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- PDB-9ip3: Cryo-EM structure of the RNA-dependent RNA polymerase complex in ... -

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Basic information

Entry
Database: PDB / ID: 9ip3
TitleCryo-EM structure of the RNA-dependent RNA polymerase complex in a compact conformation from Ebola virus
Components
  • Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35
  • Maltose/maltodextrin-binding periplasmic protein,RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / RNA-dependent RNA polymerase complex
Function / homology
Function and homology information


RNA polymerase activity / negative stranded viral RNA transcription / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / NNS virus cap methyltransferase / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / GDP polyribonucleotidyltransferase / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II ...RNA polymerase activity / negative stranded viral RNA transcription / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / NNS virus cap methyltransferase / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / GDP polyribonucleotidyltransferase / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / positive regulation of protein sumoylation / negative stranded viral RNA replication / detection of maltose stimulus / maltose transport complex / carbohydrate transport / viral transcription / molecular sequestering activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / viral genome replication / cell chemotaxis / virion component / outer membrane-bounded periplasmic space / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / viral nucleocapsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / periplasmic space / symbiont-mediated suppression of host innate immune response / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / negative regulation of gene expression / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / GTPase activity / DNA damage response / RNA binding / ATP binding / membrane
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. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Maltose/maltodextrin-binding periplasmic protein / Polymerase cofactor VP35 / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Ebola virus - Eckron (Zaire
1976)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLi, G. / Du, T. / Wang, J. / Wu, S. / Ru, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32371344 China
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into the RNA-dependent RNA polymerase complexes from highly pathogenic Marburg and Ebola viruses.
Authors: Guobao Li / Tianjiao Du / Jiening Wang / Kaiyue Jie / Zhuolu Ren / Xiaokang Zhang / Long Zhang / Shan Wu / Heng Ru /
Abstract: The Ebola and the Marburg viruses belong to the Filoviridae family, a group of filamentous, single-stranded, negative-sensed RNA viruses. Upon infection, uncontrolled propagation of the Ebola and the ...The Ebola and the Marburg viruses belong to the Filoviridae family, a group of filamentous, single-stranded, negative-sensed RNA viruses. Upon infection, uncontrolled propagation of the Ebola and the Marburg viruses causes severe hemorrhagic fevers with high mortality rates. The replication and transcription of viral genomes are mediated by a polymerase complex consisting of two proteins: L and its cofactor VP35. However, the molecular mechanism of filovirus RNA synthesis remains understudied due to the lack of high-resolution structures of L and VP35 complexes from these viruses. Here, we present the cryo-EM structures of the polymerase complexes for the Marburg virus and the Ebola virus at 2.7 Å and 3.1 Å resolutions respectively. Despite the similar assembly and overall structures between these two viruses, we identify virus-specific L-VP35 interactions. Our data show that intergeneric exchange of VP35 would diminish these interactions and prevent the formation of a functional chimeric polymerase complex between L protein and heterologous VP35. Additionally, we identify a contracted conformation of the Ebola virus polymerase structure, revealing the structural dynamics of the polymerase during RNA synthesis. These insights enhance our understanding of filovirus RNA synthesis mechanisms and may facilitate the development of antiviral drugs targeting filovirus polymerase.
History
DepositionJul 10, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Maltose/maltodextrin-binding periplasmic protein,RNA-directed RNA polymerase L
B: Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35
C: Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35
D: Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35
E: Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)496,1626
Polymers496,0975
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 Maltose/maltodextrin-binding periplasmic protein,RNA-directed RNA polymerase L / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Protein L / Large structural ...MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 207335.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Sequence reference for Ebola virus - Eckron (Zaire, 1976) (12900) is not available in UniProt at the time of biocuration. Current sequence reference is from UniProt ID Q05318.
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Ebola virus - Eckron (Zaire, 1976)
Gene: malE, b4034, JW3994 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P0AEX9, UniProt: Q05318, RNA-directed RNA polymerase, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides, GDP polyribonucleotidyltransferase, NNS virus cap methyltransferase
#2: Protein
Maltose/maltodextrin-binding periplasmic protein,Polymerase cofactor VP35 / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Ebola VP35 / eVP35


Mass: 72190.336 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Sequence reference for Ebola virus - Eckron (Zaire, 1976) (12900) is not available in UniProt at the time of biocuration. Current sequence reference is from UniProt ID Q05127.
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Ebola virus - Eckron (Zaire, 1976)
Gene: malE, b4034, JW3994, VP35 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0AEX9, UniProt: Q05127
#3: 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
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

ComponentName: Ternary complex of L-VP35 core region from Ebola virus
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Ebola virus - Eckron (Zaire, 1976)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5 / Details: 25 mM HEPES, 300 mM NaCl, 1 mM TCEP, 6 mM MgCl2
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample is monodisperse.
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77587 / Symmetry type: POINT
RefinementResolution: 3.1→238.08 Å / SU ML: 0.31 / σ(F): 0.72 / Phase error: 31.36 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
obs0.2456 948510 99.97 %
Rfree-2064 22 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01313782
ELECTRON MICROSCOPYf_angle_d1.21518711
ELECTRON MICROSCOPYf_dihedral_angle_d9.9741836
ELECTRON MICROSCOPYf_chiral_restr0.0652109
ELECTRON MICROSCOPYf_plane_restr0.0062391
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.170.41571440.430563948ELECTRON MICROSCOPY100
3.17-3.250.35741320.407362374ELECTRON MICROSCOPY100
3.25-3.340.41741200.382762764ELECTRON MICROSCOPY100
3.34-3.440.35021680.36863614ELECTRON MICROSCOPY100
3.44-3.550.34951200.336662932ELECTRON MICROSCOPY100
3.55-3.680.33081200.316463265ELECTRON MICROSCOPY100
3.68-3.820.25561200.288862924ELECTRON MICROSCOPY100
3.82-40.24221080.250563289ELECTRON MICROSCOPY100
4-4.210.22041680.221663197ELECTRON MICROSCOPY100
4.21-4.470.21781200.202862843ELECTRON MICROSCOPY100
4.47-4.820.2011680.189663182ELECTRON MICROSCOPY100
4.82-5.30.20651200.190963321ELECTRON MICROSCOPY100
5.3-6.070.21551440.211462871ELECTRON MICROSCOPY100
6.07-7.640.2111440.217463205ELECTRON MICROSCOPY100
7.65-238.080.18161680.175362717ELECTRON MICROSCOPY99

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