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- EMDB-2073: Structure of the dengue virus glycoprotein non-structural protein... -

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Basic information

Entry
Database: EMDB / ID: EMD-2073
TitleStructure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis
Map data3D reconstruction of recombinant DENV-2 sNS1 protein
Sample
  • Sample: Secreted form of DENV-2 NS1
  • Protein or peptide: Non structural protein 1
KeywordsFlavivirus / Dengue / NS1 / glycoprotein
Function / homologyFlavivirus non-structural protein NS1
Function and homology information
Biological speciesDengue virus 2
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsMuller DA / Landsberg MJ / Bletchly C / Rothnagel R / Waddington L / Hankamer B / Young PR
CitationJournal: J Gen Virol / Year: 2012
Title: Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis.
Authors: David A Muller / Michael J Landsberg / Cheryl Bletchly / Rosalba Rothnagel / Lynne Waddington / Ben Hankamer / Paul R Young /
Abstract: The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted ...The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.
History
DepositionApr 13, 2012-
Header (metadata) releaseApr 17, 2012-
Map releaseApr 17, 2012-
UpdateApr 17, 2012-
Current statusApr 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2073.jpg

Downloads & links

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Map

FileDownload / File: emd_2073.map.gz / Format: CCP4 / Size: 4.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of recombinant DENV-2 sNS1 protein
Voxel sizeX=Y=Z: 1.86 Å
Density
Contour LevelBy AUTHOR: 1.04 / Movie #1: 1.04
Minimum - Maximum-3.1992631 - 6.28529167
Average (Standard dev.)0.02490416 (±0.49955314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-135-135-135
Dimensions108108108
Spacing108108108
CellA=B=C: 200.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.861.861.86
M x/y/z108108108
origin x/y/z0.0000.0000.000
length x/y/z200.880200.880200.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS-135-135-135
NC/NR/NS108108108
D min/max/mean-3.1996.2850.025

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Supplemental data

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Sample components

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Entire : Secreted form of DENV-2 NS1

EntireName: Secreted form of DENV-2 NS1
Components
  • Sample: Secreted form of DENV-2 NS1
  • Protein or peptide: Non structural protein 1

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Supramolecule #1000: Secreted form of DENV-2 NS1

SupramoleculeName: Secreted form of DENV-2 NS1 / type: sample / ID: 1000 / Details: Monodisperse sample as assessed by SEC / Oligomeric state: Homohexamer / Number unique components: 1
Molecular weightTheoretical: 246 KDa

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Macromolecule #1: Non structural protein 1

MacromoleculeName: Non structural protein 1 / type: protein_or_peptide / ID: 1 / Name.synonym: NS1 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Dengue virus 2 / synonym: Dengue virus 2 / Location in cell: Secreted
Molecular weightTheoretical: 41 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths) / Recombinant plasmid: pFastBac
SequenceInterPro: Flavivirus non-structural protein NS1

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8 / Details: 20 mM Tris, 300 mM NaCl
StainingType: NEGATIVE
Details: Grids with adsorbed protein washed with an aqueous solution of 2% (w/v) uranyl acetate for 30 seconds.
GridDetails: 400 mesh copper grid with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 68000
Sample stageSpecimen holder: FEI single tilt room temperature / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 295 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 93,000 times magnification
DateSep 17, 2003
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: 40 / Average electron dose: 60 e/Å2 / Bits/pixel: 8

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Image processing

Final two d classificationNumber classes: 174
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, EMAN, XMIPP
Details: An exhaustive evaluation of point symmetry was performed as described in the associated manuscript
Number images used: 3523
DetailsParticles selected semi-automatically using the SWARM-PS software

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