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- PDB-7elc: Structure of monomeric complex of MACV L bound to Z and 3'-vRNA -

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Basic information

Entry
Database: PDB / ID: 7elc
TitleStructure of monomeric complex of MACV L bound to Z and 3'-vRNA
Components
  • 3'-vRNA promoter
  • RING finger protein Z
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN/RNA / Machupo virus / Polymerase / Z protein / replication regulation / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


negative stranded viral RNA replication / cap snatching / viral budding via host ESCRT complex / viral budding from plasma membrane / virion component / host cell / Hydrolases; Acting on ester bonds / host cell cytoplasm / hydrolase activity / host cell perinuclear region of cytoplasm ...negative stranded viral RNA replication / cap snatching / viral budding via host ESCRT complex / viral budding from plasma membrane / virion component / host cell / Hydrolases; Acting on ester bonds / host cell cytoplasm / hydrolase activity / host cell perinuclear region of cytoplasm / RNA-directed RNA polymerase / nucleotide binding / RNA-directed RNA polymerase activity / host cell plasma membrane / RNA binding / zinc ion binding / metal ion binding / membrane
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: 3.1 Å
AuthorsXu, X. / Peng, R. / Peng, Q. / Shi, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB29010000 China
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
Revision 1.3Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)267,4638
Polymers267,1463
Non-polymers3175
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4270 Å2
ΔGint-74 kcal/mol
Surface area61470 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Machupo mammarenavirus / Description: baculovirus expression system
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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Machupo mammarenavirus / Production host: Escherichia coli (E. coli) / References: UniProt: Q6UY77
#3: RNA chain 3'-vRNA promoter


Mass: 6069.649 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Machupo mammarenavirus
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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 matrix protein and 3'-vRNA in monomeric formCOMPLEX#1-#30MULTIPLE SOURCES
2Machupo virus polymerseCOMPLEX#11RECOMBINANT
3Machupo virus Z martix proteinCOMPLEX#21RECOMBINANT
43'-vRNA promoterCOMPLEX#31SYNTHETIC
Molecular weightValue: 0.27 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Machupo mammarenavirus11628
32Machupo mammarenavirus11628
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)2449148
32Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: BASIC
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
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
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.17model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3930000
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180190 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16KLD

6kld

16KLD1PDBexperimental model
25I72

5i72

15I722PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311970
ELECTRON MICROSCOPYf_angle_d0.53516166
ELECTRON MICROSCOPYf_dihedral_angle_d24.8684472
ELECTRON MICROSCOPYf_chiral_restr0.0391858
ELECTRON MICROSCOPYf_plane_restr0.0032014

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