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- PDB-6klh: Dimeric structure of Machupo virus polymerase bound to vRNA promoter -

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Basic information

Entry
Database: PDB / ID: 6klh
TitleDimeric structure of Machupo virus polymerase bound to vRNA promoter
Components
  • RNA (5'-R(*GP*CP*CP*UP*AP*GP*GP*AP*UP*CP*CP*AP*CP*UP*GP*UP*GP*CP*G)-3')
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN/RNA / polymerase / RNA virus / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


negative stranded viral RNA replication / cap snatching / virion component / host cell cytoplasm / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / metal ion binding
Similarity search - Function
RNA polymerase, arenaviral / RNA endonuclease, cap-snatching / Arenavirus RNA polymerase / Arenavirus cap snatching domain / : / 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 speciesMachupo virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPeng, R. / Xu, X. / Jing, J. / Peng, Q. / Gao, G.F. / Shi, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81622031 China
CitationJournal: Nature / Year: 2020
Title: Structural insight into arenavirus replication machinery.
Authors: Ruchao Peng / Xin Xu / Jiamei Jing / Min Wang / Qi Peng / Sheng Liu / Ying Wu / Xichen Bao / Peiyi Wang / Jianxun Qi / George F Gao / Yi Shi /
Abstract: Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis ...Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.
History
DepositionJul 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Apr 8, 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_PubMed / _citation.title
Revision 2.0Dec 8, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / database_2 ...atom_site / database_2 / em_software / entity_name_com / entity_src_gen / pdbx_audit_support / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_struct_assembly_prop / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / refine_ls_restr / software / struct_conf / struct_conn / struct_sheet / struct_sheet_order / struct_sheet_range / struct_site / struct_site_gen
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_software.category / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_audit_support.funding_organization / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _software.version
Description: Model completeness
Details: Some poorly resolved regions are updated based on higher resolution maps obtained in our follow-up study.
Provider: author / Type: Coordinate replacement

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Structure visualization

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  • Deposited structure unit
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  • EMDB-0710
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: RNA (5'-R(*GP*CP*CP*UP*AP*GP*GP*AP*UP*CP*CP*AP*CP*UP*GP*UP*GP*CP*G)-3')
C: RNA-directed RNA polymerase L
D: RNA (5'-R(*GP*CP*CP*UP*AP*GP*GP*AP*UP*CP*CP*AP*CP*UP*GP*UP*GP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)513,2128
Polymers512,9714
Non-polymers2414
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: 250416.062 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Machupo virus
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q6IVU0, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (5'-R(*GP*CP*CP*UP*AP*GP*GP*AP*UP*CP*CP*AP*CP*UP*GP*UP*GP*CP*G)-3')


Mass: 6069.649 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: Machupo virus polymerase bound to vRNA promoter / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.54 MDa / Experimental value: YES
Source (natural)Organism: Machupo mammarenavirus
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Cell: HiFive
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / 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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4Gctf0.5CTF correction
7UCSF Chimera1.11model fitting
8Coot0.8.9model fitting
10PHENIX1.11model refinement
11RELION2initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1600000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 403000 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6KLD

6kld

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00329318
ELECTRON MICROSCOPYf_angle_d0.72439716
ELECTRON MICROSCOPYf_dihedral_angle_d13.76810546
ELECTRON MICROSCOPYf_chiral_restr0.0454666
ELECTRON MICROSCOPYf_plane_restr0.0045024

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