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.
Name: RNA (5'-R(P*UP*UP*U)-3') / Class: RNA Details: A poly-U has been built but the RNA corresponds to heterogeneous insect cell RNA that bind to nucleoproteins during expression. Structure: OTHER / Synthetic: No Sequence:
UUU
Mass
Theoretical: 0.87354 kDa
Source
Species: Fall armyworm (fall armyworm)
+
Component #5: protein, Nucleoprotein
Protein
Name: Nucleoprotein / Number of Copies: 1 / Recombinant expression: No
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 % 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
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 4 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Magnification: 46860.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen Holder
Model: FEI TITAN KRIOS AUTOGRID HOLDER
Camera
Detector: GATAN K2 SUMMIT (4k x 4k)
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Image acquisition
Image acquisition
Number of digital images: 4328 / Sampling size: 5 microns
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Image processing
Processing
Method: helical reconstruction
3D reconstruction
Software: RELION / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF Euler angles: 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.
FSC plot (resolution estimation)
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Atomic model buiding
Modeling #1
Refinement space: REAL Details: Fiber model was iteratively rebuilt and all-atom refined using stereochemical and NCS restraints Input PDB model: 5FSG
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