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Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure–function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity (https://github.com/bHimes/emClarity/wiki), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software.

The structure of chromatin plays pivotal roles in regulating gene transcription, DNA replication and repair, and chromosome segregation. This structure, however, remains elusive. Here, using cryo-FIB and cryo-ET, we delineate the 3D architecture of native chromatin fibres in intact interphase human T-lymphoblasts and determine the in situ structures of nucleosomes in different conformations. These chromatin fibres are not structured as uniform 30 nm one-start or two-start filaments but are composed of relaxed, variable zigzag organizations of nucleosomes connected by straight linker DNA. Nucleosomes with little H1 and linker DNA density are distributed randomly without any spatial preference. This work will inspire future high-resolution investigations on native chromatin structures in situ at both a single-nucleosome level and a population level under many different cellular conditions in health and disease. Here, using cryo-FIB and cryo-ET, the authors delineate the architecture of native chromatin fibers and decipher the in situ nucleosomes structure, inspiring future chromatin research.

SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2’ cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2’ cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike’s stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies. This study delineates the full sequence of SARS-CoV-2 spike-mediated membrane fusion and shows how S2-targeting antibodies inhibit this process, using a near-native functional fusion system and cryo-electron tomography.

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.Cryo-electron tomography with subtomogram averaging is useful for the structural analysis of heterogeneous protein complexes. emClarity is graphics processing unit-accelerated image-processing software for subtomogram averaging and classification at high resolution.

Defects-rich heterointerfaces integrated with adjustable crystalline phases and atom vacancies, as well as veiled dielectric-responsive character, are instrumental in electromagnetic dissipation. Conventional methods, however, constrain their delicate constructions. Herein, an innovative alternative is proposed: carrageenan-assistant cations-regulated (CACR) strategy, which induces a series of sulfides nanoparticles rooted in situ on the surface of carbon matrix. This unique configuration originates from strategic vacancy formation energy of sulfides and strong sulfides-carbon support interaction, benefiting the delicate construction of defects-rich heterostructures in M x S y /carbon composites (M-CAs). Impressively, these generated sulfur vacancies are firstly found to strengthen electron accumulation/consumption ability at heterointerfaces and, simultaneously, induct local asymmetry of electronic structure to evoke large dipole moment, ultimately leading to polarization coupling, i.e., defect-type interfacial polarization. Such “Janus effect” (Janus effect means versatility, as in the Greek two-headed Janus) of interfacial sulfur vacancies is intuitively confirmed by both theoretical and experimental investigations for the first time. Consequently, the sulfur vacancies-rich heterostructured Co/Ni-CAs displays broad absorption bandwidth of 6.76 GHz at only 1.8 mm, compared to sulfur vacancies-free CAs without any dielectric response. Harnessing defects-rich heterostructures, this one-pot CACR strategy may steer the design and development of advanced nanomaterials, boosting functionality across diverse application domains beyond electromagnetic response. Highlights A series of sulfides/carbon composites with sulfur vacancies-rich sulfides heterointerfaces are well-designed and developed via a simple one-pot carrageenan-assistant cations-regulated strategy. “Janus effect” of interfacial sulfur vacancies, which triggers strong defect-type interfacial polarization, are firstly intuitively confirmed by both theoretical and experimental investigations. Optimized Co/Ni-carbon composites (CAs) imbued with sulfur vacancies-rich heterointerfaces displays broad absorption bandwidth of 6.76 GHz at only 1.8 mm, compared to sulfur vacancies-free CAs without any dielectric response.

The structure of the HIV-1 capsid is analysed by cryo-electron microscopy and cryo-electron tomography, allowing presentation of an all-atom molecular dynamics model of the entire capsid. Atomic structure of the HIV-1 capsid Human immunodeficiency virus-1 (HIV-1), the predominant AIDS virus, contains a spheroidal capsid enclosing the viral RNA genome. As the retrovirus matures, the capsid forms through spontaneous oligomerization of the capsid protein CA. Using cryo-electron microscopy and cryo-electron tomography, combined with all-atom large-scale molecular dynamics simulations, Gongpu Zhao et al . have determined a complete atomic structure of the HIV-1 capsid. The resulting structural models reveal elements that are essential for capsid formation, stability and viral infectivity. Of special interest are the hydrophobic interactions evident in a novel three-fold interface between the carboxy-terminal domains of CA protein, a feature that appears to be unique to the mature capsid and which has previously been suggested as a potentially attractive therapeutic target. Retroviral capsid proteins are conserved structurally but assemble into different morphologies 1 . The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a ‘fullerene cone’ model 2 , 3 , in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin 4 , 5 , a carboxy-terminal domain (CTD) comprising four α-helices 6 , 7 , and a flexible linker with a 3 10 -helix connecting the two structural domains 8 . Structures of the capsid-protein assembly units have been determined by X-ray crystallography 9 , 10 ; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface 11 , a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

Understanding of the construction and function of the HIV capsid has advanced considerably in the last decade. This is due in large part to the development of more sophisticated structural techniques, particularly cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). The capsid is known to be a pleomorphic fullerene cone comprised of capsid protein monomers arranged into 200–250 hexamers and 12 pentamers. The latter of these induce high curvature necessary to close the cone at both ends. CryoEM/cryoET, NMR, and X-ray crystallography have collectively described these interactions to atomic or near-atomic resolutions. Further, these techniques have helped to clarify the role the HIV capsid plays in several parts of the viral life cycle, from reverse transcription to nuclear entry and integration into the host chromosome. This includes visualizing the capsid bound to host factors. Multiple proteins have been shown to interact with the capsid. Cyclophilin A, nucleoporins, and CPSF6 promote viral infectivity, while MxB and Trim5α diminish the viral infectivity. Finally, structural insights into the intra- and intermolecular interactions that govern capsid function have enabled development of small molecules, peptides, and truncated proteins to disrupt or stabilize the capsid to inhibit HIV replication. The most promising of these, GS6207, is now in clinical trial.

Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å 2 ) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 μm), thus making it suitable for studies of many biologically important structures. Cryo-electron microscopy is widely employed in structural biology and uses phase contrast imaging. Here, the authors employ electron ptychography, a quantitative phase retrieval method for high-contrast, low-dose phase imaging of cryo-state rotavirus and immature HIV-1 virus-like particles, and show that electron ptychography is more efficient for phase recovery than conventional phase contrast imaging.

Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions. Carboxysomes are bacterial microcompartments encapsulating Rubisco and carbonic anhydrase for carbon fixation. Here, authors determine the organization of Rubisco and its interaction with the linker protein CsoS2 within two distant α-carboxysomes.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules. In this study, Peijun Zhang and colleagues use cryoFIB/SEM volume imaging and soft x-ray cryo-tomography with cryo-electron tomography (cryoET) of cellular periphery, lamellae, and subtomogram averaging to place critical structural events in the SARS-CoV-2 infection cycle in the context of whole-cell images.