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- PDB-8p1l: Structure of hantaan orthohantavirus (HTNV) polymerase - Apo full... -

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Basic information

Entry
Database: PDB / ID: 8p1l
TitleStructure of hantaan orthohantavirus (HTNV) polymerase - Apo full length
ComponentsRNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / RNA-dependent RNA polymerase / HTNV / Hantaan orthohantavirus / Bunyavirales / Hantaviridae.
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 / nucleotide binding / RNA-directed RNA polymerase activity / 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 orthohantavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKeown, J.R. / Carrique, L. / Grimes, J.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
University of Oxford, Medical Sciences Internal Fund (MSIF)BDR00281 United Kingdom
CitationJournal: PLoS Pathog / Year: 2024
Title: Structural characterization of the full-length Hantaan virus polymerase.
Authors: Jeremy R Keown / Loïc Carrique / Benjamin E Nilsson-Payant / Ervin Fodor / Jonathan M Grimes /
Abstract: Hantaviridae are a family of segmented negative-sense RNA viruses that contain important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and ...Hantaviridae are a family of segmented negative-sense RNA viruses that contain important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome. Here we establish the expression and purification of the polymerase from the Old World Hantaan virus and characterise the structure using Cryo-EM. We determine a series of structures at resolutions between 2.7 and 3.3 Å of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerases. We further describe structures with 5' vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including β-sheet, two helical arrangements, and nucleotide primed arrangement. The insights gained here guide future mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.
History
DepositionMay 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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)251,8061
Polymers251,8061
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 251806.016 Da / Num. of mol.: 1 / Mutation: D97A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hantaan orthohantavirus / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P23456, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
Has protein modificationN

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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: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.247 MDa / Experimental value: YES
Source (natural)Organism: Hantaan orthohantavirus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
2500 mMsodium chlorideNaCl1
31 mMdithiothreitolDTT1
45 %GlycerolGlycerol1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 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
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2Topaz1.5particle selection
3EPU3.1image acquisition
5cryoSPARC4CTF correction
8UCSF ChimeraX1.5model fitting
9Coot0.9.6model fitting
11PHENIX1.19.2model refinement
12cryoSPARC4initial Euler assignment
13cryoSPARC4final Euler assignment
14cryoSPARC4classification
15cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.5 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002516794
ELECTRON MICROSCOPYf_angle_d0.488222706
ELECTRON MICROSCOPYf_chiral_restr0.03982529
ELECTRON MICROSCOPYf_plane_restr0.00362867
ELECTRON MICROSCOPYf_dihedral_angle_d4.14372213

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