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- PDB-8r6y: Structure of the SFTSV L protein stalled in a transcription-speci... -

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Basic information

Entry
Database: PDB / ID: 8r6y
TitleStructure of the SFTSV L protein stalled in a transcription-specific early elongation state with bound capped RNA [TRANSCRIPTION-EARLY-ELONGATION]
Components
  • RNA (5'-R(*(M7G)*AP*AP*A)-3')
  • RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')
  • RNA (5'-R(P*AP*CP*A)-3')
  • RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / SFTSV / RNA-DEPENDENT RNA POLYMERASE / VIRAL RNA
Function / homology
Function and homology information


host cell endoplasmic reticulum / endoplasmic reticulum-Golgi intermediate compartment / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent 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: 3.4 Å
AuthorsWilliams, H.M. / Thorkelsson, S.R. / Vogel, D. / Busch, C. / Milewski, M. / Cusack, S. / Grunewald, K. / Quemin, E.R.J. / Rosenthal, M.
Funding support Germany, 3items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017 Germany
German Research Foundation (DFG)NST 152/772-1, 774-1, 775-1 and 776-1 Germany
German Federal Ministry for Education and Research01KI2019 Germany
Citation
Journal: Nucleic Acids Res / Year: 2024
Title: Structural snapshots of phenuivirus cap-snatching and transcription.
Authors: Harry M Williams / Sigurdur R Thorkelsson / Dominik Vogel / Carola Busch / Morlin Milewski / Stephen Cusack / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by ...Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.
#1: Journal: BiorXiv / Year: 2023
Title: Structural snapshots of phenuivirus cap-snatching and transcription
Authors: Williams, H.M. / Thorkelsson, S.R. / Vogel, D. / Busch, C. / Milewski, M. / Cusack, S. / Grunewald, K. / Quemin, E.R.J. / Rosenthal, M.
History
DepositionNov 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Revision 1.2May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
P: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')
T: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')
G: RNA (5'-R(P*AP*CP*A)-3')
C: RNA (5'-R(*(M7G)*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,9217
Polymers255,4145
Non-polymers5072
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein RNA-directed RNA polymerase L


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

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RNA chain , 4 types, 4 molecules PTGC

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


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


Mass: 6480.872 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Synthesised by Biomers. / Source: (synth.) SFTS virus AH12
#4: RNA chain RNA (5'-R(P*AP*CP*A)-3')


Mass: 918.636 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Produced by the viral polymerase - not synthesised.
Source: (synth.) SFTS virus AH12
#5: RNA chain RNA (5'-R(*(M7G)*AP*AP*A)-3')


Mass: 5863.617 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Synthesised by ChemGenes. / Source: (synth.) SFTS virus AH12

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Non-polymers , 2 types, 2 molecules

#6: 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
#7: Chemical ChemComp-MG / MAGNESIUM ION


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

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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

ComponentName: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA acting as a primer.
Type: COMPLEX
Details: SFTSV L protein expressed recombinantly in complex with several RNA species.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)Organism: SFTS virus AH12
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportFilm material: CARBON / Grid material: GOLD / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 27095

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2998852
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264083 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317094
ELECTRON MICROSCOPYf_angle_d0.82923261
ELECTRON MICROSCOPYf_dihedral_angle_d12.7132656
ELECTRON MICROSCOPYf_chiral_restr0.0532605
ELECTRON MICROSCOPYf_plane_restr0.0052822

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