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- PDB-8z98: Cryo-EM structure of Thogoto virus polymerase in a transcription ... -

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Basic information

Entry
Database: PDB / ID: 8z98
TitleCryo-EM structure of Thogoto virus polymerase in a transcription reception conformation
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(P*GP*CP*AP*AP*AP*AP*AP*CP*A)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsTRANSCRIPTION / RNA polymerase
Function / homology
Function and homology information


cap snatching / 7-methylguanosine mRNA capping / virion component / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / host cell nucleus / RNA binding
Similarity search - Function
: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain ...: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-V9G / RNA / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein / Polymerase basic protein 2
Similarity search - Component
Biological speciesThogoto virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsXue, L. / Chang, T. / Li, Z. / Zhao, H. / Li, M. / He, J. / Chen, X. / Xiong, X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82341085 to X.X. China
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses.
Authors: Lu Xue / Tiancai Chang / Zimu Li / Chenchen Wang / Heyu Zhao / Mei Li / Peng Tang / Xin Wen / Mengmeng Yu / Jiqin Wu / Xichen Bao / Xiaojun Wang / Peng Gong / Jun He / Xinwen Chen / Xiaoli Xiong /
Abstract: Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV ...Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5' end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses' broad-infectivity range.
History
DepositionApr 22, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: 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
Revision 1.2Jun 19, 2024Group: Data collection / Structure summary
Category: audit_author / em_author_list ...audit_author / em_author_list / em_entity_assembly / struct
Item: _em_entity_assembly.name / _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
D: RNA (5'-R(P*GP*CP*AP*AP*AP*AP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,1625
Polymers250,3614
Non-polymers8011
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Polymerase acidic protein / PA / RNA-directed RNA polymerase subunit P3


Mass: 71623.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogoto virus (isolate SiAr 126) / Gene: Segment 3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P27194
#2: Protein RNA-directed RNA polymerase catalytic subunit / RNA-directed RNA polymerase subunit P2


Mass: 81432.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogoto virus (isolate SiAr 126) / Gene: Segment 2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O41353, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2 / PB2 / RNA-directed RNA polymerase subunit P3


Mass: 94418.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogoto virus (isolate SiAr 126) / Gene: Segment 1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9YNA4
#4: RNA chain RNA (5'-R(P*GP*CP*AP*AP*AP*AP*AP*CP*A)-3')


Mass: 2885.847 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thogoto virus (isolate SiAr 126)
#5: Chemical ChemComp-V9G / 7-METHYL-GUANOSINE-5'-TRIPHOSPHATE-5'-(2'-O-METHYL)-ADENOSINE


Mass: 801.468 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H32N10O17P3 / 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: Cryo-EM structure of Thogoto virus polymerase in a transcription reception conformation
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162604 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.21 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002612657
ELECTRON MICROSCOPYf_angle_d0.525717185
ELECTRON MICROSCOPYf_chiral_restr0.03981919
ELECTRON MICROSCOPYf_plane_restr0.00422158
ELECTRON MICROSCOPYf_dihedral_angle_d5.41481751

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