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- EMDB-24199: Localized reconstruction of EEEV -

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Basic information

Entry
Database: EMDB / ID: EMD-24199
TitleLocalized reconstruction of EEEV
Map dataEEEV_alone
Sample
  • Virus: Eastern equine encephalitis virus
Biological speciesEastern equine encephalitis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsChen C-L / Kuhn RJ / Klose T
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI095366 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI142790 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI095436 United States
Defense Threat Reduction Agency (DTRA)HDTRA1-15-1-0047 United States
Defense Threat Reduction Agency (DTRA)HDTRA1-15-1-0013 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states.
Authors: Chun-Liang Chen / Thomas Klose / Chengqun Sun / Arthur S Kim / Geeta Buda / Michael G Rossmann / Michael S Diamond / William B Klimstra / Richard J Kuhn /
Abstract: Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for ...Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.
History
DepositionJun 7, 2021-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24199.png

Downloads & links

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Map

FileDownload / File: emd_24199.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEEEV_alone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.62 Å/pix.
x 256 pix.
= 414.208 Å		1.62 Å/pix.
x 256 pix.
= 414.208 Å		1.62 Å/pix.
x 256 pix.
= 414.208 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.618 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-6.864348 - 16.462105
Average (Standard dev.)5.0803775e-09 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 414.208 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Eastern equine encephalitis virus

EntireName: Eastern equine encephalitis virus
Components
  • Virus: Eastern equine encephalitis virus

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Supramolecule #1: Eastern equine encephalitis virus

SupramoleculeName: Eastern equine encephalitis virus / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 11021 / Sci species name: Eastern equine encephalitis virus / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Equus caballus (horse)
Host systemOrganism: Cricetinae gen. sp. (mammal) / Recombinant cell: Baby hamster kidney cells
Virus shellShell ID: 1 / Diameter: 660.0 Å

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
20.0 mMC4H11NO3Tris
0.1 mMNa2HPO4EDTA

Details: pH 8
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / Details: blot for 3.3 seconds before plunging.
DetailsEstimate E2 protein concentration by SDS-PAGE with BSA ranging from 0.1 to 1 microgram.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 38000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Details: Reconstruction from 848700 asymmetric units / Number images used: 848700
Initial angle assignmentType: COMMON LINE / Software - Name: jspr (ver. 2017)
Final angle assignmentType: COMMON LINE / Software - Name: jspr (ver. 2017)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mx4

RefinementProtocol: RIGID BODY FIT

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