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- PDB-8ci5: Structure of the SNV L protein bound to 5' RNA -

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Basic information

Entry
Database: PDB / ID: 8ci5
TitleStructure of the SNV L protein bound to 5' RNA
Components
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*U)-3')
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Negative-strand viruses / Bunyaviruses / Hantaviruses / RNA-dependent RNA polymerase / viral genome replication and transcription / VIRUS PROTEIN
Function / homology
Function and homology information


cap snatching / endonuclease activity / host cell perinuclear region of cytoplasm / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription
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 speciesSin Nombre orthohantavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMeier, K. / Thorkelsson, S.R. / Durieux Trouilleton, Q. / Vogel, D. / Yu, D. / Kosinski, J. / Cusack, S. / Malet, H. / Grunewald, K. / Quemin, E.R.J. / Rosenthal, M.
Funding support Germany, France, 5items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017) Germany
German Research Foundation (DFG)5979/2-1, INST 152/772-1, 774-1, 775-1, 776-1 Germany
Alexander von Humboldt Foundation Germany
German Federal Ministry for Education and Research01KI2019 Germany
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INBS-05-02 France
CitationJournal: PLoS Pathog / Year: 2023
Title: Structural and functional characterization of the Sin Nombre virus L protein.
Authors: Kristina Meier / Sigurdur R Thorkelsson / Quentin Durieux Trouilleton / Dominik Vogel / Dingquan Yu / Jan Kosinski / Stephen Cusack / Hélène Malet / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus ...The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.
History
DepositionFeb 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
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 / _citation_author.identifier_ORCID

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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*U)-3')


Theoretical massNumber of molelcules
Total (without water)252,6432
Polymers252,6432
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, Conducted native gel electrophoresis confirms complex formation
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2820 Å2
ΔGint-17 kcal/mol
Surface area54070 Å2

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Components

#1: Protein RNA-directed RNA polymerase L


Mass: 246846.797 Da / Num. of mol.: 1 / Mutation: K124A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sin Nombre orthohantavirus / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A0P0ALW1
#2: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*U)-3')


Mass: 5796.515 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Sin Nombre orthohantavirus

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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: Structure of Sin nombre virus L protein bound to 5' RNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.254 MDa / Experimental value: YES
Source (natural)Organism: Sin Nombre orthohantavirus
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 514913 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 46.26 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01410613
ELECTRON MICROSCOPYf_angle_d1.127414389
ELECTRON MICROSCOPYf_chiral_restr0.06711611
ELECTRON MICROSCOPYf_plane_restr0.00771772
ELECTRON MICROSCOPYf_dihedral_angle_d10.51631488

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