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- PDB-7el9: Structure of Machupo virus L polymerase in complex with Z protein... -

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Basic information

Entry
Database: PDB / ID: 7el9
TitleStructure of Machupo virus L polymerase in complex with Z protein and 3'-vRNA (dimeric complex)
Components
  • Machupo virus 3'-vRNA promoter
  • RING finger protein Z
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN/RNA / RNA virus / polymerase / replication / transcription / matrix protein / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


negative stranded viral RNA replication / viral budding via host ESCRT complex / cap snatching / viral budding from plasma membrane / virion component / host cell cytoplasm / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / hydrolase activity / RNA-directed RNA polymerase ...negative stranded viral RNA replication / viral budding via host ESCRT complex / cap snatching / viral budding from plasma membrane / virion component / host cell cytoplasm / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / host cell plasma membrane / RNA binding / zinc ion binding / membrane / metal ion binding
Similarity search - Function
RING finger protein Z, zinc finger / RING finger protein Z, arenaviridae / Protein Z, RING-type zinc finger superfamily / P-11 zinc finger / 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 / RING finger protein Z
Similarity search - Component
Biological speciesMachupo mammarenavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPeng, R. / Xu, X. / Peng, Q. / Shi, Y.
CitationJournal: Nat Microbiol / Year: 2021
Title: Cryo-EM structures of Lassa and Machupo virus polymerases complexed with cognate regulatory Z proteins identify targets for antivirals.
Authors: Xin Xu / Ruchao Peng / Qi Peng / Min Wang / Ying Xu / Sheng Liu / Xiaolin Tian / Haiteng Deng / Yimin Tong / Xiaoyou Hu / Jin Zhong / Peiyi Wang / Jianxun Qi / George F Gao / Yi Shi /
Abstract: Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, ...Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, the multifunctional Z protein interacts with the L polymerase to shut down RNA synthesis and initiate virion assembly. However, the mechanism by which the Z protein regulates the activity of L polymerase is unclear. Here, we used cryo-electron microscopy to resolve the structures of both Lassa and Machupo virus L polymerases in complex with their cognate Z proteins, and viral RNA, to 3.1-3.9 Å resolutions. These structures reveal that Z protein binding induces conformational changes in two catalytic motifs of the L polymerase, and restrains their conformational dynamics to inhibit RNA synthesis, which is supported by hydrogen-deuterium exchange mass spectrometry analysis. Importantly, we show, by in vitro polymerase reactions, that Z proteins of Lassa and Machupo viruses can cross-inhibit their L polymerases, albeit with decreased inhibition efficiencies. This cross-reactivity results from a highly conserved determinant motif at the contacting interface, but is affected by other variable auxiliary motifs due to the divergent evolution of Old World and New World arenaviruses. These findings could provide promising targets for developing broad-spectrum antiviral drugs.
History
DepositionApr 9, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed
Revision 1.2Jul 14, 2021Group: 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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-31177
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: RING finger protein Z
C: Machupo virus 3'-vRNA promoter
D: RNA-directed RNA polymerase L
E: RING finger protein Z
F: Machupo virus 3'-vRNA promoter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)534,92516
Polymers534,2926
Non-polymers63310
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14520 Å2
ΔGint-86 kcal/mol
Surface area154990 Å2

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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 mammarenavirus
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q6IVU0, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#2: Protein RING finger protein Z / Protein Z / Zinc-binding protein


Mass: 10660.265 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Machupo mammarenavirus / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q6UY77
#3: RNA chain Machupo virus 3'-vRNA promoter


Mass: 6069.649 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Machupo mammarenavirus
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn / 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

Component
IDNameTypeEntity IDParent-IDSource
1Machupo virus polymerase in complex with Z martix protein and 3'-vRNA in dimeric form.COMPLEX#1-#30MULTIPLE SOURCES
2Machupo virus polymeraseCOMPLEX#11RECOMBINANT
3Machupo virus 3'-vRNA promoterCOMPLEX#21SYNTHETIC
4Machupo virus Z matrix proteinCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.58 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Machupo mammarenavirus11628
33Machupo mammarenavirus11628
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)2449148
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in.
VitrificationCryogen name: ETHANE

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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 FIELD / Cs: 2.7 mm / 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)
Image scansMovie frames/image: 32 / Used frames/image: 3-18

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFIND4.1CTF correction
7UCSF Chimera1.11model fitting
10RELION3final Euler assignment
11RELION3classification
12RELION3D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 328964 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16KLH

6klh

1
25I72

5i72

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00228630
ELECTRON MICROSCOPYf_angle_d0.57338732
ELECTRON MICROSCOPYf_dihedral_angle_d24.05810520
ELECTRON MICROSCOPYf_chiral_restr0.044506
ELECTRON MICROSCOPYf_plane_restr0.0034850

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