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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 / 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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsArragain, B. / Reguera, J. / Desfosses, A. / Gutsche, I. / Schoehn, G. / Malet, H.
Funding support France, 1items
OrganizationGrant numberCountry
G7H-IRS17H50 France
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, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Data collection / Source and taxonomy / Category: em_admin / entity_src_gen / pdbx_database_proc
Item: _em_admin.last_update / _entity_src_gen.pdbx_host_org_cell_line
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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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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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
Polymers49,1362
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
Polymers343,95114
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation6
Buried area900 Å2
ΔGint-8 kcal/mol
Surface area20000 Å2
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 / Source method: isolated from a natural source
Details: 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 Nucleoprotein / / Nucleocapsid protein / Protein N


Mass: 48262.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hantaan orthohantavirus / Variant: KHF / Plasmid: 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 / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RNA-bound Hantaan virus nucleocapsidCOMPLEXall0MULTIPLE SOURCES
2Hantaan virus nucleoproteinCOMPLEX#21RECOMBINANT
3RNACOMPLEX#11NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
112.554 kDa/nmNO
210.0482 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
13Spodoptera frugiperda (fall armyworm)7108
22Hantaan orthohantavirus1980471
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF21 / Plasmid: pFastBac
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris1
2300 mMSodium chlorideNaClSodium chloride1
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 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
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
MicroscopyModel: 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 X / 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) / Num. of grids imaged: 1 / Num. of real images: 4328
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansSampling size: 5 µm / 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
6RELION3CTF correctionCTF-refinement
9UCSF Chimeramodel fittinginitial rigid fitting
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
14RELION33D reconstruction
15PHENIX1.13model refinement
16Coot0.8.9model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -99.95 ° / Axial rise/subunit: 18.87 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 168709
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.
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105665 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 103 / Protocol: AB INITIO MODEL / Space: REAL
Details: Fiber model was iteratively rebuilt and all-atom refined using stereochemical and NCS restraints
Atomic model buildingPDB-ID: 5FSG

5fsg


Pdb chain residue range: 113-429

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