Structure of the Infective Getah Virus at 2.8 Å-resolution Determined by Cryo-electron Microscopy

Getah virus (GETV) is a mosquito-borne pathogen that can cause a mild illness and reproductive losses in animals. Although antibodies to GETV have been found in humans, there are no reports of clinical symptom associated with GETV. However, antivirals or vaccine against GETV is still unavailable due to lack of knowledge of the structure of GETV virion. Here, we present the structure of mature GETV at a resolution of 2.8 Å with capsid protein, envelope glycoproteins E1 and E2. Glycosylation and S-acylation sites in E1 and E2 are identified. The surface-exposed glycans demonstrated their impact on the viral immune evasion and host cell invasion. The S-acylation sites strongly stabilize the virion. In addition, a cholesterol and phospholipid molecule are observed in transmembrane hydrophobic pocket, together with two more cholesterols surround the pocket. These structural information are helpful for structure-based antivirals and vaccine design.

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A resolution record for cryoEM

Faculty Reviews ◽

10.12703/r-01-000002 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jonathan Ashmore ◽

Bridget Carragher ◽

Peter B Rosenthal ◽

William Weis

Keyword(s):

Electron Microscopy ◽

Image Analysis ◽

Structure Determination ◽

Test Specimen ◽

Structural Information ◽

Atomic Resolution ◽

Fast Growing ◽

Cryo Electron Microscopy ◽

New Instrument ◽

Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

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Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation

eLife ◽

10.7554/elife.62772 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Xiaowei Hou ◽

Ian R Outhwaite ◽

Leanne Pedi ◽

Stephen Barstow Long

Keyword(s):

Electron Microscopy ◽

Amino Acids ◽

Calcium Channel ◽

Calcium Release ◽

Structural Information ◽

Excitable Cells ◽

X Ray ◽

Cryo Electron Microscopy ◽

Open Conformation ◽

Hydrophobic Amino Acids

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel’s gate. Structured M1–M2 turrets, not evident previously, form the channel’s extracellular entrance.

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Template-free 13-protofilament microtubule–MAP assembly visualized at 8 Å resolution

The Journal of Cell Biology ◽

10.1083/jcb.201007081 ◽

2010 ◽

Vol 191 (3) ◽

pp. 463-470 ◽

Cited By ~ 91

Author(s):

Franck J. Fourniol ◽

Charles V. Sindelar ◽

Béatrice Amigues ◽

Daniel K. Clare ◽

Geraint Thomas ◽

...

Keyword(s):

Electron Microscopy ◽

Structural Information ◽

Binding Mode ◽

Microtubule Associated Proteins ◽

Cryo Electron Microscopy ◽

Associated Proteins ◽

Template Free ◽

Map Function ◽

Insight Into

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

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Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase fromBacteroides fragilis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14012722 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1206-1210 ◽

Cited By ~ 2

Author(s):

Chongyun Cheng ◽

Jianhua Gu ◽

Jing Su ◽

Wei Ding ◽

Jie Yin ◽

...

Keyword(s):

Electron Microscopy ◽

Analytical Ultracentrifugation ◽

Structural Information ◽

Three Dimensional ◽

Size Exclusion ◽

Diffusion Method ◽

Bifunctional Enzyme ◽

X Rays ◽

Cell Parameters ◽

Cryo Electron Microscopy

Fucokinase/L-fucose-1-P-guanylyltransferase (FKP) is a bifunctional enzyme which converts L-fucose to Fuc-1-P and thence to GDP-L-fucose through a salvage pathway. The molecular weights of full-length FKP (F-FKP) and C-terminally truncated FKP (C-FKP, residues 300–949) are 105.7 and 71.7 kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space groupP212121and diffracted synchrotron X-rays to 3.7 Å resolution. The crystal unit-cell parameters area= 91.36,b= 172.03,c= 358.86 Å, α = β = γ = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19 Å3 Da−1with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.

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Unconventional Specimen Preparation Techniques Using High Resolution Low Voltage Field Emission Scanning Electron Microscopy to Study Cell Motility, Host Cell Invasion, and Internal Cell Structures in Toxoplasma gondii

Microscopy and Microanalysis ◽

10.1017/s1431927601020025 ◽

2002 ◽

Vol 8 (2) ◽

pp. 94-103 ◽

Cited By ~ 9

Author(s):

Heide Schatten ◽

Hans Ris

Keyword(s):

Electron Microscopy ◽

Cell Division ◽

Host Cell ◽

Cell Invasion ◽

Low Voltage ◽

Cytoskeletal Proteins ◽

Specimen Preparation ◽

Host Cell Invasion ◽

Apicomplexan Parasites ◽

Preparation Techniques

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

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The potential of cryo-electron microscopy for structure-based drug design

Essays in Biochemistry ◽

10.1042/ebc20170032 ◽

2017 ◽

Vol 61 (5) ◽

pp. 543-560 ◽

Cited By ~ 21

Author(s):

Andreas Boland ◽

Leifu Chang ◽

David Barford

Keyword(s):

Electron Microscopy ◽

Drug Discovery ◽

Drug Design ◽

Structural Information ◽

Biological Molecules ◽

Ligand Complex ◽

Structure Based Drug Design ◽

Technical Developments ◽

Cryo Electron Microscopy ◽

Therapeutic Development

Structure-based drug design plays a central role in therapeutic development. Until recently, protein crystallography and NMR have dominated experimental approaches to obtain structural information of biological molecules. However, in recent years rapid technical developments in single particle cryo-electron microscopy (cryo-EM) have enabled the determination to near-atomic resolution of macromolecules ranging from large multi-subunit molecular machines to proteins as small as 64 kDa. These advances have revolutionized structural biology by hugely expanding both the range of macromolecules whose structures can be determined, and by providing a description of macromolecular dynamics. Cryo-EM is now poised to similarly transform the discipline of structure-based drug discovery. This article reviews the potential of cryo-EM for drug discovery with reference to protein ligand complex structures determined using this technique.

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Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement

10.1101/2021.12.28.474380 ◽

2021 ◽

Author(s):

Matthew McCallum ◽

Nadine Czudnochowski ◽

Laura E Rosen ◽

Samantha K Zepeda ◽

John E Bowen ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Crystal Structures ◽

Immune Evasion ◽

Marked Reduction ◽

Structural Basis ◽

X Ray ◽

Structural Framework ◽

Cryo Electron Microscopy ◽

Antigenic Shift

The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.

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Structural basis of σ appropriation

Nucleic Acids Research ◽

10.1093/nar/gkz682 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9423-9432 ◽

Cited By ~ 6

Author(s):

Jing Shi ◽

Aijia Wen ◽

Minxing Zhao ◽

Linlin You ◽

Yu Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Transcription Factors ◽

Structural Information ◽

Bacteriophage T4 ◽

Transcription Activation ◽

Structural Basis ◽

Concerted Effort ◽

Double Stranded Dna ◽

Cryo Electron Microscopy ◽

Large Conformational Change

Abstract Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.

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Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

Communications Biology ◽

10.1038/s42003-019-0604-2 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 16

Author(s):

Herve Celia ◽

Istvan Botos ◽

Xiaodan Ni ◽

Tara Fox ◽

Natalia De Val ◽

...

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

High Resolution ◽

Outer Membrane ◽

Motor Function ◽

Structural Information ◽

Molecular Motor ◽

Single Copy ◽

Proton Motive Force ◽

Cryo Electron Microscopy

Abstract The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

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