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- PDB-7oe7: Apo-structure of Lassa virus L protein (well-resolved alpha ribbo... -

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Basic information

Entry
Database: PDB / ID: 7oe7
TitleApo-structure of Lassa virus L protein (well-resolved alpha ribbon) [APO-RIBBON]
ComponentsRNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Lassa virus RNA-dependent RNA polymerase viral RNA
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: 3.73 Å
AuthorsKouba, T. / Vogel, D. / Thorkelsson, S. / Quemin, E. / Williams, H. / Milewski, M. / Busch, C. / Gunther, S. / Grunewald, K. / Rosenthal, M. / Cusack, S.
Funding support Germany, 2items
OrganizationGrant numberCountry
Leibniz AssociationK27/2017 Germany
German Research Foundation (DFG)INST 152/777-1 FUGG Germany
CitationJournal: Nat Commun / Year: 2021
Title: Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity.
Authors: Tomas Kouba / Dominik Vogel / Sigurdur R Thorkelsson / Emmanuelle R J Quemin / Harry M Williams / Morlin Milewski / Carola Busch / Stephan Günther / Kay Grünewald / Maria Rosenthal / Stephen Cusack /
Abstract: Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we ...Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.
History
DepositionMay 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _audit_author.name / _citation.country ..._audit_author.name / _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.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)253,7473
Polymers253,6571
Non-polymers902
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area120 Å2
ΔGint-9 kcal/mol
Surface area70230 Å2

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Components

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


Mass: 253656.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa mammarenavirus / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: A0A3S8NV63, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
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: RNA-directed RNA polymerase L Lassa mammarenavirus / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.253346 MDa / Experimental value: NO
Source (natural)Organism: Lassa mammarenavirus
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 49.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35400 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211322
ELECTRON MICROSCOPYf_angle_d0.4715269
ELECTRON MICROSCOPYf_dihedral_angle_d3.7171490
ELECTRON MICROSCOPYf_chiral_restr0.0381735
ELECTRON MICROSCOPYf_plane_restr0.0031928

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