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- EMDB-2967: Cryo-EM structure of Dengue virus serotype 2 in complex with anti... -

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Entry
Database: EMDB / ID: 2967
TitleCryo-EM structure of Dengue virus serotype 2 in complex with antigen-binding fragments of human antibody 2D22
Map dataReconstruction of Dengue virus 2 strain PVP94/07 (a clinical isolate) in complex with human antibody 2D22 Fab
SampleDengue virus serotype 2 strain PVP94/07 (a clinical isolate) complexed with Fab fragments of human antibody 2D22.
  • virus
  • Antigen-binding fragment of human antibody 2D22
Keywordsdengue virus / human antibody / cryo-EM / neutralization
Function / homologyFlaviviral glycoprotein E, central domain, subdomain 2 / Flavivirus glycoprotein E, immunoglobulin-like domain / RNA-directed RNA polymerase, catalytic domain / Immunoglobulin-like domain / Peptidase S1, PA clan / DEAD box, Flavivirus / Flavivirus glycoprotein central and dimerisation domain / Immunoglobulin V-set domain / Flaviviral glycoprotein E, central domain, subdomain 1 / Flavivirus non-structural protein NS4A ...Flaviviral glycoprotein E, central domain, subdomain 2 / Flavivirus glycoprotein E, immunoglobulin-like domain / RNA-directed RNA polymerase, catalytic domain / Immunoglobulin-like domain / Peptidase S1, PA clan / DEAD box, Flavivirus / Flavivirus glycoprotein central and dimerisation domain / Immunoglobulin V-set domain / Flaviviral glycoprotein E, central domain, subdomain 1 / Flavivirus non-structural protein NS4A / Immunoglobulin-like fold / Helicase superfamily 1/2, ATP-binding domain / Genome polyprotein, Flavivirus / Immunoglobulin E-set / Flavivirus envelope glycoprotein E, Stem/Anchor domain / mRNA cap 0/1 methyltransferase / P-loop containing nucleoside triphosphate hydrolase / Immunoglobulin C1-set / Flavivirus glycoprotein, central and dimerisation domains / Flavivirus envelope glycoprotein M / Flavivirus capsid protein C / Flavivirus non-structural protein NS2B / Flavivirus RNA-directed RNA polymerase / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus non-structural Protein NS1 / Flavivirus polyprotein propeptide superfamily / S-adenosyl-L-methionine-dependent methyltransferase / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Envelope glycoprotein M superfamily, flavivirus / Flaviviral glycoprotein E, dimerisation domain / Flavivirus capsid protein C superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Immunoglobulin-like domain superfamily / Immunoglobulin subtype / Immunoglobulin/major histocompatibility complex, conserved site / Flavivirus non-structural protein NS4B / Flavivirus glycoprotein, immunoglobulin-like domain / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus NS3 protease (NS3pro) domain profile. / Flavivirus NS2B domain profile. / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / Ig-like domain profile. / RdRp of positive ssRNA viruses catalytic domain profile. / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin V-set domain / Immunoglobulin C1-set domain / Flavivirus DEAD domain / Ribosomal RNA methyltransferase FtsJ domain / Flavivirus non-structural protein NS2A / FtsJ-like methyltransferase / Flavivirus non-structural protein NS2A / Flavivirus polyprotein propeptide / Flavivirus NS3, petidase S7 / Helicase, C-terminal / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS1 / Flavivirus polyprotein propeptide / Flavivirus capsid protein C / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS4A / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / RNA-directed RNA polymerase, flavivirus / Envelope glycoprotein M, flavivirus / suppression by virus of host innate immune response / host cell endoplasmic reticulum / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA (guanine-N7-)-methyltransferase activity / ATP-dependent helicase activity / host cell membrane / RNA helicase activity / double-stranded RNA binding / viral capsid / induction by virus of host autophagy / viral RNA genome replication / fusion of virus membrane with host endosome membrane / RNA-directed 5'-3' RNA polymerase activity / viral envelope / protein dimerization activity / virion attachment to host cell / virion membrane / structural molecule activity / serine-type endopeptidase activity / integral component of membrane / ATP binding / metal ion binding / Envelope protein / Genome polyprotein / Genome polyprotein / IgG L chain
Function and homology information
SourceDengue Virus serotype 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 6.5 Å resolution
AuthorsFibriansah G / Ibarra KD / Ng TS / Smith SA / Tan JL / Lim XN / Ooi JSG / Kostyuchenko VA / Wang J / de Silva AM / Harris E / Crowe JE / Lok SM
CitationJournal: Science / Year: 2015
Title: DENGUE VIRUS. Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers.
Authors: Guntur Fibriansah / Kristie D Ibarra / Thiam-Seng Ng / Scott A Smith / Joanne L Tan / Xin-Ni Lim / Justin S G Ooi / Victor A Kostyuchenko / Jiaqi Wang / Aravinda M de Silva / Eva Harris / James E Crowe / Shee-Mei Lok
Abstract: There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. ...There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)-specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo-electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 "locks" two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.
Validation ReportPDB-ID: 4uif

