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- EMDB-24205: Pre-fusion state 1 of EEEV with localized reconstruction -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-24205
TitlePre-fusion state 1 of EEEV with localized reconstruction
Map dataEEEV early prefusion state
Sample
  • Virus: Eastern equine encephalitis virus
    • Protein or peptide: Spike glycoprotein E1
    • Protein or peptide: Spike glycoprotein E2
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 (strain Florida 91-469) / Eastern equine encephalitis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 14.3 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_24205.png

Downloads & links

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Map

FileDownload / File: emd_24205.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEEEV early prefusion state
Voxel sizeX=Y=Z: 5.312 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-5.569626 - 6.2539067
Average (Standard dev.)-1.6348719e-09 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-54-54-54
Dimensions108108108
Spacing108108108
CellA=B=C: 573.696 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
    • Protein or peptide: Spike glycoprotein E1
    • Protein or peptide: Spike glycoprotein E2

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Supramolecule #1: Eastern equine encephalitis virus

SupramoleculeName: Eastern equine encephalitis virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / 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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Macromolecule #1: Spike glycoprotein E1

MacromoleculeName: Spike glycoprotein E1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Eastern equine encephalitis virus (strain Florida 91-469)
Strain: Florida 91-469
Molecular weightTheoretical: 47.938141 KDa
Recombinant expressionOrganism: Cricetinae gen. sp. (mammal)
SequenceString: YEHTAVMPNK VGIPYKALVE RPGYAPVHLQ IQLVNTRIIP STNLEYITCK YKTKVPSPVV KCCGATQCTS KPHPDYQCQV FTGVYPFMW GGAYCFCDTE NTQMSEAYVE RSEECSIDHA KAYKVHTGTV QAMVNITYGS VSWRSADVYV NGETPAKIGD A KLIIGPLS ...String:
YEHTAVMPNK VGIPYKALVE RPGYAPVHLQ IQLVNTRIIP STNLEYITCK YKTKVPSPVV KCCGATQCTS KPHPDYQCQV FTGVYPFMW GGAYCFCDTE NTQMSEAYVE RSEECSIDHA KAYKVHTGTV QAMVNITYGS VSWRSADVYV NGETPAKIGD A KLIIGPLS SAWSPFDNKV VVYGHEVYNY DFPEYGTGKA GSFGDLQSRT STSNDLYANT NLKLQRPQAG IVHTPFTQAP SG FERWKRD KGAPLNDVAP FGCSIALEPL RAENCAVGSI PISIDIPDAA FTRISETPTV SDLECKITEC TYASDFGGIA TVA YKSSKA GNCPIHSPSG VAVIKENDVT LAESGSFTFH FSTANIHPAF KLQVCTSAVT CKGDCKPPKD HIVDYPAQHT ESFT SAISA TAWSWLKVLV GGTSAFIVLG LIATAVVALV LFFHRH

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Macromolecule #2: Spike glycoprotein E2

MacromoleculeName: Spike glycoprotein E2 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Eastern equine encephalitis virus (strain Florida 91-469)
Strain: Florida 91-469
Molecular weightTheoretical: 47.046953 KDa
Recombinant expressionOrganism: Cricetinae gen. sp. (mammal)
SequenceString: DLDTHFTQYK LARPYIADCP NCGHSRCDSP IAIEEVRGDA HAGVIRIQTS AMFGLKTDGV DLAYMSFMNG KTQKSIKIDN LHVRTSAPC SLVSHHGYYI LAQCPPGDTV TVGFHDGPNR HTCTVAHKVE FRPVGREKYR HPPEHGVELP CNRYTHKRAD Q GHYVEMHQ ...String:
DLDTHFTQYK LARPYIADCP NCGHSRCDSP IAIEEVRGDA HAGVIRIQTS AMFGLKTDGV DLAYMSFMNG KTQKSIKIDN LHVRTSAPC SLVSHHGYYI LAQCPPGDTV TVGFHDGPNR HTCTVAHKVE FRPVGREKYR HPPEHGVELP CNRYTHKRAD Q GHYVEMHQ PGLVADHSLL SIHSAKVKIT VPSGAQVKYY CKCPDVREGI TSSDHTTTCT DVKQCRAYLI DNKKWVYNSG RL PRGEGDT FKGKLHVPFV PVKAKCIATL APEPLVEHKH RTLILHLHPD HPTLLTTRSL GSDANPTRQW IERPTTVNFT VTG EGLEYT WGNHPPKRVW AQESGEGNPH GWPHEVVVYY YNRYPLTTII GLCTCVAIIM VSCVTSVWLL CRTRNLCITP YKLA PNAQV PILLALLCCI KPTRA

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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: 5.5
Component:
ConcentrationFormulaName
140.0 mMNaClSodium chloridesodium chloride
10.0 mMKClpotassium chloride
8.0 mMNa2HPO4sodium phosphate
2.0 mMKH2PO4potassium phosphate
50.0 mMNa3C6H5O7sodium citrate

Details: pH 5.5
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7573
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: COMMON LINE / Software - Name: jspr (ver. 2017)
Final angle assignmentType: COMMON LINE / Software - Name: jspr (ver. 2017)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 14.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM
Details: 135660 asymmetric units were used for localized reconstruction.
Number images used: 135660
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mx4

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7n6a:
Pre-fusion state 1 of EEEV with localized reconstruction

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