+Open data
-Basic information
Entry | Database: PDB / ID: 6i2n | ||||||
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Title | Helical RNA-bound Hantaan virus nucleocapsid | ||||||
Components |
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Keywords | VIRAL PROTEIN / Nucleoprotein / Nucleocapsid / RNA-binding | ||||||
Function / homology | Function and homology information symbiont-mediated perturbation of host apoptosis / symbiont-mediated suppression of host apoptosis / helical viral capsid / viral nucleocapsid / host cell Golgi apparatus / endonuclease activity / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / ribonucleoprotein complex / RNA binding Similarity search - Function | ||||||
Biological species | Hantaan orthohantavirus Spodoptera frugiperda (fall armyworm) | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å | ||||||
Authors | Arragain, B. / Reguera, J. / Desfosses, A. / Gutsche, I. / Schoehn, G. / Malet, H. | ||||||
Funding support | France, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6i2n.cif.gz | 74.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6i2n.ent.gz | 52.4 KB | Display | PDB format |
PDBx/mmJSON format | 6i2n.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/i2/6i2n ftp://data.pdbj.org/pub/pdb/validation_reports/i2/6i2n | HTTPS FTP |
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-Related structure data
Related structure data | 0333MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: RNA chain | 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 |
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#2: Protein | 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 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
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Molecular weight |
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Source (natural) |
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Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF21 / Plasmid: pFastBac | ||||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Details: 30 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: 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 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 46860 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron 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 optics | Energyfilter name: GIF Quantum LS |
Image scans | Sampling size: 5 µm / Width: 3840 / Height: 3712 / Movie frames/image: 28 / Used frames/image: 3-18 |
-Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -99.95 ° / Axial rise/subunit: 18.87 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. 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 reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105665 / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B 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 building | PDB-ID: 5FSG Accession code: 5FSG / Pdb chain residue range: 113-429 / Source name: PDB / Type: experimental model |