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- PDB-8c4s: Apo Hantaan virus polymerase core -

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Basic information

Entry
Database: PDB / ID: 8c4s
TitleApo Hantaan virus polymerase core
ComponentsRNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Polymerase / replication / bunyavirus
Function / homology
Function and homology information


RNA-templated viral transcription / negative stranded viral RNA replication / cap snatching / endonuclease activity / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, hantavirus / RNA-directed RNA polymerase, hantavirus, N-terminal / : / RNA dependent RNA polymerase / Cap-snatching endonuclease / : / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHantaan virus 76-118
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsDurieux trouilleton, Q. / Arragain, B. / Malet, H.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0024 France
CitationJournal: Nat Commun / Year: 2023
Title: Structures of active Hantaan virus polymerase uncover the mechanisms of Hantaviridae genome replication.
Authors: Quentin Durieux Trouilleton / Sergio Barata-García / Benoît Arragain / Juan Reguera / Hélène Malet /
Abstract: Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here ...Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here we describe the structure of the Hantaan virus polymerase core and establish conditions for in vitro replication activity. The apo structure adopts an inactive conformation that involves substantial folding rearrangement of polymerase motifs. Binding of the 5' viral RNA promoter triggers Hantaan virus polymerase reorganization and activation. It induces the recruitment of the 3' viral RNA towards the polymerase active site for prime-and-realign initiation. The elongation structure reveals the formation of a template/product duplex in the active site cavity concomitant with polymerase core widening and the opening of a 3' viral RNA secondary binding site. Altogether, these elements reveal the molecular specificities of Hantaviridae polymerase structure and uncover the mechanisms underlying replication. They provide a solid framework for future development of antivirals against this group of emerging pathogens.
History
DepositionJan 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L


Theoretical massNumber of molelcules
Total (without water)249,4321
Polymers249,4321
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area53370 Å2
MethodPISA

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Components

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / RdRp / Replicase / Transcriptase


Mass: 249432.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hantaan virus 76-118 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Hi5
References: UniProt: P23456, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Apo Hantaan virus polymerase core / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.246 MDa / Experimental value: NO
Source (natural)Organism: Hantaan virus 76-118 / Strain: 76-118
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: Hi 5
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPESC8H18N2O4S1
2250 mMSodium ChlorideNaCl1
35 mMTCEPC9H15O6P1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 276 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Calibrated magnification: 43668 X / Nominal defocus max: 800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 2200 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 5.5 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2856
Image scansSampling size: 5 µm / Width: 3840 / Height: 3712 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.2particle selectionBlobPicker
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correctionPatch CTF estimation
5RELION4CTF correctionCTF refinement
8Coot0.9.2model fitting
10cryoSPARC3.3.2initial Euler assignmentNon homogeneous refinement
11RELION4final Euler assignment3D auto-refine
12RELION4classification3D classification
13RELION43D reconstruction
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1116690
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105717 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 65.75 / Protocol: AB INITIO MODEL / Space: REAL

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