scholarly journals Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein

2021 ◽  
Vol 118 (48) ◽  
pp. e2112703118
Author(s):  
Linhua Tai ◽  
Guoliang Zhu ◽  
Minnan Yang ◽  
Lei Cao ◽  
Xiaorui Xing ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Structural Information ◽  
Electron Tomography ◽  
Host Cells ◽  
Spike Protein ◽  
Fusion Pore ◽  
Nanometer Resolution ◽  
Glycosylation Sites ◽  
Entry Mechanism

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in β-propiolactone–inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.

scholarly journals Nanometer resolution in situ structure of SARS-CoV-2 post-fusion spike

2021 ◽  
Author(s):  
Yun Zhu ◽  
Fei Sun ◽  
Xiangxi Wang ◽  
Linhua Tai ◽  
Guoliang Zhu ◽  
...  
Keyword(s):  
Pore Formation ◽  
Transmembrane Domain ◽  
Structural Information ◽  
Protein S ◽  
Fusion Pore ◽  
Electron Microscopic ◽  
Nanometer Resolution ◽  
Entry Mechanism ◽  
Resolution Structure

Abstract The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

scholarly journals In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shrawan Kumar Mageswaran ◽  
Amandine Guérin ◽  
Liam M. Theveny ◽  
William David Chen ◽  
Matthew Martinez ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Electron Tomography ◽  
Secretion System ◽  
Host Cells ◽  
Apical Region ◽  
Secretion Systems ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Secretory Apparatus

AbstractParasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite’s apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.

scholarly journals In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

2022 ◽  
Author(s):  
Matthew Martinez ◽  
William David Chen ◽  
Marta Cova ◽  
Petra Andrea Molnár ◽  
Shrawan Kumar Mageswaran ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Secretion System ◽  
Host Cells ◽  
Apicomplexan Parasites ◽  
Significant Difference ◽  
Cryo Electron Tomography ◽  
Parasite Plasmodium Falciparum ◽  
Priming Process

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

scholarly journals Deciphering the O-Glycosylation of HKU1 Spike Protein With the Dual-Functional Hydrophilic Interaction Chromatography Materials

2021 ◽  
Vol 9 ◽  
Author(s):  
Yun Cui ◽  
Xuefang Dong ◽  
Xiaofei Zhang ◽  
Cheng Chen ◽  
Dongmei Fu ◽  
...  
Keyword(s):  
Protein S ◽  
Host Cells ◽  
Spike Protein ◽  
Biological Functions ◽  
Hydrophilic Interaction ◽  
Protein Surface ◽  
Enrichment Method ◽  
Dual Functional ◽  
Glycosylation Sites ◽  
Comprehensive Study

HKU1 is a human beta coronavirus and infects host cells via highly glycosylated spike protein (S). The N-glycosylation of HKU1 S has been reported. However, little is known about its O-glycosylation, which hinders the in-depth understanding of its biological functions. Herein, a comprehensive study of O-glycosylation of HKU1 S was carried out based on dual-functional histidine-bonded silica (HBS) materials. The enrichment method for O-glycopeptides with HBS was developed and validated using standard proteins. The application of the developed method to the HKU1 S1 subunit resulted in 46 novel O-glycosylation sites, among which 55.6% were predicted to be exposed on the outer protein surface. Moreover, the O-linked glycans and their abundance on each HKU1 S1 site were analyzed. The obtained O-glycosylation dataset will provide valuable insights into the structure of HKU1 S.

scholarly journals Fine details in complex environments: the power of cryo-electron tomography

2018 ◽  
Vol 46 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Joshua Hutchings ◽  
Giulia Zanetti
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Molecular Structures ◽  
Complex Environments ◽  
Single Particle Reconstruction ◽  
Wide Range ◽  
Cryo Electron Tomography ◽  
Recent Developments

Cryo-electron tomography (CET) is uniquely suited to obtain structural information from a wide range of biological scales, integrating and bridging knowledge from molecules to cells. In particular, CET can be used to visualise molecular structures in their native environment. Depending on the experiment, a varying degree of resolutions can be achieved, with the first near-atomic molecular structures becoming recently available. The power of CET has increased significantly in the last 5 years, in parallel with improvements in cryo-EM hardware and software that have also benefited single-particle reconstruction techniques. In this review, we cover the typical CET pipeline, starting from sample preparation, to data collection and processing, and highlight in particular the recent developments that support structural biology in situ. We provide some examples that highlight the importance of structure determination of molecules embedded within their native environment, and propose future directions to improve CET performance and accessibility.

A Combined In-situ and Electron Tomography Holder for (S)TEM

2007 ◽  
Vol 15 (1) ◽  
pp. 34-35
Author(s):  
C. Mitterbauer ◽  
N.D. Browning ◽  
Pushkarraj V. Deshmukh
Keyword(s):  
Scanning Transmission Electron Microscopy ◽  
Structural Information ◽  
Electron Tomography ◽  
Analytical Techniques ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
3D Information ◽  
Electron Energy Loss

Recent advances in in-situ and tomographic methods using (scanning) transmission electron microscopy ((S)TEM) provide the unique possibility of obtaining 3-dimensional (3D) information and in-situ studies of gas-solid chemical reactions on the nanometer scale. In combination with the well-known analytical techniques, electron energy-loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) we can obtain 3D, dynamic, analytical, and structural information down to the atomic level.The JEOL JEM-2500SE STEM/TEM at UC Davis (CHMS) is equipped with a Schottky field-emission source operated at 200 kV, a post column Gatan imaging filter (863 GIF Tridiem) for EELS and a Thermo System Six for EDXS.

scholarly journals Cryo-electron tomography: The challenge of doing structural biology in situ

2013 ◽  
Vol 202 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Vladan Lučić ◽  
Alexander Rigort ◽  
Wolfgang Baumeister
Keyword(s):  
Cell Biology ◽  
Structural Information ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Molecular Structures ◽  
New Techniques ◽  
Preparation Methods ◽  
Technical Advances ◽  
Macromolecular Organization

Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.

scholarly journals Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

scholarly journals In situ cryo-ET structure of phycobilisome–photosystem II supercomplex from red alga

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Meijing Li ◽  
Jianfei Ma ◽  
Xueming Li ◽  
Sen-Fang Sui
Keyword(s):  
Photosystem Ii ◽  
Structural Information ◽  
Electron Tomography ◽  
Light Energy ◽  
Porphyridium Purpureum ◽  
Cellular Environment ◽  
Interaction Interface ◽  
Subtomogram Averaging ◽  
Light Harvesting Antenna

Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS–PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS–PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three ‘connector’ proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS–PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS–PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.

scholarly journals Structural remodeling of bacteriophage T4 and host membranes during infection initiation

2015 ◽  
Vol 112 (35) ◽  
pp. E4919-E4928 ◽  
Author(s):  
Bo Hu ◽  
William Margolin ◽  
Ian J. Molineux ◽  
Jun Liu
Keyword(s):  
Cell Surface ◽  
Structural Changes ◽  
Structural Information ◽  
Electron Tomography ◽  
Bacteriophage T4 ◽  
Host Cells ◽  
Irreversible Adsorption ◽  
Structural Remodeling ◽  
Host Cytoplasm ◽  
Mechanistic Pathway

The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinnyE. coliminicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection.

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