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- EMDB-5190: West Nile Virus in complex with Fab fragments of the neutralizing... -

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Basic information

Entry
Database: EMDB / ID: EMD-5190
TitleWest Nile Virus in complex with Fab fragments of the neutralizing monoclonal antibody CR4354
Map dataCryoEM map of West Nile virus in complex with Fab fragments of the neutralizing antibody CR4354
Sample
  • Sample: West Nile virus NY99 complexed with Fab fragments of the neutralizing monoclonal antibody CR4354
  • Virus: West Nile virus HNY99
  • Protein or peptide: CR4354 Fab fragment
KeywordsWest Nile Virus / infectious virus / flavivirus / neutralizing antibody / Fab fragment / CR4354 / complex / envelope protein
Function / homology
Function and homology information


flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral capsid / nucleoside-triphosphate phosphatase / double-stranded RNA binding / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity ...flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral capsid / nucleoside-triphosphate phosphatase / double-stranded RNA binding / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / 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 / symbiont entry into host cell / 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 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 / DNA/RNA polymerase superfamily / Peptidase S1, PA clan / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Envelope glycoprotein / Genome polyprotein
Similarity search - Component
Biological speciesHomo sapiens (human) / West Nile virus HNY99
Methodsingle particle reconstruction / cryo EM / Resolution: 13.7 Å
AuthorsKaufmann B / Vogt MR / Holdaway HA / Goudsmit J / Chipman PR / Kuhn RJ / Diamond MS / Rossmann MG
CitationJournal: Proc Natl Acad Sci U S A / Year: 2010
Title: Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354.
Authors: Bärbel Kaufmann / Matthew R Vogt / Jaap Goudsmit / Heather A Holdaway / Anastasia A Aksyuk / Paul R Chipman / Richard J Kuhn / Michael S Diamond / Michael G Rossmann /
Abstract: Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a ...Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.
History
DepositionApr 21, 2010-
Header (metadata) releaseNov 18, 2010-
Map releaseNov 18, 2010-
UpdateNov 18, 2010-
Current statusNov 18, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iyw
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3iyw
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5190_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5190.map.gz / Format: CCP4 / Size: 99.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of West Nile virus in complex with Fab fragments of the neutralizing antibody CR4354
Voxel sizeX=Y=Z: 2.7237 Å
Density
Contour LevelBy AUTHOR: 1.8 / Movie #1: 1.8
Minimum - Maximum-1.43001 - 4.59224
Average (Standard dev.)-0.0000000163666 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-149-149-149
Dimensions299299299
Spacing299299299
CellA=B=C: 814.386 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72369899665552.72369899665552.7236989966555
M x/y/z299299299
origin x/y/z0.0000.0000.000
length x/y/z814.386814.386814.386
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-149-149-149
NC/NR/NS299299299
D min/max/mean-1.4304.592-0.000

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Supplemental data

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Sample components

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Entire : West Nile virus NY99 complexed with Fab fragments of the neutrali...

EntireName: West Nile virus NY99 complexed with Fab fragments of the neutralizing monoclonal antibody CR4354
Components
  • Sample: West Nile virus NY99 complexed with Fab fragments of the neutralizing monoclonal antibody CR4354
  • Virus: West Nile virus HNY99
  • Protein or peptide: CR4354 Fab fragment

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Supramolecule #1000: West Nile virus NY99 complexed with Fab fragments of the neutrali...

SupramoleculeName: West Nile virus NY99 complexed with Fab fragments of the neutralizing monoclonal antibody CR4354
type: sample / ID: 1000
Details: complex was formed by incubating infectious virus with Fab (ratio of about 4 Fab fragments per E molecule) at 37degC for 30min, followed by a 2h incubation at RT
Oligomeric state: T1 icosahedron with three E monomers and two Fab per asymmetric unit
Number unique components: 2
Molecular weightTheoretical: 23 MDa

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Supramolecule #1: West Nile virus HNY99

SupramoleculeName: West Nile virus HNY99 / type: virus / ID: 1 / Name.synonym: West Nile virus
Details: The infectious virus (180 molecules of envelope glycoprotein E form outer protein shell of virion) is complexed with Fab fragments of the neutralizing antibody CR4354 (120 Fab molecules per virion)
Sci species name: West Nile virus HNY99 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No / Syn species name: West Nile virus
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 17 MDa
Virus shellShell ID: 1 / Name: E protein shell / Diameter: 495 Å / T number (triangulation number): 1

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Macromolecule #1: CR4354 Fab fragment

MacromoleculeName: CR4354 Fab fragment / type: protein_or_peptide / ID: 1 / Name.synonym: CR4354 Fab fragment
Details: Fab fragments of the neutralizing monoclonal antibody CR4354 are complexed with West Nile Virus (120 Fab molecules per virion)
Number of copies: 120 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: peripheral blood / Cell: B cells
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: mammalian cells (unknown)

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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 mg/mL
BufferpH: 8 / Details: 12mM Tris-HCl, 120mM NaCl, 1mM EDTA
GridDetails: holey carbon 400 mesh copper grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: guillotine-style plunge freezing device
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

MicroscopeFEI/PHILIPS CM300FEG/T
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47244 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.53 µm / Nominal defocus min: 1.45 µm / Nominal magnification: 45000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 98 K
Alignment procedureLegacy - Astigmatism: live FFT at 200K magnification
Detailslow dose imaging
DateSep 9, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 69 / Average electron dose: 22 e/Å2 / Details: scanned images binned 2x2 / Od range: 1 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: AUTO3DEM
Details: final map includes data to 13.0 Ang resolution (FCS at about 0.2 cut-off)
Number images used: 5006
DetailsThe particles were selected interactively at the computer terminal.

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