scholarly journals Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yuanzhu Gao ◽  
Shanshan Liu ◽  
Jiamiao Huang ◽  
Qianqian Wang ◽  
Kunpeng Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Structure ◽  
Structural Features ◽  
Economic Losses ◽  
Scylla Paramamosain ◽  
Multiple Infections ◽  
Mud Crab ◽  
Cryo Electron Microscopy ◽  
Novel Virus ◽  
Icosahedral Capsid

ABSTRACT Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus. IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Wenxin Hu ◽  
Hongjin Zheng
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Molecular Basis ◽  
Iron Uptake ◽  
Structural Features ◽  
Biological Processes ◽  
Type Ii ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
Translocation Pathway

AbstractAs one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer’s ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

scholarly journals High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants

2020 ◽  
Vol 21 (22) ◽  
pp. 8643
Author(s):  
Alessandro Grinzato ◽  
Pascal Albanese ◽  
Roberto Marotta ◽  
Paolo Swuec ◽  
Guido Saracco ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Photosystem Ii ◽  
Structural Rearrangement ◽  
Structural Features ◽  
Thylakoid Membranes ◽  
Variable Number ◽  
High Light ◽  
Low Light ◽  
Cryo Electron Microscopy ◽  
Light Effects

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

3D Reconstruction of Smooth Muscle A-Actinin by Cryo Electron Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 244-245
Author(s):  
Jun Liu ◽  
Dianne Taylor ◽  
Kenneth A. Taylor
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Calcium Binding ◽  
3D Structure ◽  
Unit Cell Dimension ◽  
Lipid Monolayer ◽  
Actin Binding ◽  
Cryo Electron Microscopy ◽  
Ef Hand ◽  
Polypeptide Chains

α-Actinin is an actin crosslinking protein identified in a wide variety of cells. Both muscle and nonmuscle isoforms of α-actinin have been characterized [1]. The molecule consists of two polypeptide chains that form a rod-shaped antiparallel dimer. Each polypeptide is composed of three distinct structural regions: actin-binding domain, four triple helical repeats that form a central rod, and a carboxyl terminal domain that contains two EF hand calcium-binding sites. Here we present the 3D structure of the smooth muscle α-actinin, as determined by cryo electron microscopy at 2.0 nm resolution.Two dimensional crystals of chicken gizzard α-actinin were formed on positively charged lipid monolayer [2] and preserved frozen hydrated for TEM. The crystal has a unit cell dimension of a = 26.31 nm, b = 20.37 nm, γ= 107.10°, based on internal calibration against TMV.

Cryo-Electron Microscopy and Image Analysis of SV40

1985 ◽  
Vol 43 ◽  
pp. 316-317
Author(s):  
T. S. Baker ◽  
J. Drak ◽  
M. Bina
Keyword(s):  
Electron Microscopy ◽  
Simian Virus 40 ◽  
Carbon Support ◽  
Simian Virus ◽  
Thin Layers ◽  
Sv40 Virus ◽  
Virus Particles ◽  
Frozen Solution ◽  
Cryo Electron Microscopy ◽  
Icosahedral Capsid

The discovery that the T=7 icosahedral capsid of polyoma virus is composed of 72 pentameric capsomers rather than 12 pentamers and 60 hexamers as predicted by constraints of quasi-equivalence has prompted an examination of SV40 virus by electron microscopy to determine whether the capsids of other members of the papovavirus family are similarly constructed.Thin layers of buffered aqueous solutions (∼4 mg/ml) of Simian virus 40 (strain WT776) were prepared for cryo-microscopy using recently developed procedures. Images of virus particles suspended in thin layers of vitreous ice over holes in the carbon support film and maintained at -170°C were recorded using minimal irradiation conditions. Figure 1 shows a typical field in which the frozen solution is similar in thickness to the virus particles (∼49 nm diameter). Particles appear to be excluded or squeezed away from the thinnest regions of solution (e.g., the clear region bordered by particles at the top of Fig. 1).

scholarly journals Antibody-induced uncoating of human rhinovirus B14

2017 ◽  
Vol 114 (30) ◽  
pp. 8017-8022 ◽  
Author(s):  
Yangchao Dong ◽  
Yue Liu ◽  
Wen Jiang ◽  
Thomas J. Smith ◽  
Zhikai Xu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Virus Infection ◽  
Conformational Changes ◽  
Neutralizing Antibody ◽  
Antigen Binding ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
Virus Complex ◽  
Positive Strand Rna ◽  
Icosahedral Capsid

Rhinoviruses (RVs) are the major causes of common colds in humans. They have a nonenveloped, icosahedral capsid surrounding a positive-strand RNA genome. Here we report that the antigen-binding (Fab) fragment of a neutralizing antibody (C5) can trigger genome release from RV-B14 to form emptied particles and neutralize virus infection. Using cryo-electron microscopy, structures of the C5 Fab in complex with the full and emptied particles have been determined at 2.3 Å and 3.0 Å resolution, respectively. Each of the 60 Fab molecules binds primarily to a region on viral protein 3 (VP3). Binding of the C5 Fabs to RV-B14 results in significant conformational changes around holes in the capsid through which the viral RNA might exit. These results are so far the highest resolution view of an antibody–virus complex and elucidate a mechanism whereby antibodies neutralize RVs and related viruses by inducing virus uncoating.

