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- PDB-8qhd: Hantaan virus polymerase in hexameric state -

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Basic information

Entry
Database: PDB / ID: 8qhd
TitleHantaan virus polymerase in hexameric state
ComponentsRNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Bunyavirus / Hantaan virus / polymerase / dimer
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 / : / 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: 3.6 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: 2024
Title: Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers.
Authors: Quentin Durieux Trouilleton / Dominique Housset / Paco Tarillon / Benoît Arragain / Hélène Malet /
Abstract: Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete ...Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer's interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.
History
DepositionSep 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: RNA-directed RNA polymerase L
B: RNA-directed RNA polymerase L
C: RNA-directed RNA polymerase L
F: RNA-directed RNA polymerase L
A: RNA-directed RNA polymerase L
E: RNA-directed RNA polymerase L


Theoretical massNumber of molelcules
Total (without water)1,496,8596
Polymers1,496,8596
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
RNA-directed RNA polymerase L


Mass: 249476.484 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hantaan virus 76-118 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P23456

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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: Hantaan virus polymerase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.5 MDa / Experimental value: NO
Source (natural)Organism: Hantaan virus 76-118
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPESC8H18N2O4S1
2250 mMNaClSodium chlorideNaClSodium chloride1
310 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: 77 K / Details: blot force 1 3s

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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 / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.45 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14650
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionblob picker
2EPUimage acquisition
4cryoSPARC4.2.1CTF correction
7Coot0.9.8.1model fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
11cryoSPARC4.2.1classification
12cryoSPARC4.2.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3354153
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28695
Details: This is a chimeric map of Hantaan polymerase hexamer. It originates from the global map in which the tow internal protomers are defined and a focused map that comprises one internal protomer ...Details: This is a chimeric map of Hantaan polymerase hexamer. It originates from the global map in which the tow internal protomers are defined and a focused map that comprises one internal protomer and two external protomers. The chimeric map has a C2 symmetry
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 8C4S

8c4s


Pdb chain-ID: A / Accession code: 8C4S / Source name: PDB / Type: experimental model

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