Cryo-electron microscopy structure of the filamentous bacteriophage IKe

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

Journal of Virology ◽

10.1128/jvi.02255-18 ◽

2019 ◽

Vol 93 (7) ◽

Cited By ~ 3

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.

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

Communications Biology ◽

10.1038/s42003-021-02916-2 ◽

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21228643 ◽

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.

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

Virology ◽

10.1016/j.virol.2017.11.016 ◽

2018 ◽

Vol 514 ◽

pp. 165-169 ◽

Cited By ~ 3

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

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

10.1101/275131 ◽

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.

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Cryo-EM structure of VASH1-SVBP bound to microtubules

eLife ◽

10.7554/elife.58157 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Faxiang Li ◽

Yang Li ◽

Xuecheng Ye ◽

Haishan Gao ◽

Zhubing Shi ◽

...

Keyword(s):

Electron Microscopy ◽

Active Site ◽

Binding Protein ◽

Substrate Preference ◽

Structural Basis ◽

Globular Domain ◽

Specific Interactions ◽

Cryo Electron Microscopy ◽

Positively Charged

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

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Gating the pore of the calcium-activated chloride channel TMEM16A

Nature Communications ◽

10.1038/s41467-020-20787-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Andy K. M. Lam ◽

Jan Rheinberger ◽

Cristina Paulino ◽

Raimund Dutzler

Keyword(s):

Electron Microscopy ◽

Conformational Change ◽

Physical Contact ◽

Open Probability ◽

Hydrophobic Residues ◽

Closed Conformation ◽

Basal Activity ◽

Cryo Electron Microscopy ◽

Selective Channel ◽

Α Helix

AbstractThe binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.

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A 3.8 Å Resolution Cryo-EM Structure of a Small Protein Bound to a Modular Imaging Scaffold

10.1101/505792 ◽

2018 ◽

Author(s):

Yuxi Liu ◽

Duc Huynh ◽

Todd O Yeates

Keyword(s):

Electron Microscopy ◽

Amino Acid ◽

Amino Acid Sequence ◽

Structural Features ◽

Cryo Electron Microscopy ◽

Cargo Protein ◽

Size Limitation ◽

Protein Molecules ◽

Lower Size ◽

General Method

Proteins smaller than about 50 kDa are currently too small to be imaged by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein. We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where basic structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.

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Immuno-electron microscopy at sub-nanometer resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123015 ◽

1992 ◽

Vol 50 (1) ◽

pp. 522-523

Author(s):

Alasdair C. Steven ◽

William W. Newcomb ◽

Frank P. Booy ◽

Jay C. Brown ◽

Benes L. Trus

Keyword(s):

Electron Microscopy ◽

Major Capsid Protein ◽

Three Dimensional ◽

Structural Features ◽

Protein Species ◽

Cryo Electron Microscopy ◽

Immuno Electron Microscopy ◽

Simplex Virus ◽

Hsv 1

We have been studying the structure and assembly properties of the capsid of herpes simplex virus, type 1 (HSV-1), whose icosahedral shell (T=16) is ∽ 125 nm in diameter, has a mass of ∽ 200 MDa, and is composed of at least six protein species, ranging from 12 kDa to 149 kDa per monomer. To this end, the structures of empty and full purified capsids have been visualized in three-dimensional reconstructions from cryo-electron micrographs, at a resolution of ∽ 3.5 nm. With the intent of identifying specific structural features of the capsid in terms of particular proteins, or particular segments of these proteins, we have begun to explore the potentialities of cryo-reconstructions of capsids decorated with monoclonal antibodies.B-capsids were purified from BHK cells infected with the MP strain of HSV-1. Some material was analyzed directly by cryo-electron microscopy and image reconstruction as described. Other samples were incubated with approximately equimolar amounts of a monoclonal antibody (8F5) raised against purified VP5 (the major capsid protein, 149 kDa). The resulting precipitate was then washed with buffer to flush out unbound antibodies, and then analyzed in the same way, i.e. by observation in a Philips EM400T equipped with a Gatan 626 cryo-holder. The specificity of Mab.8F5 for VP5 was confirmed by an ELISA with purified VP5.

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Cryo-electron Microscopy Structure of the Swine Acute Diarrhea Syndrome Coronavirus Spike Glycoprotein Provides Insights into Evolution of Unique Coronavirus Spike Proteins

Journal of Virology ◽

10.1128/jvi.01301-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Hongxin Guan ◽

Youwang Wang ◽

Vanja Perčulija ◽

Abdullah F. U. H. Saeed ◽

Yichang Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Immune Evasion ◽

Acute Diarrhea ◽

Structural Features ◽

The Other ◽

Spike Protein ◽

Interspecies Transmission ◽

Cryo Electron Microscopy ◽

Diarrhea Syndrome ◽

Spike Proteins

ABSTRACT Coronaviruses (CoV) have caused a number of major epidemics in humans and animals, including the current pandemic of coronavirus disease 2019 (COVID-19), which has brought a renewed focus on the evolution and interspecies transmission of coronaviruses. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was recently identified in piglets in southern China, is an alphacoronavirus that originates from the same genus of horseshoe bats as severe acute respiratory syndrome CoV (SARS-CoV) and that was reported to be capable of infecting cells from a broad range of species, suggesting a considerable potential for interspecies transmission. Given the importance of the coronavirus spike (S) glycoprotein in host range determination and viral entry, we report a cryo-electron microscopy (cryo-EM) structure of the SADS-CoV S trimer in the prefusion conformation at a 3.55-Å resolution. Our structure reveals that the SADS-CoV S trimer assumes an intrasubunit quaternary packing mode in which the S1 subunit N-terminal domain (S1-NTD) and the S1 subunit C-terminal domain (S1-CTD) of the same protomer pack together by facing each other in the lying-down state. SADS-CoV S has several distinctive structural features that may facilitate immune escape, such as a relatively compact architecture of the S trimer and epitope masking by glycan shielding. Comparison of SADS-CoV S with the spike proteins of the other coronavirus genera suggested that the structural features of SADS-CoV S are evolutionarily related to those of the spike proteins of the other genera rather than to the spike protein of a typical alphacoronavirus. These data provide new insights into the evolutionary relationship between spike glycoproteins of SADS-CoV and those of other coronaviruses and extend our understanding of their structural and functional diversity. IMPORTANCE In this article, we report the atomic-resolution prefusion structure of the spike protein from swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV is a pathogenic alphacoronavirus that was responsible for a large-scale outbreak of fatal disease in pigs and that was reported to be capable of interspecies transmission. We describe the overall structure of the SADS-CoV spike protein and conducted a detailed analysis of its main structural elements. Our results and analyses are consistent with those of previous phylogenetic studies and suggest that the SADS-CoV spike protein is evolutionarily related to the spike proteins of betacoronaviruses, with a strong similarity in S1-NTDs and a marked divergence in S1-CTDs. Moreover, we discuss the possible immune evasion strategies used by the SADS-CoV spike protein. Our study provides insights into the structure and immune evasion strategies of the SADS-CoV spike protein and broadens the understanding of the evolutionary relationships between coronavirus spike proteins of different genera.

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