The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography.

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.

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

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.

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

Practical Guide for Implementing Cryogenic Electron Microscopy Structure Determination in Dermatology Research.

Cryogenic electron microscopy (cryo-EM) provides powerful insights into the study of biological systems by revealing molecular structures in their close-to-native environments.Single particle analysis (SPA) has enabled structure determination of purified macromolecular complexes up to atomic resolution [1][2].Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging (STA) [3][4] has been demonstrated to offer distinct perspectives of the arrangement and function of structural proteins within intact cells.Compared to SPA, cryo-ET with STA, despite its powerful potential to offer much richer information, is still an emerging technique that has only achieved high resolution for a handful of biomolecular structures thus far.At the Chan Zuckerberg Institute for Advanced Biological Imaging (CZ Imaging Institute), our objective is to contribute to the ongoing refinement of this technique through joint efforts.This includes collaborative hardware development such as the laser phase plate [5] and direct electron detectors, and algorithm development, including but not limited to machine-learning-based denoising and automated annotation, and the establishment of a data portal to catalyze the development of machine-learning methods [6].Currently, one another challenge that cryo-ET faces is reduced resolution when imaging thick cellular specimens, where increased inelastic scattered signals result in increased chromatic blurring.Energy-filtered TEM (EFTEM) removes this blur caused by inelastic scattering but reduces signal potential.In response, we are exploring the potential advantages of an emerging technique enabled by 4D scanning transmission electron microscopy (4D-STEM): tilt-corrected bright-field (tcBF-)STEM [7-8] (also known as parallax [9]).In tcBF, each pixel within the bright-field (BF) disk functions as a virtual coherent detector with a small collection angle.The resulting virtual BF images, depending on the aberration function, exhibit image shifts, and these shifts are measured and corrected on a pixel-by-pixel basis.In principle, tcBF has an advantage over EFTEM for thick samples.It allows a coherent image to be retained and uses almost all the forward-scattered incident beam -i.e., a similar dose efficiency to TEM.The post-specimen lenses for TEM are the image-formation lenses, so chromatic aberrations there degrade the image resolution.But for tcBF the post-specimen lenses simply transfer an image of the diffraction pattern, so chromatic aberrations result in a small loss of angular resolution instead -i.e., a reduction in coherence.In our preliminary observations, we noticed improved contrast in large organelles and intact bacterial cells when imaged with tcBF-STEM as compared to EFTEM.Additionally, on a thin specimen of purified proteins, we demonstrate sub-nanometer resolution SPA maps with tcBF-STEM at a nominal resolution of 7 .This suggests the potential feasibility of employing this technique for biomolecular structure determination using cryo-ET and STA.However, more efforts are needed in both algorithm development and experimental design to match the state-of-the-art performance of EFTEM on thin samples at high resolution.In summary, our observations on both thick and thin samples indicate that combining 4D-STEM phase imaging techniques with cryo-ET for studying biological systems may provide additional information by enabling imaging on thicker and denser samples.[10]

Electron cryo-tomography (cryo-ET) is a powerful imaging tool that allows three-dimensional visualization of subcellular and molecular architecture without chemical fixation. Tomogram quality varies widely, particularly during large high-throughput data collections, and the most common strategy for initial quality assessment is empirical judgment by an expert. Tomograms may be collected for two distinct purposes: annotation of subcellular features and cellular morphology, typically performed at lower magnifications and higher defocus, and subtomogram averaging, at high magnifications, closer to focus. For the first purpose, contrast and the ability to distinguish cellular features of interest are key, whereas for subtomogram averaging, recoverable signal at high resolution is the key factor. We have developed "TomoScore" a deep-learning based tomogram screening tool targeting cellular annotation. This tool provides a single quantitative measure of the suitability of a tomogram for annotation of subcellular features, in terms of the scale of features that can be readily distinguished. We further explore the relationship between accumulated electron dose and resulting quality, suggesting an optimum dose range for cryo-ET data collection. Overall, our study streamlines data processing and reduces the need for human involvement during pre-selection for tomogram segmentation.

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

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.

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.