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- PDB-6uen: Cryo-EM structure of the respiratory syncytial virus RNA polymerase -

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Basic information

Entry
Database: PDB / ID: 6uen
TitleCryo-EM structure of the respiratory syncytial virus RNA polymerase
Components
  • RNA-directed RNA polymerase L
  • the phosphoprotein (P) of human respiratory syncytial virus
KeywordsVIRAL PROTEIN/Transferase / Viral protein complexes / VIRAL PROTEIN / VIRAL PROTEIN-Transferase complex
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / virion component => GO:0044423 / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral life cycle / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / : / mRNA 5'-cap (guanine-N7-)-methyltransferase activity ...NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / virion component => GO:0044423 / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral life cycle / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / : / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase ...RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
Phosphoprotein / RNA-directed RNA polymerase L / Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHuman respiratory syncytial virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsCao, D. / Gao, Y. / Liang, B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM130950 United States
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-EM structure of the respiratory syncytial virus RNA polymerase.
Authors: Dongdong Cao / Yunrong Gao / Claire Roesler / Samantha Rice / Paul D'Cunha / Lisa Zhuang / Julia Slack / Mason Domke / Anna Antonova / Sarah Romanelli / Shayon Keating / Gabriela Forero / ...Authors: Dongdong Cao / Yunrong Gao / Claire Roesler / Samantha Rice / Paul D'Cunha / Lisa Zhuang / Julia Slack / Mason Domke / Anna Antonova / Sarah Romanelli / Shayon Keating / Gabriela Forero / Puneet Juneja / Bo Liang /
Abstract: The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide ...The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (P) and C-terminal domain (P) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (P) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.
History
DepositionSep 22, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: the phosphoprotein (P) of human respiratory syncytial virus
C: the phosphoprotein (P) of human respiratory syncytial virus
D: the phosphoprotein (P) of human respiratory syncytial virus
E: the phosphoprotein (P) of human respiratory syncytial virus


Theoretical massNumber of molelcules
Total (without water)282,2505
Polymers282,2505
Non-polymers00
Water0
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 / Replicase / Transcriptase


Mass: 173586.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human respiratory syncytial virus / Gene: L, MZ07_64039gpL, MZ07_64040gpL / Plasmid: pET / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: G8EJ12, UniProt: P28887*PLUS, RNA-directed RNA polymerase, mRNA (guanine-N7)-methyltransferase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, ...References: UniProt: G8EJ12, UniProt: P28887*PLUS, RNA-directed RNA polymerase, mRNA (guanine-N7)-methyltransferase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, GDP polyribonucleotidyltransferase
#2: Protein
the phosphoprotein (P) of human respiratory syncytial virus / Phosphoprotein


Mass: 27165.838 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human respiratory syncytial virus / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: G3C7Q7, UniProt: P03421*PLUS

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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: the L protein of the human respiratory syncytial virus; the phosphoprotein (P) of human respiratory syncytial virus
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.360 MDa / Experimental value: YES
Source (natural)Organism: Human respiratory syncytial virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf21
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 91 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 253372 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213792
ELECTRON MICROSCOPYf_angle_d0.67618631
ELECTRON MICROSCOPYf_dihedral_angle_d10.058465
ELECTRON MICROSCOPYf_chiral_restr0.0422159
ELECTRON MICROSCOPYf_plane_restr0.0042353

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