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- PDB-3ixx: The pseudo-atomic structure of West Nile immature virus in comple... -

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Basic information

Entry
Database: PDB / ID: 3ixx
TitleThe pseudo-atomic structure of West Nile immature virus in complex with Fab fragments of the anti-fusion loop antibody E53
Components
  • E53 Fab Fragment (chain H)
  • E53 Fab Fragment (chain L)
  • Envelope protein E
  • Peptide pr
KeywordsVIRUS / West Nile Virus / WNV / immature / fusion loop / Fab / E53 / ATP-binding / Envelope protein / Helicase / Hydrolase / Membrane / Nucleotide-binding / RNA replication / Transmembrane / Virion / Icosahedral virus
Function / homology
Function and homology information


suppression by virus of host complement activation / methyltransferase cap1 / mRNA (guanine-N7)-methyltransferase / suppression by virus of host STAT2 activity / flavivirin / positive stranded viral RNA replication / suppression by virus of host innate immune response / mRNA (nucleoside-2'-O-)-methyltransferase activity / suppression by virus of host STAT1 activity / mRNA (guanine-N7-)-methyltransferase activity ...suppression by virus of host complement activation / methyltransferase cap1 / mRNA (guanine-N7)-methyltransferase / suppression by virus of host STAT2 activity / flavivirin / positive stranded viral RNA replication / suppression by virus of host innate immune response / mRNA (nucleoside-2'-O-)-methyltransferase activity / suppression by virus of host STAT1 activity / mRNA (guanine-N7-)-methyltransferase activity / host cell endoplasmic reticulum membrane / double-stranded RNA binding / host cell perinuclear region of cytoplasm / viral capsid / suppression by virus of host type I interferon-mediated signaling pathway / nucleoside-triphosphate phosphatase / RNA helicase / induction by virus of host autophagy / viral RNA genome replication / RNA-directed RNA polymerase / protein dimerization activity / RNA helicase activity / RNA-directed 5'-3' RNA polymerase activity / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / serine-type endopeptidase activity / virion membrane / endoplasmic reticulum membrane / structural molecule activity / host cell nucleus / integral component of membrane / extracellular region / ATP binding / metal ion binding / nucleus
Flavivirus glycoprotein, immunoglobulin-like domain / Helicase superfamily 1/2, ATP-binding domain / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS2A / Flavivirus capsid protein C / Flavivirus non-structural protein NS1 / Flavivirus non-structural protein NS4B / Helicase, C-terminal / Flavivirus NS3, petidase S7 ...Flavivirus glycoprotein, immunoglobulin-like domain / Helicase superfamily 1/2, ATP-binding domain / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS2A / Flavivirus capsid protein C / Flavivirus non-structural protein NS1 / Flavivirus non-structural protein NS4B / Helicase, C-terminal / Flavivirus NS3, petidase S7 / Flavivirus polyprotein propeptide / Ribosomal RNA methyltransferase FtsJ domain / RNA-directed RNA polymerase, catalytic domain / Peptidase S1, PA clan / DEAD box, Flavivirus / Flavivirus glycoprotein central and dimerisation domain / Genome polyprotein, Flavivirus / Envelope glycoprotein M, flavivirus / Flaviviral glycoprotein E, dimerisation domain / Flavivirus glycoprotein, central and dimerisation domains / Peptidase S1, PA clan, chymotrypsin-like fold / DNA/RNA polymerase superfamily / Flavivirus polyprotein propeptide superfamily / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Envelope glycoprotein M superfamily, flavivirus / Flavivirus capsid protein C superfamily / Immunoglobulin E-set / Flavivirus glycoprotein, central and dimerisation domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase / P-loop containing nucleoside triphosphate hydrolase / Flavivirus glycoprotein E, immunoglobulin-like domain / mRNA cap 0/1 methyltransferase / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / RNA-directed RNA polymerase, flavivirus
Core protein / Core protein / Genome polyprotein / Core protein
Biological speciesWest Nile virus
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 15 Å
AuthorsCherrier, M.V. / Kaufmann, B. / Nybakken, G.E. / Lok, S.M. / Warren, J.T. / Nelson, C.A. / Kostyuchenko, V.A. / Holdaway, H.A. / Chipman, P.R. / Kuhn, R.J. ...Cherrier, M.V. / Kaufmann, B. / Nybakken, G.E. / Lok, S.M. / Warren, J.T. / Nelson, C.A. / Kostyuchenko, V.A. / Holdaway, H.A. / Chipman, P.R. / Kuhn, R.J. / Diamond, M.S. / Rossmann, M.G. / Fremont, D.H.
CitationJournal: EMBO J / Year: 2009
Title: Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody.
Authors: Mickaël V Cherrier / Bärbel Kaufmann / Grant E Nybakken / Shee-Mei Lok / Julia T Warren / Beverly R Chen / Christopher A Nelson / Victor A Kostyuchenko / Heather A Holdaway / Paul R ...Authors: Mickaël V Cherrier / Bärbel Kaufmann / Grant E Nybakken / Shee-Mei Lok / Julia T Warren / Beverly R Chen / Christopher A Nelson / Victor A Kostyuchenko / Heather A Holdaway / Paul R Chipman / Richard J Kuhn / Michael S Diamond / Michael G Rossmann / Daved H Fremont /
Abstract: Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have ...Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have defined the structure of the flavivirus cross-reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo-electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion-loop-specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibody-dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 26, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 27, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Envelope protein E
B: Envelope protein E
C: Envelope protein E
D: Peptide pr
E: Peptide pr
F: Peptide pr
G: E53 Fab Fragment (chain H)
H: E53 Fab Fragment (chain L)
I: E53 Fab Fragment (chain H)
J: E53 Fab Fragment (chain L)


