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- PDB-8jsn: The structure of EBOV L-VP35-RNA complex (conformation 2) -

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Basic information

Entry
Database: PDB / ID: 8jsn
TitleThe structure of EBOV L-VP35-RNA complex (conformation 2)
Components
  • Polymerase cofactor VP35
  • RNA-directed RNA polymerase L
  • The leader sequence of EBOV genome
KeywordsVIRAL PROTEIN/RNA / EBOV / Polymerase / Replication / cryo-EM / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


GDP polyribonucleotidyltransferase / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / symbiont-mediated suppression of host toll-like receptor signaling pathway / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm ...GDP polyribonucleotidyltransferase / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / symbiont-mediated suppression of host toll-like receptor signaling pathway / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / cytoplasm
Similarity search - Function
RNA-directed RNA polymerase L, filovirus / Filoviruses VP35 interferon inhibitory domain, beta-sheet subdomain / Filoviridae VP35 protein / Filoviruses VP35 interferon inhibitory domain / Filoviruses VP35 interferon inhibitory domain, helical subdomain / Filoviridae VP35 / Filoviruses VP35 interferon inhibitory domain profile. / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V ...RNA-directed RNA polymerase L, filovirus / Filoviruses VP35 interferon inhibitory domain, beta-sheet subdomain / Filoviridae VP35 protein / Filoviruses VP35 interferon inhibitory domain / Filoviruses VP35 interferon inhibitory domain, helical subdomain / Filoviridae VP35 / Filoviruses VP35 interferon inhibitory domain profile. / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA-directed RNA polymerase L / Polymerase cofactor VP35
Similarity search - Component
Biological speciesEbola virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsQi, P. / Yi, S.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32192452 China
CitationJournal: Nature / Year: 2023
Title: Molecular mechanism of de novo replication by the Ebola virus polymerase.
Authors: Qi Peng / Bin Yuan / Jinlong Cheng / Min Wang / Siwei Gao / Suran Bai / Xuejin Zhao / Jianxun Qi / George F Gao / Yi Shi /
Abstract: Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and ...Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide. Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We present the high-resolution structures of the EBOV L-VP35-RNA complex and show that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Using mutagenesis assays, we confirm that the bend conformation of the RNA is required for the de novo replication activity and reveal the key residues of the L protein that stabilize the RNA conformation. These findings provide a new mechanistic understanding of RNA synthesis for polymerases of non-segmented negative-strand RNA viruses, and reveal important targets for the development of antiviral drugs.
History
DepositionJun 20, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: 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

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: Polymerase cofactor VP35
C: Polymerase cofactor VP35
D: Polymerase cofactor VP35
E: Polymerase cofactor VP35
G: The leader sequence of EBOV genome
hetero molecules


Theoretical massNumber of molelcules
Total (without water)408,5067
Polymers408,4416
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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: 252863.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus / Gene: L / Production host: Spodoptera (butterflies/moths)
References: UniProt: A0A1C4HDB0, RNA-directed RNA polymerase, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides, GDP polyribonucleotidyltransferase, Transferases; ...References: UniProt: A0A1C4HDB0, RNA-directed RNA polymerase, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides, GDP polyribonucleotidyltransferase, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein
Polymerase cofactor VP35


Mass: 37489.430 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus / Gene: VP35 / Production host: Spodoptera (butterflies/moths) / References: UniProt: A0A1C4HDK9
#3: RNA chain The leader sequence of EBOV genome


Mass: 5619.235 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Ebola virus
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / 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: The structure of EBOV L-VP35-RNA complex (conformation 2)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Ebola virus
Source (recombinant)Organism: Spodoptera (butterflies/moths)
Buffer solutionpH: 7.8
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 FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 183964 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315209
ELECTRON MICROSCOPYf_angle_d0.57820719
ELECTRON MICROSCOPYf_dihedral_angle_d15.0045608
ELECTRON MICROSCOPYf_chiral_restr0.0392383
ELECTRON MICROSCOPYf_plane_restr0.0042616

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