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

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Basic information

Entry
Database: PDB / ID: 6i2n
TitleHelical RNA-bound Hantaan virus nucleocapsid
Components
  • Nucleoprotein
  • RNA (5'-R(P*UP*UP*U)-3')
KeywordsVIRAL PROTEIN / Nucleoprotein / Nucleocapsid / RNA-binding
Function / homologyHantavirus nucleocapsid protein / Hantavirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / RNA binding / Nucleoprotein
Function and homology information
Specimen sourceHantaan orthohantavirus
Spodoptera frugiperda (fall armyworm)
MethodELECTRON MICROSCOPY / helical 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 Report
SummaryFull reportAbout validation report
DateDeposition: Nov 1, 2018 / Release: Jan 23, 2019

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Structure visualization

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  • Biological unit as author_and_software_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-0333
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Structure viewerMolecule:
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Assembly

Deposited unit
U: RNA (5'-R(P*UP*UP*U)-3')
D: Nucleoprotein


Theoretical massNumber of molelcules
Total (without water)49,1362
Polyers49,1362
Non-polymers00
Water0
1
U: RNA (5'-R(P*UP*UP*U)-3')
D: Nucleoprotein
x 7


Theoretical massNumber of molelcules
Total (without water)343,95114
Polyers343,95114
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation6
Buried area (Å2)900
ΔGint (kcal/M)-8.0
Surface area (Å2)20000
MethodPISA

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Components

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


Mass: 873.540 Da / Num. of mol.: 1
Details: A poly-U has been built but the RNA corresponds to heterogeneous insect cell RNA that bind to ...A poly-U has been built but the RNA corresponds to heterogeneous insect cell RNA that bind to nucleoproteins during expression.
Source: (natural) Spodoptera frugiperda (fall armyworm) / Cell line: SF21
#2: Protein/peptide Nucleoprotein / / Nucleocapsid protein / Protein N


Mass: 48262.266 Da / Num. of mol.: 1 / Source: (gene. exp.) Hantaan orthohantavirus / Variant: KHF / Plasmid name: pFastBac / Cell line (production host): SF21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P05133

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1RNA-bound Hantaan virus nucleocapsidCOMPLEX1, 20MULTIPLE SOURCES
2Hantaan virus nucleoproteinCOMPLEX21RECOMBINANT
3RNACOMPLEX11NATURAL
Molecular weight
IDValueEntity assembly IDExperimental value
12.554 kDa/nm1NO
20.0482 MDa1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
137108Spodoptera frugiperda (fall armyworm)
221980471Hantaan orthohantavirus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: pFastBac / Strain: SF21
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
120 mMTris-HCl1
2300 mMSodium chlorideNaCl1
310 mMMagnesium chlorideMgCl21
42 mMBeta-mercapto-ethanol1
520 mMImidazole1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA / Grid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 kelvins
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 46860 / Calibrated defocus min: 800 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 4328
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansSampling size: 5 microns / Width: 3840 / Height: 3712 / Movie frames/image: 28 / Used frames/image: 3-18

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2.1particle selectionmanual picking
2EPUimage acquisition
4GctfCTF correctionglobal
5GctfCTF correctionlocal CTF-determination was calculated for each segment
6RELION3.0CTF correctionCTF-refinement
9UCSF Chimeramodel fittinginitial rigid fitting
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
14RELION3.03D reconstruction
15PHENIX1.13model refinement
16Coot0.8.9model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -99.95 deg. / Axial rise/subunit: 18.87 Å / Axial symmetry: C1
Particle selectionDetails: 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.
Number of particles selected: 168709
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 105665 / Number of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingDetails: Fiber model was iteratively rebuilt and all-atom refined using stereochemical and NCS restraints
Overall b value: 103 / Ref protocol: AB INITIO MODEL / Ref space: REAL
Atomic model buildingPDB-ID: 5FSG
Pdb chain residue range: 113-429

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