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- PDB-8p1m: Structure of hantaan orthohantavirus (HTNV) polymerase bound to 5... -

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Basic information

Entry
Database: PDB / ID: 8p1m
TitleStructure of hantaan orthohantavirus (HTNV) polymerase bound to 5'vRNA and NTP Mg
Components
  • RNA (5'-R(P*AP*GP*UP*AP*G)-3')
  • RNA-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 / 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
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHantaan orthohantavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 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: To Be Published
Title: Structure of hantaan orthohantavirus (HTNV) polymerase bound to 5'vRNA and NTP Mg
Authors: Keown, J.R. / Carrique, L. / Grimes, J.M.
History
DepositionMay 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 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
D: RNA (5'-R(P*AP*GP*UP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)257,7665
Polymers257,2102
Non-polymers5563
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
#2: RNA chain RNA (5'-R(P*AP*GP*UP*AP*G)-3')


Mass: 5404.245 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Hantaan orthohantavirus
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: #1-#2 / 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: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144000 / 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: 40.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003410841
ELECTRON MICROSCOPYf_angle_d0.507114678
ELECTRON MICROSCOPYf_chiral_restr0.03861635
ELECTRON MICROSCOPYf_plane_restr0.0041826
ELECTRON MICROSCOPYf_dihedral_angle_d5.03881468

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