The hexameric lattices of these capsids were intact or nearly intact. These findings argued against the current models of uncoating in the cytosol or at the NPC and rather supported translocation of intact HIV-1 capsids through the NPC. Instead, HIV-1 capsids uncoat in the nucleus of T cells. Inside the nucleus, tube-shape fragments still containing few lattice elements were observed. The lack of density at their inside suggested that the viral genome was released from these complexes. Uncoating may thus occur through the partial opening and remodeling of the hexameric lattices of HIV-1 capsids, rather than a step-wise disassembly. To address how an intact HIV-1 capsid can enter the central channel of NPC, the human NPC from T cells was structurally analyzed in cellulo by SA. The NPC overall structure was not altered upon infection but rather dilated in comparison to the structure previously obtained from purified envelopes. The diameter of the central channel of NPC in T cells corresponded to ~64 nm, suggesting that the translocation of intact HIV-1 capsid through the NPC is geometrically possible. The dilated conformation of NPCs in T cells is independent of HIV-1 infection, but represents a physiological condition of actively transporting NPCs.

Quantifying resolution limiting factors in subtomogram averaged cryo-electron tomography using simulations.

Background Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Methods Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. Results We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ∼15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. Discussion This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ~ 15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Determining the three-dimensional structure of biomacromolecules at high resolution in their native cellular environment is a major challenge for structural biology. Toward this end, electron cryotomography (ECT) allows large bio-macromolecular assemblies to be imaged directly in their hydrated physiological milieu to ~4nm resolution. Combining ECT with other techniques like fluorescent imaging, immunogold labeling, and genetic manipulation has allowed the in situ investigation of complex biological processes at macromolecular resolution. Furthermore, the advent of cryogenic focused ion beam (FIB) milling has extended the domain of ECT to include regions even deep within thick eukaryotic cells. Anticipating two audiences (scientists who just want to understand the potential and general workflow involved and scientists who are learning how to do the work themselves), here we present both a broad overview of this kind of work and a step-by-step example protocol for ECT and subtomogram averaging using the Legionella pneumophila Dot/Icm type IV secretion system (T4SS) as a case study. While the general workflow is presented in step-by-step detail, we refer to online tutorials, user's manuals, and other training materials for the essential background understanding needed to perform each step.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR—a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent (“white box”) approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal-loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We have developed a method called “TomographY-Guided 3D REconstruction of Subcellular Structures” (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio/contrast and minimal radiation damage) and subtomogram averaging (3D-alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to 12 Å resolution. These results reveal many structural details that were not visible by cryo-ET. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging, bringing us a step closer to (pseudo-)atomic models of cells.

M-free: scoring the reference bias in sub-tomogram averaging and template matching.

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.