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- EMDB-36624: The structure of EBOV L-VP35-RNA complex (conformation 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-36624
TitleThe structure of EBOV L-VP35-RNA complex (conformation 2)
Map data
Sample
  • Complex: The structure of EBOV L-VP35-RNA complex (conformation 2)
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Polymerase cofactor VP35
    • RNA: The leader sequence of EBOV genome
  • Ligand: ZINC ION
KeywordsEBOV / Polymerase / Replication / cryo-EM / VIRAL PROTEIN/RNA / 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-directed RNA polymerase L / Polymerase cofactor VP35
Similarity search - Component
Biological speciesEbola virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsQi P / Yi S
Funding support China, 1 items
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, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36624.png

Downloads & links

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Map

FileDownload / File: emd_36624.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.037974384 - 0.077960275
Average (Standard dev.)0.00005670045 (±0.0013226275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36624_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36624_half_map_2.map
Projections & Slices
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Sample components

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Entire : The structure of EBOV L-VP35-RNA complex (conformation 2)

EntireName: The structure of EBOV L-VP35-RNA complex (conformation 2)
Components
  • Complex: The structure of EBOV L-VP35-RNA complex (conformation 2)
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Polymerase cofactor VP35
    • RNA: The leader sequence of EBOV genome
  • Ligand: ZINC ION

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Supramolecule #1: The structure of EBOV L-VP35-RNA complex (conformation 2)

SupramoleculeName: The structure of EBOV L-VP35-RNA complex (conformation 2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Ebola virus

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Macromolecule #1: RNA-directed RNA polymerase L

MacromoleculeName: RNA-directed RNA polymerase L / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 252.863734 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString: MATQHTQYPD ARLSSPIVLD QCDLVTRACG LYSSYSLNPQ LRNCKLPKHI YRLKYDVTVT KFLSDVPVAT LPIDFIVPIL LKALSGNGF CPVEPRCQQF LDEIIKYTMQ DALFLKYYLK NVGAQEDCVD DHFQEKILSS IQGNEFLHQM FFWYDLAILT R RGRLNRGN ...String:
MATQHTQYPD ARLSSPIVLD QCDLVTRACG LYSSYSLNPQ LRNCKLPKHI YRLKYDVTVT KFLSDVPVAT LPIDFIVPIL LKALSGNGF CPVEPRCQQF LDEIIKYTMQ DALFLKYYLK NVGAQEDCVD DHFQEKILSS IQGNEFLHQM FFWYDLAILT R RGRLNRGN SRSTWFVHDD LIDILGYGDY VFWKIPISLL PLNTQGIPHA AMDWYQTSVF KEAVQGHTHI VSVSTADVLI MC KDLITCR FNTTLISKIA EVEDPVCSDY PNFKIVSMLY QSGDYLLSIL GSDGYKIIKF LEPLCLAKIQ LCSKYTERKG RFL TQMHLA VNHTLEEITE IRALKPSQAH KIREFHRTLI RLEMTPQQLC ELFSIQKHWG HPVLHSETAI QKVKKHATVL KALR PIVIF ETYCVFKYSI AKHYFDSQGS WYSVTSDRNL TPGLNSYIKR NQFPPLPMIK ELLWEFYHLD HPPLFSTKII SDLSI FIKD RATAVERTCW DAVFEPNVLG YNPPHKFSTK RVPEQFLEQE NFSIENVLSY AQKLEYLLPQ YRNFSFSLKE KELNVG RTF GKLPYPTRNV QTLCEALLAD GLAKAFPSNM MVVTEREQKE SLLHQASWHH TSDDFGEHAT VRGSSFVTDL EKYNLAF RY EFTAPFIEYC NRCYGVKNVF NWMHYTIPQC YMHVSDYYNP PHNLTLENRN NPPEGPSSYR GHMGGIEGLQ QKLWTSIS C AQISLVEIKT GFKLRSAVMG DNQCITVLSV FPLETDADEQ EQSAEDNAAR VAASLAKVTS ACGIFLKPDE TFVHSGFIY FGKKQYLNGV QLPQSLKTAT RMAPLSDAIF DDLQGTLASI GTAFERSISE TRHIFPCRIT AAFHTFFSVR ILQYHHLGFN KGFDLGQLT LGKPLDFGTI SLALAVPQVL GGLSFLNPEK CFYRNLGDPV TSGLFQLKTY LRMIEMDDLF LPLIAKNPGN C TAIDFVLN PSGLNVPGSQ DLTSFLRQIV RRTITLSAKN KLINTLFHAS ADFEDEMVCK WLLSSTPVMS RFAADIFSRT PS GKRLQIL GYLEGTRTLL ASKIINNNTE TPVLDRLRKI TLQRWSLWFS YLDHCDNILA EALTQITCTV DLAQILREYS WAH ILEGRP LIGATLPCMI EQFKVVWLKP YEQCPQCSNA KQPGGKPFVS VAVKKHIVSA WPNASRISWT IGDGIPYIGS RTED KIGQP AIKPKCPSAA LREAIELASR LTWVTQGSSN SDLLIKPFLE ARVNLSVQEI LQMTPSHYSG NIVHRYNDQY SPHSF MANR MSNSATRLIV STNTLGEFSG GGQSARDSNI IFQNVINYAV ALFDIKFRNT EATDIQYNRA HLHLTKCCTR EVPAQY LTY TSTLDLDLTR YRENELIYDN NPLKGGLNCN ISFDNPFFQG KQLNIIEDDL IRLPHLSGWE LAKTIMQSII SDSNNSS TD PISSGETRSF TTHFLTYPKI GLLYSFGAFV SYYLGNTILR TKKLTLDNFL YYLTTQIHNL PHRSLRILKP TFKHASVM S RLMSIDPHFS IYIGGAAGDR GLSDAARLFL RTSISSFLTF VKEWIINRGT IVPLWIVYPL EGQNPTPVNN FLHQIVELL VHDSSRHQAF KTTINDHVHP HDNLVYTCKS TASNFFHASL AYWRSRHRNS NRKDLTRNSS TGSSTNNSDG HIKRSQEQTT RDPHDGTER SLVLQMSHEI KRTTIPQENT HQGPSFQSFL SDSACGTANP KLNFDRSRHN VKSQDHNSAS KREGHQIISH R LVLPFFTL SQGTRQLTSS NESQTQDEIS KYLRQLRSVI DTTVYCRFTG IVSSMHYKLD EVLWEIENFK SAVTLAEGEG AG ALLLIQK YQVKTLFFNT LATESSIESE IVSGMTTPRM LLPVMSKFHN DQIEIILNNS ASQITDITNP TWFKDQRARL PRQ VEVITM DAETTENINR SKLYEAVHKL ILHHVDPSVL KAVVLKVFLS DTEGMLWLND NLAPFFATGY LIKPITSSAR SSEW YLCLT NFLSTTRKMP HQNHLSCKQV ILTALQLQIQ RSPYWLSHLT QYADCDLHLS YIRLGFPSLE KVLYHRYNLV DSKRG PLVS VTQHLAHLRA EIRELTNDYN QQRQSRTQTY HFIRTAKGRI TKLVNDYLKF FLIVQALKHN GTWQAEFKKL PELISV CNR FYHIRDCNCE ERFLVQTLYL HRMQDSEVKL IERLTGLLSL FPDGLYRFD

