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- EMDB-2369: The Respiratory Syncytial Virus nucleoprotein-RNA complex forms a... -

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Basic information

Entry
Database: EMDB / ID: EMD-2369
TitleThe Respiratory Syncytial Virus nucleoprotein-RNA complex forms a left-handed helical nucleocapsid.
Map dataSub-tomogram average of purified RSV nucleocapsid
Sample
  • Sample: Purified recombinant RSV nucleocapsids.
  • Virus: Respiratory syncytial virus
KeywordsRespiratory Syncytial Virus / nucleocapsid / cryo-electron tomography / sub-tomogram averaging
Function / homology
Function and homology information


Respiratory syncytial virus genome transcription / Translation of respiratory syncytial virus mRNAs / symbiont-mediated suppression of host PKR/eIFalpha signaling / protein serine/threonine kinase inhibitor activity / Respiratory syncytial virus genome replication / helical viral capsid / RSV-host interactions / Assembly and release of respiratory syncytial virus (RSV) virions / Maturation of hRSV A proteins / Respiratory syncytial virus (RSV) attachment and entry ...Respiratory syncytial virus genome transcription / Translation of respiratory syncytial virus mRNAs / symbiont-mediated suppression of host PKR/eIFalpha signaling / protein serine/threonine kinase inhibitor activity / Respiratory syncytial virus genome replication / helical viral capsid / RSV-host interactions / Assembly and release of respiratory syncytial virus (RSV) virions / Maturation of hRSV A proteins / Respiratory syncytial virus (RSV) attachment and entry / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / PKR-mediated signaling / Evasion by RSV of host interferon responses / viral capsid / symbiont-mediated suppression of host NF-kappaB cascade / viral nucleocapsid / host cell cytoplasm / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / ribonucleoprotein complex / virus-mediated perturbation of host defense response / RNA binding
Similarity search - Function
Pneumovirus nucleocapsid protein / Pneumovirus nucleocapsid protein
Similarity search - Domain/homology
Biological speciesRespiratory syncytial virus
Methodsubtomogram averaging / negative staining
AuthorsBakker SE / Duquerroy S / Galloux M / Loney C / Conner E / Eleouet JF / Rey FA / Bhella D
CitationJournal: J Gen Virol / Year: 2013
Title: The respiratory syncytial virus nucleoprotein-RNA complex forms a left-handed helical nucleocapsid.
Authors: Saskia E Bakker / Stéphane Duquerroy / Marie Galloux / Colin Loney / Edward Conner / Jean-François Eléouët / Félix A Rey / David Bhella /
Abstract: Respiratory syncytial virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical ...Respiratory syncytial virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of an NC-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N-N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N-N-protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription.
History
DepositionApr 25, 2013-
Header (metadata) releaseMay 8, 2013-
Map releaseMay 29, 2013-
UpdateJul 24, 2013-
Current statusJul 24, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.66
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.66
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bkk
  • Surface level: 0.66
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4bkk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2369.png

Downloads & links

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Map

FileDownload / File: emd_2369.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram average of purified RSV nucleocapsid
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.5 Å/pix.
x 64 pix.
= 416. Å		6.5 Å/pix.
x 64 pix.
= 416. Å		6.5 Å/pix.
x 64 pix.
= 416. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.66 / Movie #1: 0.66
Minimum - Maximum-2.29873204 - 1.89033198
Average (Standard dev.)-0.0025243 (±0.4238916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 416.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.56.56.5
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z416.000416.000416.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-2.2991.890-0.003

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Supplemental data

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Sample components

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Entire : Purified recombinant RSV nucleocapsids.

EntireName: Purified recombinant RSV nucleocapsids.
Components
  • Sample: Purified recombinant RSV nucleocapsids.
  • Virus: Respiratory syncytial virus

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Supramolecule #1000: Purified recombinant RSV nucleocapsids.

SupramoleculeName: Purified recombinant RSV nucleocapsids. / type: sample / ID: 1000 / Oligomeric state: variable helices / Number unique components: 1

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Supramolecule #1: Respiratory syncytial virus

SupramoleculeName: Respiratory syncytial virus / type: virus / ID: 1 / Name.synonym: RSV / Details: Purified recombinant nucleocapsid / NCBI-ID: 12814 / Sci species name: Respiratory syncytial virus / Virus type: OTHER / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No / Syn species name: RSV
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf21 / Recombinant plasmid: Rbac69

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Experimental details

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Structure determination

Methodnegative staining
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7.4 / Details: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA
StainingType: NEGATIVE
Details: Protein preparations were mixed with 10 nm colloidal gold and loaded onto freshly glow-discharged perforated C-flat carbon films (CF-22-2C, Protochips Inc.). Grids were then washed in 0.1% ...Details: Protein preparations were mixed with 10 nm colloidal gold and loaded onto freshly glow-discharged perforated C-flat carbon films (CF-22-2C, Protochips Inc.). Grids were then washed in 0.1% trehalose, and stained using 5% ammonium molybdate (pH 7.4) and 0.1% trehalose, drained and allowed to dry in air
GridDetails: Glow-discharged perforated C-flat carbon films (CF-22-2C, Protochips Inc.)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2200FSC
TemperatureMin: 97 K / Max: 293 K
Alignment procedureLegacy - Astigmatism: Corrected at 100k times mag in digital micrograph
Specialist opticsEnergy filter - Name: Jeol / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 30.0 eV
DetailsRoom temperature stained grids imaged at LN2 temperatures.
DateJul 15, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 9 / Average electron dose: 131 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °

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Image processing

DetailsSubtomograms were manually picked. A starting model was obtained by manually aligning 100 subtomograms, all subtomograms were aligned to this starting model. To mitigate missing wedge artefacts, the orientations around the helical axis were randomised before further alignment.
Final reconstructionAlgorithm: OTHER / Software - Name: Etomo, Dynamo / Details: Subtomograms were picked from nine tomograms. / Number subtomograms used: 911
Final 3D classificationNumber classes: 1

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Atomic model buiding 1

Initial modelPDB ID:

2wj8

RefinementSpace: REAL
Output model

PDB-4bkk:
The Respiratory Syncytial Virus nucleoprotein-RNA complex forms a left-handed helical nucleocapsid.

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