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- EMDB-6146: Cryoelectron microscopy of dengue-Fab E104 complex at pH 5.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-6146
TitleCryoelectron microscopy of dengue-Fab E104 complex at pH 5.5
Map dataReconstruction of low-pH Dengue virus complexed with DENV2 Fab E104
Sample
  • Sample: Dengue virus complexed with Fab E104 at pH 5.5
  • Virus: Dengue virus 2 Thailand/16681/84
  • Protein or peptide: Fab E104
KeywordsDengue virus / DENV2 Fab E104 / Low pH / Fusion trimer
Function / homology
Function and homology information


host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / : / viral capsid / nucleoside-triphosphate phosphatase / protein complex oligomerization / double-stranded RNA binding ...host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / : / viral capsid / nucleoside-triphosphate phosphatase / protein complex oligomerization / double-stranded RNA binding / monoatomic ion channel activity / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / clathrin-dependent endocytosis of virus by host cell / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / protein dimerization activity / RNA helicase / induction by virus of host autophagy / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / structural molecule activity / virion attachment to host cell / virion membrane / ATP hydrolysis activity / proteolysis / extracellular region / ATP binding / membrane / metal ion binding
Similarity search - Function
: / Flavivirus envelope glycoprotein E, stem/anchor domain / RNA-directed RNA polymerase, thumb domain, Flavivirus / Flavivirus RNA-directed RNA polymerase, thumb domain / Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Flavivirus NS3 helicase, C-terminal helical domain / Genome polyprotein, Flavivirus / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B ...: / Flavivirus envelope glycoprotein E, stem/anchor domain / RNA-directed RNA polymerase, thumb domain, Flavivirus / Flavivirus RNA-directed RNA polymerase, thumb domain / Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Flavivirus NS3 helicase, C-terminal helical domain / Genome polyprotein, Flavivirus / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A / Flavivirus NS2B domain profile. / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS2A / Flavivirus non-structural protein NS2A / Flavivirus NS3, petidase S7 / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus NS3 protease (NS3pro) domain profile. / Envelope glycoprotein M, flavivirus / Flavivirus envelope glycoprotein M / RNA-directed RNA polymerase, flavivirus / Flavivirus RNA-directed RNA polymerase, fingers and palm domains / Flavivirus non-structural Protein NS1 / Flavivirus non-structural protein NS1 / Envelope glycoprotein M superfamily, flavivirus / Flavivirus polyprotein propeptide / Flavivirus polyprotein propeptide superfamily / Flavivirus polyprotein propeptide / Flaviviral glycoprotein E, central domain, subdomain 1 / Flaviviral glycoprotein E, central domain, subdomain 2 / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus glycoprotein E, immunoglobulin-like domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Flavivirus glycoprotein, immunoglobulin-like domain / Flavivirus glycoprotein central and dimerisation domain / Flavivirus glycoprotein, central and dimerisation domains / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / DEAD box, Flavivirus / Flavivirus DEAD domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Genome polyprotein / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesunidentified (others) / Dengue virus 2 Thailand/16681/84
Methodsingle particle reconstruction / cryo EM / Resolution: 26.0 Å
AuthorsZhang XZ / Sheng J / Austin SK / Hoornweg T / Smit JM / Kuhn RJ / Diamond MS / Rossmann MG
CitationJournal: J Virol / Year: 2015
Title: Structure of acidic pH dengue virus showing the fusogenic glycoprotein trimers.
Authors: Xinzheng Zhang / Ju Sheng / S Kyle Austin / Tabitha E Hoornweg / Jolanda M Smit / Richard J Kuhn / Michael S Diamond / Michael G Rossmann /
Abstract: Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid ...Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer.
IMPORTANCE: The structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the ...IMPORTANCE: The structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130° to the postfusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" postfusion conformation, thus inhibiting fusion.
History
DepositionOct 13, 2014-
Header (metadata) releaseNov 12, 2014-
Map releaseNov 12, 2014-
UpdateDec 24, 2014-
Current statusDec 24, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.4
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  • Surface view with fitted model
  • Atomic models: PDB-3j8d
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j8d
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6146.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of low-pH Dengue virus complexed with DENV2 Fab E104
Voxel sizeX=Y=Z: 5.2 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-2.13442278 - 2.60052752
Average (Standard dev.)-0.00569008 (±0.34362599)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 935.99994 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.25.25.2
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z936.000936.000936.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-2.1342.601-0.006

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Supplemental data

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Sample components

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Entire : Dengue virus complexed with Fab E104 at pH 5.5

EntireName: Dengue virus complexed with Fab E104 at pH 5.5
Components
  • Sample: Dengue virus complexed with Fab E104 at pH 5.5
  • Virus: Dengue virus 2 Thailand/16681/84
  • Protein or peptide: Fab E104

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Supramolecule #1000: Dengue virus complexed with Fab E104 at pH 5.5

SupramoleculeName: Dengue virus complexed with Fab E104 at pH 5.5 / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Dengue virus 2 Thailand/16681/84

SupramoleculeName: Dengue virus 2 Thailand/16681/84 / type: virus / ID: 1 / NCBI-ID: 31634 / Sci species name: Dengue virus 2 Thailand/16681/84 / Sci species strain: 16681 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: unidentified (others)
Virus shellShell ID: 1 / Name: E / Diameter: 600 Å / T number (triangulation number): 3

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Macromolecule #1: Fab E104

MacromoleculeName: Fab E104 / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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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

Concentration1.0 mg/mL
BufferpH: 5.5 / Details: 120 mM NaCl, 20 mM Tris HCl, 1 mM EDTA
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJan 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1000 / Average electron dose: 24 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN / Number images used: 528

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: EMFit
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

PDB-3j8d:
Cryoelectron microscopy of dengue-Fab E104 complex at pH 5.5

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: EMFit
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

PDB-3j8d:
Cryoelectron microscopy of dengue-Fab E104 complex at pH 5.5

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