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: Polymerase cofactor VP35

MacromoleculeName: Polymerase cofactor VP35 / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 37.48943 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString: MTTRTKGRGH TVATTQNDRM PGPELSGWIS EQLMTGRIPV NDIFCDIENN PGLCYASQMQ QTKPNPKMRN SQTQTDPICN HSFEEVVQT LASLATVVQQ QTIASESLEQ RITSLENGLK PVYDMAKTIS SLNRVCAEMV AKYDLLVMTT GRATATAAAT E AYWAEHGQ ...String:
MTTRTKGRGH TVATTQNDRM PGPELSGWIS EQLMTGRIPV NDIFCDIENN PGLCYASQMQ QTKPNPKMRN SQTQTDPICN HSFEEVVQT LASLATVVQQ QTIASESLEQ RITSLENGLK PVYDMAKTIS SLNRVCAEMV AKYDLLVMTT GRATATAAAT E AYWAEHGQ PPPGPSLYEE SAIRGKIESR DETVPQSVRE AFNNLDSTTS LTEENFGKPD ISAKDLRNIM YDHLPGFGTA FH QLVQVIC KLGKDSNSLD IIHAEFQASL AEGDSPQCAL IQITKRVPIF QDAAPPVIHI RSRGDIPRAC QKSLRPVPPS PKI DRGWVC VFQLQDGKTL GLKI

UniProtKB: Polymerase cofactor VP35

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Macromolecule #3: The leader sequence of EBOV genome

MacromoleculeName: The leader sequence of EBOV genome / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 5.619235 KDa
SequenceString:
UUUCUUUUUG UGUGUCCG

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

33775

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 183964
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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