SummaryFull reportAbout validation report
DateDeposition: Mar 29, 2015 / Header (metadata) release: Apr 15, 2015 / Map release: Jul 15, 2015 / Last update: Jul 15, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-4uif
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4uif
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2967.map.gz (map file in CCP4 format, 524289 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
512 pix
1.69 Å/pix.
= 864.256 Å
512 pix
1.69 Å/pix.
= 864.256 Å
512 pix
1.69 Å/pix.
= 864.256 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.688 Å
Density
Contour Level:2 (by author), 3 (movie #1):
Minimum - Maximum-10.67656708 - 14.26177883
Average (Standard dev.)-0.05019815 (0.96412933)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions512512512
Origin-256-256-256
Limit255255255
Spacing512512512
CellA=B=C: 864.256 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.6881.6881.688
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z864.256864.256864.256
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-10.67714.262-0.050

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Supplemental data

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Sample components

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Entire Dengue virus serotype 2 strain PVP94/07 (a clinical isolate) comp...

EntireName: Dengue virus serotype 2 strain PVP94/07 (a clinical isolate) complexed with Fab fragments of human antibody 2D22.
Number of components: 2

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Component #1: virus, Dengue Virus serotype 2

VirusName: Dengue Virus serotype 2 / Class: VIRION / Enveloped: Yes / Empty: No / Isolate: STRAIN
SpeciesSpecies: Dengue Virus serotype 2 / Strain: PVP94/07
Source (engineered)Expression System: Aedes albopictus (Asian tiger mosquito) / Cell of expression system: C6/36
Source (natural)Host Species: Homo sapiens (human) / Host category: VERTEBRATES

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Component #2: protein, Antigen-binding fragment of human antibody 2D22

ProteinName: Antigen-binding fragment of human antibody 2D22
Details: 180 copies of Fab 2D22 molecule bind to virus surface
Recombinant expression: No / Number of Copies: 180
MassTheoretical: 50 kDa
SourceSpecies: Homo sapiens (human)
Source (natural)Cell: memory B-cell

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 10 mM Tris-HCl pH 8.0, 120 mM NaCl and 1 mM EDTA
pH: 8
Support filmultra-thin carbon-coated lacey carbon grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Method: blotted with filter paper for 2 seconds prior to snap freezing

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Feb 5, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 47000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1.5 - 4.7 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 94

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2485 / Details: The particles were manually selected
3D reconstructionAlgorithm: Cross-common lines / Software: EMAN, EMAN2, MPSA / CTF correction: Each particle / Resolution: 6.5 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Chimera, NAMD/MDFF / Refinement protocol: flexible / Target criteria: real-space correlation / Refinement space: REAL
Details: Initially fitted in Chimera and refined using NAMD/MDFF
Input PDB model: 3J27
Chain ID: A, B, C, D, E, F
Output model

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