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- PDB-8as7: Structure of the SFTSV L protein stalled at early elongation [EAR... -

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Basic information

Entry
Database: PDB / ID: 8as7
TitleStructure of the SFTSV L protein stalled at early elongation [EARLY-ELONGATION]
Components
  • RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*A)-3')
  • RNA (5'-R(P*AP*CP*A)-3')
  • RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')
  • RNA-dependent RNA-polymerase L protein
KeywordsVIRAL PROTEIN / SFTSV RNA-DEPENDENT RNA POLYMERASE / VIRAL RNA
Function / homology
Function and homology information


host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / 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
Chem-2KH / RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesSFTS virus AH12
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsWilliams, H.M. / Thorkelsson, S.R. / Vogel, D. / Milewski, M. / Busch, C. / Cusack, S. / Grunewald, K. / Quemin, E.R.J. / Rosenthal, M.
Funding support Germany, 3items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017 Germany
German Research Foundation (DFG)INST 152/772-1, 774-1, 775-1 and 776-1 Germany
German Federal Ministry for Education and Research01KI2019 Germany
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.
Authors: Harry M Williams / Sigurdur R Thorkelsson / Dominik Vogel / Morlin Milewski / Carola Busch / Stephen Cusack / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA- ...Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.
History
DepositionAug 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-dependent RNA-polymerase L protein
P: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*A)-3')
T: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')
G: RNA (5'-R(P*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,5158
Polymers258,7454
Non-polymers7704
Water18010
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: There is no protein assembly here, it is a single polypeptide chain.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein RNA-dependent RNA-polymerase L protein / Replicase / Transcriptase


Mass: 235698.500 Da / Num. of mol.: 1 / Mutation: D112A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SFTS virus AH12 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: U3GU88, RNA-directed RNA polymerase

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RNA chain , 3 types, 3 molecules PTG

#2: RNA chain RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*A)-3')


Mass: 6451.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SFTS virus AH12
#3: RNA chain RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')


Mass: 8386.002 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Genome end with 6 additional A's added. / Source: (synth.) SFTS virus AH12
#4: RNA chain RNA (5'-R(P*AP*CP*A)-3')


Mass: 8208.922 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Additional 6A's added to template RNA (nt 21 - 26) so poly-U stretch here is artificial.
Source: (synth.) SFTS virus AH12

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Non-polymers , 4 types, 14 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#7: Chemical ChemComp-2KH / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1SFTSV L protein stalled at early elongation [EARLY-ELONGATION]COMPLEX#1-#40MULTIPLE SOURCES
2RNA-dependent RNA-polymerase L proteinCOMPLEX#11RECOMBINANT
3RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*A)-3')COMPLEX#21RECOMBINANT
4RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')COMPLEX#31RECOMBINANT
5RNA (5'-R(P*AP*CP*A)-3')COMPLEX#41RECOMBINANT
Molecular weightValue: 0.238 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12SFTS virus AH12992210
23SFTS virus AH12992210
34SFTS virus AH12992210
45SFTS virus AH12992210
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Synthetic construct (others)32630
34Synthetic construct (others)32630
45Synthetic construct (others)32630
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313782
ELECTRON MICROSCOPYf_angle_d0.58218788
ELECTRON MICROSCOPYf_dihedral_angle_d11.1422170
ELECTRON MICROSCOPYf_chiral_restr0.042102
ELECTRON MICROSCOPYf_plane_restr0.0042253

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