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

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Basic information

Entry
Database: EMDB / ID: EMD-0333
TitleHelical RNA-bound Hantaan virus nucleocapsid
Map dataHelical RNA-bound Hantaan virus nucleocapsid structure
Sample
  • Complex: RNA-bound Hantaan virus nucleocapsid
    • Complex: Hantaan virus nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA
      • RNA: RNA (5'-R(P*UP*UP*U)-3')
Function / homology
Function and homology information


: / helical viral capsid / : / host cell Golgi apparatus / viral nucleocapsid / endonuclease activity / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / ribonucleoprotein complex / RNA binding
Similarity search - Function
Hantavirus nucleocapsid protein / Hantavirus nucleocapsid protein
Similarity search - Domain/homology
Biological speciesHantaan orthohantavirus / Spodoptera frugiperda (fall armyworm) / Fall armyworm (fall armyworm)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
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.
History
DepositionNov 1, 2018-
Header (metadata) releaseDec 19, 2018-
Map releaseJan 23, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • 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

FileDownload / File: emd_0333.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical RNA-bound Hantaan virus nucleocapsid structure
Voxel sizeX=Y=Z: 1.067 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.06770787 - 0.12958607
Average (Standard dev.)0.00007050361 (±0.0019500765)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 426.80002 Å
α=β=γ: 90.0 °

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
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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Half map: Half map 2

Fileemd_0333_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_0333_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RNA-bound Hantaan virus nucleocapsid

EntireName: RNA-bound Hantaan virus nucleocapsid
Components
  • Complex: RNA-bound Hantaan virus nucleocapsid
    • Complex: Hantaan virus nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA
      • RNA: RNA (5'-R(P*UP*UP*U)-3')

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Supramolecule #1: RNA-bound Hantaan virus nucleocapsid

SupramoleculeName: RNA-bound Hantaan virus nucleocapsid / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 48.2 KDa

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Supramolecule #2: Hantaan virus nucleoprotein

SupramoleculeName: Hantaan virus nucleoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Hantaan orthohantavirus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: SF21 / Recombinant plasmid: pFastBac

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: RNA (5'-R(P*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*UP*U)-3') / type: rna / ID: 1
Details: A poly-U has been built but the RNA corresponds to heterogeneous insect cell RNA that bind to nucleoproteins during expression.
Number of copies: 1
Source (natural)Organism: Fall armyworm (fall armyworm)
Molecular weightTheoretical: 873.54 Da
SequenceString:
UUU

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Macromolecule #2: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Hantaan orthohantavirus
Molecular weightTheoretical: 48.262266 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMATMEELQR EINAHEGQLV IARQKVRDAE KQYEKDPDEL NKRTLTDREG VAVSIQAKID ELKRQLADRI ATGKNLGKEQ DPTGVEPGD HLKERSMLSY GNVLDLNHLD IDEPTGQTAD WLSIIVYLTS FVVPILLKAL YMLTTRGRQT TKDNKGTRIR F KDDSSFED ...String:
GMATMEELQR EINAHEGQLV IARQKVRDAE KQYEKDPDEL NKRTLTDREG VAVSIQAKID ELKRQLADRI ATGKNLGKEQ DPTGVEPGD HLKERSMLSY GNVLDLNHLD IDEPTGQTAD WLSIIVYLTS FVVPILLKAL YMLTTRGRQT TKDNKGTRIR F KDDSSFED VNGIRKPKHL YVSLPNAQSS MKAEEITPGR YRTAVCGLYP AQIKARQMIS PVMSVIGFLA LAKDWSDRIE QW LIEPCKL LPDTAAVSLL GGPATNRDYL RQRQVALGNM ETKESKAIRQ HAEAAGCSMI EDIESPSSIW VFAGAPDRCP PTC LFIAGI AELGAFFSIL QDMRNTIMAS KTVGTSEEKL RKKSSFYQSY LRRTQSMGIQ LGQRIIVLFM VAWGKEAVDN FHLG DDMDP ELRTLAQSLI DVKVKEISNQ EPLKL

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HClTris
300.0 mMSodium chlorideNaClSodium chloride
10.0 mMMagnesium chlorideMgCl2
2.0 mMBeta-mercapto-ethanol
20.0 mMImidazole
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Details: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 46860 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 3-18 / Number grids imaged: 1 / Number real images: 4328 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 168709 / Software - Name: RELION (ver. 2.1) / Software - details: manual picking
Details: Straight HTNV-NC were manually picked and computationally cut with an inter-box distance of 38 A along the helical axis into overlapping boxes of 400*400 pixels, resulting in 168,709 ...Details: Straight HTNV-NC were manually picked and computationally cut with an inter-box distance of 38 A along the helical axis into overlapping boxes of 400*400 pixels, resulting in 168,709 extracted segments. 2D classification was used to eliminate bad quality filaments.
CTF correctionSoftware:
Namedetails
Gctfglobal
Gctflocal CTF-determination was calculated for each segment
RELION (ver. 3.0)CTF-refinement
Startup modelType of model: OTHER
Details: 130 A diameter featureless cylinder to which helical symmetry was applied.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.1)
Details: 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.
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 18.87 Å
Applied symmetry - Helical parameters - Δ&Phi: -99.95 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 105665
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5fsg


Chain - Residue range: 113-429
DetailsFiber model was iteratively rebuilt and all-atom refined using stereochemical and NCS restraints
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 103
Output model

PDB-6i2n:
Helical RNA-bound Hantaan virus nucleocapsid

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