|Entry||Database: PDB / ID: 6i2n|
|Title||Helical RNA-bound Hantaan virus nucleocapsid|
|Keywords||VIRAL PROTEIN / Nucleoprotein / Nucleocapsid / RNA-binding|
|Function / homology||Hantavirus nucleocapsid protein / Hantavirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / RNA binding / Nucleoprotein|
Function and homology information
|Specimen source||Hantaan orthohantavirus|
Spodoptera frugiperda (fall armyworm)
|Method||ELECTRON MICROSCOPY / helical reconstruction / cryo EM / 3.3 Å resolution|
|Authors||Arragain, B. / Reguera, J. / Desfosses, A. / Gutsche, I. / Schoehn, G. / Malet, H.|
|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.
SummaryFull reportAbout validation report
|Date||Deposition: Nov 1, 2018 / Release: Jan 23, 2019|
|Structure viewer||Molecule: |
Downloads & links
U: RNA (5'-R(P*UP*UP*U)-3')
U: RNA (5'-R(P*UP*UP*U)-3')x 7
|#1: RNA chain|| |
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|| |
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
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: FILAMENT / Reconstruction method: helical reconstruction|
|Source (recombinant)||Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: pFastBac / Strain: SF21|
|Buffer solution||pH: 8|
|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 / Grid type: Quantifoil R2/1|
|Vitrification||Instrument: 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
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: 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 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) / Number of grids imaged: 1 / Number of real images: 4328|
|EM imaging optics||Energyfilter name: GIF Quantum LS|
|Image scans||Sampling size: 5 microns / Width: 3840 / Height: 3712 / Movie frames/image: 28 / Used frames/image: 3-18|
|Software||Name: PHENIX / Version: 1.13_2998: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Helical symmerty||Angular rotation/subunit: -99.95 deg. / Axial rise/subunit: 18.87 Å / Axial symmetry: C1|
|Particle selection||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.|
Number of particles selected: 168709
|3D reconstruction||Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 105665 / Number of class averages: 1 / Symmetry type: HELICAL|
|Atomic model building||Details: 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 building||PDB-ID: 5FSG|
Pdb chain residue range: 113-429
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