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- PDB-6klc: Structure of apo Lassa virus polymerase -

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Basic information

Entry
Database: PDB / ID: 6klc
TitleStructure of apo Lassa virus polymerase
ComponentsRNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / polymerase / RNA virus
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-directed RNA polymerase L
Similarity search - Component
Biological speciesLassa mammarenavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsPeng, R. / Xu, X. / Jing, J. / Peng, Q. / Gao, G.F. / Shi, Y.
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 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)253,5612
Polymers253,5061
Non-polymers551
Water00
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: 253505.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa mammarenavirus / Cell line (production host): HiFive
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: A0A097F4L1, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
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: Lassa virus polymerase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)Organism: Lassa 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 FIELD / 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: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4Gctf0.5CTF correction
7UCSF Chimera1.11model fitting
8Coot0.8.9model fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
14PHENIX1.11model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1500000
Details: approximately 1,500,000 particles were autopicked from ~2700 micrographs
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 295000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311404
ELECTRON MICROSCOPYf_angle_d0.69215422
ELECTRON MICROSCOPYf_dihedral_angle_d17.0216925
ELECTRON MICROSCOPYf_chiral_restr0.1311811
ELECTRON MICROSCOPYf_plane_restr0.0031951

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