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- PDB-7n69: Pre-fusion state 2 of EEEV with localized reconstruction -

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Basic information

Entry
Database: PDB / ID: 7n69
TitlePre-fusion state 2 of EEEV with localized reconstruction
Components
  • Spike glycoprotein E1
  • Spike glycoprotein E2
KeywordsVIRUS / EEEV / pre-fusion / localized reconstruction
Function / homology
Function and homology information


togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell ...togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / RNA binding / membrane
Similarity search - Function
Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein ...Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein / Alphavirus E1 glycoprotein / Alphavirus core protein (CP) domain profile. / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / Immunoglobulin E-set / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Structural polyprotein
Similarity search - Component
Biological speciesEastern equine encephalitis virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 14.1 Å
AuthorsChen, C.-L. / Kuhn, R.J. / Klose, T.
Funding support United States, 5items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein E1
B: Spike glycoprotein E2
C: Spike glycoprotein E1
D: Spike glycoprotein E2
E: Spike glycoprotein E1
F: Spike glycoprotein E2
G: Spike glycoprotein E1
H: Spike glycoprotein E2
I: Spike glycoprotein E1
J: Spike glycoprotein E2
K: Spike glycoprotein E1
L: Spike glycoprotein E2


Theoretical massNumber of molelcules
Total (without water)569,91112
Polymers569,91112
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40410 Å2
ΔGint-46 kcal/mol
Surface area125330 Å2

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Components

#1: Protein
Spike glycoprotein E1 / E1 envelope glycoprotein


Mass: 47938.141 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus (strain Florida 91-469)
Strain: Florida 91-469 / Production host: Cricetinae gen. sp. (mammal) / References: UniProt: Q4QXJ7
#2: Protein
Spike glycoprotein E2 / E2 envelope glycoprotein


Mass: 47046.953 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus (strain Florida 91-469)
Strain: Florida 91-469 / Production host: Cricetinae gen. sp. (mammal) / References: UniProt: Q4QXJ7

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Eastern equine encephalitis virusEastern equine encephalitis
Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Eastern equine encephalitis virus
Source (recombinant)Organism: Cricetinae gen. sp. (mammal) / Cell: Baby hamster kidney cells
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Equus caballus
Virus shellDiameter: 660 nm
Buffer solutionpH: 5.5 / Details: pH 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
1140 mMsodium chlorideNaClSodium chloride1
210 mMpotassium chlorideKCl1
38 mMsodium phosphateNa2HPO41
42 mMpotassium phosphateKH2PO41
550 mMsodium citrateNa3C6H5O71
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Estimate E2 protein concentration by SDS-PAGE with BSA ranging from 0.1 to 1 microgram.
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 298 K / Details: blot for 3.3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 100 K
Image recordingElectron dose: 32 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1RELION2particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7MDFFmodel fitting
9jspr2017initial Euler assignment
10jspr2017final Euler assignment
12cisTEM3D reconstruction
13MDFFmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 14.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192060 / Algorithm: BACK PROJECTION
Details: 192060 asymmetric units were used for reconstruction
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MX4

6mx4

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