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- PDB-8qh3: 5'vRNA-bound Hantaan virus polymerase in monomeric active state -

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Basic information

Entry
Database: PDB / ID: 8qh3
Title5'vRNA-bound Hantaan virus polymerase in monomeric active state
Components
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*A)-3')
  • RNA-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 / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHantaan virus 76-118
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 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 6, 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

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
C: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)257,5483
Polymers257,5232
Non-polymers241
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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hantaan virus 76-118 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P23456
#2: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*A)-3')


Mass: 8046.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Hantaan virus 76-118
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Hantaan virus polymeraseCOMPLEX#1-#20MULTIPLE SOURCES
2RNA-directed RNA polymerase LCOMPLEX#11RECOMBINANT
3RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*A)-3')COMPLEX#21RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Hantaan virus 76-11811602
33Hantaan virus 76-11811602
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33synthetic construct (others)32630
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 supportGrid 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: 26745
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
9PHENIX1.20.1model refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.1classification
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3285016
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 882132 / Symmetry type: POINT
Atomic model buildingB value: 8.45 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 8C4S

8c4s


Pdb chain-ID: A / Accession code: 8C4S / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311056
ELECTRON MICROSCOPYf_angle_d0.50114995
ELECTRON MICROSCOPYf_dihedral_angle_d6.5721548
ELECTRON MICROSCOPYf_chiral_restr0.0381674
ELECTRON MICROSCOPYf_plane_restr0.0041847

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