scholarly journals Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography

Virology ◽  
2018 ◽  
Vol 514 ◽  
pp. 165-169 ◽  
Author(s):  
Igor Orlov ◽  
Robert Drillien ◽  
Danièle Spehner ◽  
Max Bergoin ◽  
Adly M.M. Abd-Alla ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Salivary Gland ◽  
Structural Features ◽  
Tsetse Fly ◽  
Cryo Electron Microscopy ◽  
Salivary Gland Hypertrophy ◽  
Salivary Gland Hypertrophy Virus

scholarly journals A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

2018 ◽  
Author(s):  
A. Sicorello ◽  
G. Kelly ◽  
A. Oregioni ◽  
J. Nováček ◽  
V. Sklenář ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Magnetic Resonance ◽  
Low Complexity ◽  
Structural Features ◽  
Resonance Spectroscopy ◽  
Cryo Electron Microscopy ◽  
Per Se ◽  
Folded Proteins ◽  
First Time ◽  
Structure In Solution

AbstractIt has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions which both can contribute to function. While equally interesting, the disordered regions are difficult to study because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. Nuclear magnetic resonance spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe assignment of the spectrum of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We used a 42 kDa construct containing a globular N-terminal josephin domain and a C-terminal tail which comprises thirteen polyglutamine repeats within a low-complexity region. We developed a strategy which allowed us to achieve 87% assignment of the spectrum. We show that the C-terminal tail is flexible with extended helical regions and interacts only marginally with the rest of the protein. We could also, for the first time, deduce the structure of the polyglutamine repeats within the context of the full-length protein and show that it has a strong helical propensity stabilized by the preceding region.

Protein modeling by homology using the example of tick-borne encephalitis serine protease NS3

2020 ◽  
pp. 59-66
Author(s):  
Nikita Ilment ◽  
Ekaterina Zinina
Keyword(s):  
Electron Microscopy ◽  
Homology Modeling ◽  
Homology Modelling ◽  
Protein Structures ◽  
3D Structure ◽  
Free Software ◽  
Homologous Proteins ◽  
Cryo Electron Microscopy ◽  
Tick Borne Encephalitis ◽  
Sequences Analysis

Homology modeling is a process of obtaining a 3D structure of a protein using various algorithms based on already known structures of homologous proteins. The spatial structure of protein is required for in silico protein evaluation. 3D structures can be obtained using different methods: NMR, Xray crystallography (XRC), and cryo-electron microscopy (cryo-EM), but these methods require a lot of time and money. At the same time, the speed of nucleotide sequences analysis is increasing, thereby creating a mismatch between the number of decoded genomes and the investigated 3D protein structures that are encoded by these sequences. Also, homology modeling is the easiest and fastest way to obtain the model of the desired protein. This review describes free software for homology modelling — SWISS-MODEL and MODELLER, how to use it and how to evaluate the results.

scholarly journals Cryo-electron microscopy structure of the filamentous bacteriophage IKe

2019 ◽  
Vol 116 (12) ◽  
pp. 5493-5498 ◽  
Author(s):  
Jingwei Xu ◽  
Nir Dayan ◽  
Amir Goldbourt ◽  
Ye Xiang
Keyword(s):  
Electron Microscopy ◽  
Coat Protein ◽  
Sequence Divergence ◽  
Structural Features ◽  
Horizontal Layer ◽  
Filamentous Bacteriophage ◽  
Hydrophobic Residues ◽  
Left Handed ◽  
Cryo Electron Microscopy ◽  
Positively Charged

The filamentous bacteriophage IKe infectsEscherichia colicells bearing IncN pili. We report the cryo-electron microscopy structure of the micrometer-long IKe viral particle at a resolution of 3.4 Å. The major coat protein [protein 8 (p8)] consists of 47 residues that fold into a ∼68-Å-long helix. An atomic model of the coat protein was built. Five p8 helices in a horizontal layer form a pentamer, and symmetrically neighboring p8 layers form a right-handed helical cylinder having a rise per pentamer of 16.77 Å and a twist of 38.52°. The inner surface of the capsid cylinder is positively charged and has direct interactions with the encapsulated circular single-stranded DNA genome, which has an electron density consistent with an unusual left-handed helix structure. Similar to capsid structures of other filamentous viruses, strong capsid packing in the IKe particle is maintained by hydrophobic residues. Despite having a different length and large sequence differences from other filamentous phages, π–π interactions were found between Tyr9 of one p8 and Trp29 of a neighboring p8 in IKe that are similar to interactions observed in phage M13, suggesting that, despite sequence divergence, overall structural features are maintained.

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