scholarly journals Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry

2016 ◽  
Vol 90 (17) ◽  
pp. 7618-7627 ◽  
Author(s):  
Erin E. H. Tran ◽  
Elizabeth A. Nelson ◽  
Pranay Bonagiri ◽  
James A. Simmons ◽  
Charles J. Shoemaker ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Structural Information ◽  
Electron Tomography ◽  
West African ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Niemann Pick ◽  
Viruslike Particles

ABSTRACTZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env.IMPORTANCEThe recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed—embedded in the membrane and present at high density—on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

scholarly journals PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
Author(s):  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Gang Fu ◽  
Mary E. Porter ◽  
Daniela Nicastro ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Structural Studies ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

scholarly journals Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers

2021 ◽  
Author(s):  
Vidya Mangala Prasad ◽  
Daniel P. Leaman ◽  
Klaus N. Lovendahl ◽  
Mark A. Benhaim ◽  
Edgar A. Hodge ◽  
...  
Keyword(s):  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Structural Variation ◽  
Electron Tomography ◽  
Host Cells ◽  
Detailed Characterization ◽  
Cryo Electron Tomography ◽  
Structural Mass Spectrometry ◽  
Structural Mass ◽  
Hiv 1

SummaryHIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1Å sub-tomogram averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers plus a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

scholarly journals In SituConformational Changes of theEscherichia coliSerine Chemoreceptor in Different Signaling States

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Wen Yang ◽  
C. Keith Cassidy ◽  
Peter Ames ◽  
Christoph A. Diebolder ◽  
Klaus Schulten ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Dynamics Simulation ◽  
Structural Constraints ◽  
Content Type ◽  
Cryo Electron Tomography ◽  
Structural Differences ◽  
Coupling Protein

ABSTRACTTsr, the serine chemoreceptor inEscherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associatein vivowith the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCEInEscherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of theE. coliserine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

scholarly journals The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

Science ◽  
2013 ◽  
Vol 339 (6119) ◽  
pp. 576-579 ◽  
Author(s):  
Bo Hu ◽  
William Margolin ◽  
Ian J. Molineux ◽  
Jun Liu
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Cell Surface Receptor ◽  
Dna Ejection ◽  
Phage Tail ◽  
Cryo Electron Tomography ◽  
Surface Receptor ◽  
Host Cell Surface ◽  
Tail Fibers

Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo–electron tomography to capture T7 virions at successive stages of infection ofEscherichia coliminicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.

scholarly journals Structure of Antibody-Neutralized Murine Norovirus and Unexpected Differences from Viruslike Particles

2007 ◽  
Vol 82 (5) ◽  
pp. 2079-2088 ◽  
Author(s):  
Umesh Katpally ◽  
Christiane E. Wobus ◽  
Kelly Dryden ◽  
Herbert W. Virgin ◽  
Thomas J. Smith
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Infectious Clone ◽  
Maturation Process ◽  
Murine Norovirus ◽  
Fab Fragments ◽  
Bacterial Viruses ◽  
Capsid Maturation ◽  
Viruslike Particles ◽  
New Interactions

ABSTRACT Noroviruses (family Caliciviridae) are the major cause of epidemic nonbacterial gastroenteritis in humans, but the mechanism of antibody neutralization is unknown and no structure of an infectious virion has been reported. Murine norovirus (MNV) is the only norovirus that can be grown in tissue culture, studied in an animal model, and reverse engineered via an infectious clone and to which neutralizing antibodies have been isolated. Presented here are the cryoelectron microscopy structures of an MNV virion and the virion in complex with neutralizing Fab fragments. The most striking differences between MNV and previous calicivirus structures are that the protruding domain is lifted off the shell domain by ∼16Å and rotated ∼40° in a clockwise fashion and forms new interactions at the P1 base that create a cagelike structure engulfing the shell domains. Neutralizing Fab fragments cover the outer surface of each copy of the capsid protein P2 domains without causing any apparent conformational changes. These unique features of MNV suggest that at least some caliciviruses undergo a capsid maturation process akin to that observed with other plant and bacterial viruses.

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 Cryo-electron tomography reveals structural insights into the membrane binding and remodeling activity of dynamin-like EHDs

2021 ◽  
Author(s):  
Arthur A. Melo ◽  
Thiemo Sprink ◽  
Jeffrey K. Noel ◽  
Elena Vázquez Sarandeses ◽  
Chris van Hoorn ◽  
...  
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Spontaneous Curvature ◽  
Membrane Remodeling ◽  
Closed Conformation ◽  
Membrane Surfaces ◽  
Cryo Electron Tomography ◽  
Membrane Bound ◽  
Subtomogram Averaging ◽  
Structural Insights

AbstractDynamin-related Eps15-homology domain containing proteins (EHDs) oligomerize on membrane surfaces into filaments leading to membrane remodeling. EHD crystal structures in an open and a closed conformation were previously reported, but structural information on the membrane-bound EHD oligomeric structure has remained enigmatic. Consequently, mechanistic insight into EHD-mediated membrane remodeling is lacking. Here, by using cryo-electron tomography and subtomogram averaging, we determined the structure of an EHD4 filament on a tubular membrane template at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests the AMPPNP-bound filament has a spontaneous curvature of approximately 1/70 nm-1. Combining the available structural and functional data, we propose a model of EHD-mediated membrane remodeling.

scholarly journals Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

2017 ◽  
Author(s):  
Danielle A. Grotjahn ◽  
Saikat Chowdhury ◽  
Yiru Xu ◽  
Richard J. McKenney ◽  
Trina A. Schroer ◽  
...  
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Force Production ◽  
Cytoplasmic Dynein ◽  
Dimensional Structure ◽  
Cellular Transport ◽  
3 Dimensional ◽  
Dynein Motor ◽  
Cryo Electron Tomography ◽  
Dynactin Complex

AbstractA key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dynein’s movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.

scholarly journals Cryo-electron tomography: an ideal method to study membrane-associated proteins

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160210 ◽  
Author(s):  
Michelle A. Dunstone ◽  
Alex de Marco
Keyword(s):  
Image Processing ◽  
Structural Information ◽  
Imaging Technique ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Macromolecular Complexes ◽  
Membrane Pores ◽  
Cryo Electron Tomography ◽  
Associated Proteins ◽  
Ideal Method

Cryo-electron tomography (cryo-ET) is a three-dimensional imaging technique that makes it possible to analyse the structure of complex and dynamic biological assemblies in their native conditions. The latest technological and image processing developments demonstrate that it is possible to obtain structural information at nanometre resolution. The sample preparation required for the cryo-ET technique does not require the isolation of a protein and other macromolecular complexes from its native environment. Therefore, cryo-ET is emerging as an important tool to study the structure of membrane-associated proteins including pores. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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.

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