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- EMDB-0333: Helical RNA-bound Hantaan virus nucleocapsid -

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Basic information

Entry
Database: EMDB / ID: 0333
TitleHelical RNA-bound Hantaan virus nucleocapsid
Map dataHelical RNA-bound Hantaan virus nucleocapsid structure
SampleRNA-bound Hantaan virus nucleocapsid:
Hantaan virus nucleoprotein / RNA / nucleic-acidNucleic acid / Nucleoprotein
Function / homologyHantavirus nucleocapsid protein / Hantavirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / RNA binding / Nucleoprotein
Function and homology information
SourceHantaan orthohantavirus / Spodoptera frugiperda (fall armyworm) / Fall armyworm (fall armyworm)
Methodhelical reconstruction / cryo EM / 3.3 Å resolution
AuthorsArragain B / Reguera J / Desfosses A / Gutsche I / Schoehn G / Malet H
CitationJournal: Elife / Year: 2019
Title: High resolution cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly mechanisms.
Authors: Benoît Arragain / Juan Reguera / Ambroise Desfosses / Irina Gutsche / Guy Schoehn / Hélène Malet
Abstract: Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids ...Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids usually prevents their full-length structural characterisation at high resolution. Here, we describe purification of full-length recombinant metastable helical nucleocapsid of Hantaan virus ( family, order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerisation via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the nucleocapsid.
Validation ReportPDB-ID: 6i2n

SummaryFull reportAbout validation report
DateDeposition: Nov 1, 2018 / Header (metadata) release: Dec 19, 2018 / Map release: Jan 23, 2019 / Last update: Jan 23, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6i2n
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0333.png

Downloads & links

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Map

Fileemd_0333.map.gz (map file in CCP4 format, 256001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
400 pix
1.07 Å/pix.
= 426.8 Å		400 pix
1.07 Å/pix.
= 426.8 Å		400 pix
1.07 Å/pix.
= 426.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour Level:0.025 (by author), 0.025 (movie #1):
Minimum - Maximum-0.06770787 - 0.12958607
Average (Standard dev.)0.000070503615 (0.0019500765)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions400400400
Origin0.00.00.0
Limit399.0399.0399.0
Spacing400400400
CellA=B=C: 426.80002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0671.0671.067
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z426.800426.800426.800
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0680.1300.000

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Supplemental data

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Sample components

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Entire RNA-bound Hantaan virus nucleocapsid

EntireName: RNA-bound Hantaan virus nucleocapsid / Number of components: 5

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Component #1: protein, RNA-bound Hantaan virus nucleocapsid

ProteinName: RNA-bound Hantaan virus nucleocapsid / Recombinant expression: No
MassTheoretical: 48.2 kDa

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Component #2: protein, Hantaan virus nucleoprotein

ProteinName: Hantaan virus nucleoprotein / Recombinant expression: No
SourceSpecies: Hantaan orthohantavirus
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: pFastBac / Strain: SF21

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Component #3: protein, RNA

ProteinName: RNA / Recombinant expression: No
SourceSpecies: Spodoptera frugiperda (fall armyworm)

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Component #4: nucleic-acid, RNA (5'-R(P*UP*UP*U)-3')

Nucleic-acidName: RNA (5'-R(P*UP*UP*U)-3') / Class: RNA
Details: A poly-U has been built but the RNA corresponds to heterogeneous insect cell RNA that bind to nucleoproteins during expression.
Structure: OTHER / Synthetic: No
Sequence:
UUU
MassTheoretical: 0.87354 kDa
SourceSpecies: Fall armyworm (fall armyworm)

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Component #5: protein, Nucleoprotein

ProteinName: Nucleoprotein / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 48.262266 kDa
SourceSpecies: Hantaan orthohantavirus
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 18.87 Å / Delta phi: -99.95 deg.
Sample solutionSpecimen conc.: 0.6 mg/ml / pH: 8
Support film30 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 %
Details: 3 ul of sample were applied on the resulting glow-discharged grids and excess solution was blotted during 2.5 sec force 7.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 46860.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4328 / Sampling size: 5 microns

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: RELION / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF
Euler angles: Relion was used for helical symmetry search/refinement on the best segments (removing the ones with more than 20 degree angular tilt) with local CTF determination and CTF-refinement. Frames 3-18 were used.
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Details: Fiber model was iteratively rebuilt and all-atom refined using stereochemical and NCS restraints
Input PDB model: 5FSG

5fsg


Overall bvalue: 103
Output model

6i2n

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