Theoretical massNumber of molelcules
Total (without water)250,48010
Polymers250,48010
Non-polymers00
Water0
1
A: Envelope protein E
B: Envelope protein E
C: Envelope protein E
D: Peptide pr
E: Peptide pr
F: Peptide pr
G: E53 Fab Fragment (chain H)
H: E53 Fab Fragment (chain L)
I: E53 Fab Fragment (chain H)
J: E53 Fab Fragment (chain L)
x 60


Theoretical massNumber of molelcules
Total (without water)15,028,772600
Polymers15,028,772600
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Envelope protein E
B: Envelope protein E
C: Envelope protein E
D: Peptide pr
E: Peptide pr
F: Peptide pr
G: E53 Fab Fragment (chain H)
H: E53 Fab Fragment (chain L)
I: E53 Fab Fragment (chain H)
J: E53 Fab Fragment (chain L)
x 5


  • icosahedral pentamer
  • 1.25 MDa, 50 polymers
Theoretical massNumber of molelcules
Total (without water)1,252,39850
Polymers1,252,39850
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Envelope protein E
B: Envelope protein E
C: Envelope protein E
D: Peptide pr
E: Peptide pr
F: Peptide pr
G: E53 Fab Fragment (chain H)
H: E53 Fab Fragment (chain L)
I: E53 Fab Fragment (chain H)
J: E53 Fab Fragment (chain L)
x 6


  • icosahedral 23 hexamer
  • 1.5 MDa, 60 polymers
Theoretical massNumber of molelcules
Total (without water)1,502,87760
Polymers1,502,87760
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Envelope protein E / / Coordinate model: Cα atoms only


Mass: 43272.098 Da / Num. of mol.: 3 / Fragment: West Nile Virus envelope protein
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) West Nile virus / Strain: NY99 / References: UniProt: Q9Q6P4, UniProt: A7TZT4*PLUS
#2: Protein Peptide pr / Coordinate model: Cα atoms only


Mass: 8742.004 Da / Num. of mol.: 3 / Fragment: West Nile Virus pr peptide
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) West Nile virus / Strain: NY99 / References: UniProt: Q9WHD2, UniProt: Q3I100*PLUS
#3: Antibody E53 Fab Fragment (chain H) / Coordinate model: Cα atoms only


Mass: 23630.504 Da / Num. of mol.: 2 / Fragment: E53 Fab Fragment
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli) / Strain (production host): DE3
#4: Antibody E53 Fab Fragment (chain L) / Coordinate model: Cα atoms only


Mass: 23588.107 Da / Num. of mol.: 2 / Fragment: E53 Fab Fragment
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli) / Strain (production host): DE3

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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: Immature West Nile Virus complexed with E53 Fab / Type: VIRUS
Details: T1 icosahedron with three E monomers and two Fab per asymmetric unit
Molecular weightValue: 24.5 MDa / Experimental value: NO
Details of virusEmpty: NO / Enveloped: YES / Host category: VERTEBRATES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 8 / Details: 12 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA
SpecimenDetails: 12 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding a few microliters of the sample is held in place at the bottom of a plunger by the means of fine ...Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding a few microliters of the sample is held in place at the bottom of a plunger by the means of fine tweezers. Once the ethane in the vial is completely frozen, it needs to be slightly melted. When the liquid ethane is ready, a piece of filter paper is then pressed against the sample to blot of excess buffer, sufficient to leave a thin layer on the grid. After a predetermined time, the filter paper is removed, and the plunger is allowed to drop into the liquid ethane. Once the grid enters the liquid ethane, the sample is rapidly frozen, and the grid is transferred under liquid nitrogen to a storage box immersed liquid nitrogen for later use in the microscope.

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM300FEG/T / Date: Jan 8, 2008 / Details: low dose
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM / Electron beam tilt params: 0
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Calibrated magnification: 47244 X / Nominal defocus max: 2859 nm / Nominal defocus min: 1193 nm / Cs: 2 mm / Astigmatism: live FFT / Camera length: 0 mm
Specimen holderModel: GATAN LIQUID NITROGEN / Specimen holder type: EUCENTRIC / Temperature: 98 K / Tilt angle max: -9999 ° / Tilt angle min: -9999 °
Image recordingElectron dose: 12 e/Å2 / Film or detector model: KODAK SO-163 FILM
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1EMfitmodel fitting
2SPIDER3D reconstruction
3Xmipp3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 15 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 3927 / Actual pixel size: 2.69 Å / Magnification calibration: 47244
Details: Final maps were calculated from two averaged datasets ( Details about the particle: 400 mesh copper grid )
Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body DETAILS--Each E molecule was divided into two rigid bodies, DI-DIII and DII-pr
2RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body DETAILS--a model for WNV pr was generated by SWISS-MODEL based on DENV pr structure
Atomic model building
IDPDB-ID3D fitting-ID
12OF61
23C5X2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms2308 0 0 0 2308

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