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- EMDB-5115: West Nile virus in complex with a single-chain antibody derivativ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5115
TitleWest Nile virus in complex with a single-chain antibody derivative of the neutralizing monoclonal antibody E16
Map dataWest Nile virus in complex with a single-chain antibody derivative of the neutralizing monoclonal antibody E16.
Sample
  • Sample: West Nile virus NY99 complexed with single-chain antibody derivative of neutralizing monoclonal antibody E16
  • Virus: West Nile Virus NY99
  • Protein or peptide: E16 scFv derivative
Keywordsflavivirus / West Nile Virus / neutralizing antibody / complex / single-chain
Biological speciesunidentified (others) / West Nile Virus NY99
Methodsingle particle reconstruction / cryo EM / Resolution: 22.75 Å
AuthorsKaufmann B / Chipman PR / Holdaway HA / Johnson S / Kuhn RJ / Diamond MS / Rossmann MG
CitationJournal: PLoS Pathog / Year: 2009
Title: Capturing a flavivirus pre-fusion intermediate.
Authors: Bärbel Kaufmann / Paul R Chipman / Heather A Holdaway / Syd Johnson / Daved H Fremont / Richard J Kuhn / Michael S Diamond / Michael G Rossmann /
Abstract: During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the ...During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting approximately 60 A-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.
History
DepositionApr 13, 2009-
Header (metadata) releaseApr 20, 2009-
Map releaseMar 30, 2010-
UpdateSep 23, 2011-
Current statusSep 23, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5115_1.tif

Downloads & links

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Map

FileDownload / File: emd_5115.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWest Nile virus in complex with a single-chain antibody derivative of the neutralizing monoclonal antibody E16.
Voxel sizeX=Y=Z: 2.967 Å
Density
Contour Level1: 1.5 / Movie #1: 1.5
Minimum - Maximum-1.14105 - 4.20053
Average (Standard dev.)0.00000000567642 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-140-140-140
Dimensions280280280
Spacing280280280
CellA=B=C: 830.76 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.9672.9672.967
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z830.760830.760830.760
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-140-140-140
NC/NR/NS280280280
D min/max/mean-1.1414.2010.000

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Supplemental data

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Sample components

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Entire : West Nile virus NY99 complexed with single-chain antibody derivat...

EntireName: West Nile virus NY99 complexed with single-chain antibody derivative of neutralizing monoclonal antibody E16
Components
  • Sample: West Nile virus NY99 complexed with single-chain antibody derivative of neutralizing monoclonal antibody E16
  • Virus: West Nile Virus NY99
  • Protein or peptide: E16 scFv derivative

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Supramolecule #1000: West Nile virus NY99 complexed with single-chain antibody derivat...

SupramoleculeName: West Nile virus NY99 complexed with single-chain antibody derivative of neutralizing monoclonal antibody E16
type: sample / ID: 1000
Oligomeric state: T1 icosahedron with three E monomers and two scFv molecules per asymmetric unit
Number unique components: 2
Molecular weightTheoretical: 20 MDa

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Supramolecule #1: West Nile Virus NY99

SupramoleculeName: West Nile Virus NY99 / type: virus / ID: 1 / Name.synonym: West Nile Virus
Details: the virus is complexed with a single-chain antibody derivative of the neutralizing antibody E16 (120 scFv molecules per virion)
Sci species name: West Nile Virus NY99 / 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

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Macromolecule #1: E16 scFv derivative

MacromoleculeName: E16 scFv derivative / type: protein_or_peptide / ID: 1
Name.synonym: single-chain antibody derivative of neutralizing antibody E16
Details: single-chain antibody derivative of the neutralizing antibody E16 complexed with West Nile virus (120 scFv molecules per virion)
Number of copies: 120 / Oligomeric state: Monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 25 KDa

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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: 12 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Guillotine-style plunge freezeing 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: 47190 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.74 µm / Nominal defocus min: 1.35 µm / Nominal magnification: 45000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle min: -0
TemperatureAverage: 98 K
Alignment procedureLegacy - Astigmatism: live FFT at 200K mag
Detailslow dose imaging
DateJul 24, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 53 / Average electron dose: 17.22 e/Å2 / Details: scanned images binned 2x2 / Od range: 1 / Bits/pixel: 8
Tilt angle max0

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Image processing

CTF correctionDetails: each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 22.75 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PFTSEARCH, PO2R, P3DR / Number images used: 783
DetailsThe particles were selected interactively at the computer terminal.

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