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- PDB-7ckl: Structure of Lassa virus polymerase bound to Z matrix protein -

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Basic information

Entry
Database: PDB / ID: 7ckl
TitleStructure of Lassa virus polymerase bound to Z matrix protein
Components
  • RING finger protein Z
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Lassa virus / Polymerase / Z protein / replication regulation
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 cytoplasm / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / hydrolase activity / RNA-directed RNA polymerase ...negative stranded viral RNA replication / cap snatching / viral budding via host ESCRT complex / 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
: / RING finger protein Z / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesLassa mammarenavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.88 Å
AuthorsXu, X. / Peng, R. / 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
DepositionJul 17, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 17, 2021Group: Database references / Category: citation / citation_author / database_2
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.title / _citation.year / _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: RING finger protein Z
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,4335
Polymers264,2472
Non-polymers1863
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1800 Å2
ΔGint-22 kcal/mol
Surface area73060 Å2

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Components

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 253505.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa mammarenavirus / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: A0A097F4L1, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#2: Protein RING finger protein Z / / Protein Z / Zinc-binding protein


Mass: 10741.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa mammarenavirus / Production host: Escherichia coli (E. coli) / References: UniProt: A0A097F4I8
#3: 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
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / 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

ComponentName: Lassa virus polymerase in complex with Z matrix protein
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.26 MDa / Experimental value: YES
Source (natural)Organism: Lassa mammarenavirus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
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 / 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: 30 / Used frames/image: 3-17

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1200000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 273078 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-ID
16KLC

6klc

1
25I72

5i72

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211724
ELECTRON MICROSCOPYf_angle_d0.53815830
ELECTRON MICROSCOPYf_dihedral_angle_d21.2974318
ELECTRON MICROSCOPYf_chiral_restr0.0381818
ELECTRON MICROSCOPYf_plane_restr0.0032001

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