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- EMDB-39852: Cryo-EM structure of Thogoto virus polymerase in transcription in... -

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Entry
Database: EMDB / ID: EMD-39852
TitleCryo-EM structure of Thogoto virus polymerase in transcription initiation conformation 2
Map data
Sample
  • Complex: Cryo-EM structure of Thogoto virus polymerase in transcription initiation conformation 2
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: RNA (5'-R(*AP*GP*AP*GP*AP*AP*AP*UP*CP*AP*AP*GP*GP*CP*AP*GP*UP*U)-3')
    • RNA: RNA (5'-R(*GP*AP*CP*UP*GP*CP*CP*UP*GP*UP*UP*UP*UP*UP*GP*CP*U)-3')
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
KeywordsRNA polymerase / TRANSCRIPTION
Function / homology
Function and homology information


cap snatching / 7-methylguanosine mRNA capping / virion component / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / host cell nucleus / RNA binding
Similarity search - Function
: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain ...: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein / Polymerase basic protein 2
Similarity search - Component
Biological speciesThogoto virus (isolate SiAr 126)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsXue L / Chang T / Li Z / Zhao H / Li M / He J / Chen X / Xiong X
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82341085 to X.X. China
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses.
Authors: Lu Xue / Tiancai Chang / Zimu Li / Chenchen Wang / Heyu Zhao / Mei Li / Peng Tang / Xin Wen / Mengmeng Yu / Jiqin Wu / Xichen Bao / Xiaojun Wang / Peng Gong / Jun He / Xinwen Chen / Xiaoli Xiong /
Abstract: Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV ...Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5' end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses' broad-infectivity range.
History
DepositionApr 22, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_39852.png

Downloads & links

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Map

FileDownload / File: emd_39852.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 208 pix.
= 186.88 Å		0.9 Å/pix.
x 208 pix.
= 186.88 Å		0.9 Å/pix.
x 208 pix.
= 186.88 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.89846 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0131
Minimum - Maximum-0.041340243 - 0.08392905
Average (Standard dev.)0.00009754173 (±0.003494409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 186.87997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39852_msk_1.map
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Half map: #1

Fileemd_39852_half_map_1.map
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Half map: #2

Fileemd_39852_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of Thogoto virus polymerase in transcription in...

EntireName: Cryo-EM structure of Thogoto virus polymerase in transcription initiation conformation 2
Components
  • Complex: Cryo-EM structure of Thogoto virus polymerase in transcription initiation conformation 2
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: RNA (5'-R(*AP*GP*AP*GP*AP*AP*AP*UP*CP*AP*AP*GP*GP*CP*AP*GP*UP*U)-3')
    • RNA: RNA (5'-R(*GP*AP*CP*UP*GP*CP*CP*UP*GP*UP*UP*UP*UP*UP*GP*CP*U)-3')
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

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Supramolecule #1: Cryo-EM structure of Thogoto virus polymerase in transcription in...

SupramoleculeName: Cryo-EM structure of Thogoto virus polymerase in transcription initiation conformation 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Thogoto virus (isolate SiAr 126)

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Macromolecule #1: Polymerase acidic protein

MacromoleculeName: Polymerase acidic protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Molecular weightTheoretical: 71.623445 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTDRPDHIDS RVWELSETQE DWITQVHGHV RRVVECWKYT ICCLISNMHT HRGAPQYDVF KWQDRSTIEW ICSKKKVQYP ERDTPDLYD NERAVAYKVL LVSDLSDHSP TSGIYHDLAF NLEGEAEESC ALVLRGSQLQ DIKGFLCRAL EWVVSNNLTQ E VVETISGE ...String:
MTDRPDHIDS RVWELSETQE DWITQVHGHV RRVVECWKYT ICCLISNMHT HRGAPQYDVF KWQDRSTIEW ICSKKKVQYP ERDTPDLYD NERAVAYKVL LVSDLSDHSP TSGIYHDLAF NLEGEAEESC ALVLRGSQLQ DIKGFLCRAL EWVVSNNLTQ E VVETISGE AKLQFSVGTT FRTLLKRDTD WDVIPTPRVE PNVPRIEGRR WTQMKKLPLL KEKEGPPSPW RALLLGADSE YI VCPPGTD QEAISWIHSQ SEIECIRESK STPASVITCL TSSLQSFAEG NPVRSRIHED IIAFGINKKQ EKKQSASSSA SGE WKRAEY QVEEMSLPPW VEEEMVLLRS DQEDNWIELE KNAIYTEVDG VAEGLVDKYI EIVGRTKVAS VIEKWQIAAT RTFS QLHTD RSRITACPII TRDPSGNCQF WGMVLLGPHH VKRDTDNAPL LIAEIMGEDT EEKYPKHSVF SLKVEEKQFL LSLKI TSFS RNKLYTFSNI RRVLIQPASI YSQVVLSRAA ENNSLNLEVN PEIQLYLEGA QRGMTLYQWV RMILCLEFLM AIYNNP QME GFLANMRRLH MSRHAMMERR QVFLPFGSRP EDKVNECIIN NPIVAYLAKG WNSMPNVYY

UniProtKB: Polymerase acidic protein

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Macromolecule #2: RNA-directed RNA polymerase catalytic subunit

MacromoleculeName: RNA-directed RNA polymerase catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Molecular weightTheoretical: 81.432664 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNLFTPLSEI NPTTTQELLY AYTGPAPVAY GTRTRAVLEN IIRPYQYFYK EPNVQRALDI KTGCKEPEDI NVEGPSSGFH TASVLKLAD NFFRKYRPAM EKLKYWILVK LPKLKYAELS KGRQTYSFIH KRNLPAPIAL EETVEFLEQN LRRKIGPTLL S YCQAIADV ...String:
MNLFTPLSEI NPTTTQELLY AYTGPAPVAY GTRTRAVLEN IIRPYQYFYK EPNVQRALDI KTGCKEPEDI NVEGPSSGFH TASVLKLAD NFFRKYRPAM EKLKYWILVK LPKLKYAELS KGRQTYSFIH KRNLPAPIAL EETVEFLEQN LRRKIGPTLL S YCQAIADV MELDETTYEG ARDPRPWDIQ LEEIDSDEED PLFRQVGREE TYTIKFSREE LWDQMRTLNT MWKHLERGRL NR RTIATPS MLIRGFVKIV EDAAKEILEN VPTSGVPVGG EEKLAKLASK QTFHTAVTGE LSGDQEKFNE CLDPDAMRLM WTV FLRKLG CPDWIMELFN IPFMVFKSKL ADMGEGLVYT KGKLTDRKPL GEMPSEFDDL VRNVVGNSIS CRLGMFMGMY NLTS TLLAL ISIEREELTG SHVESSDDFI HFFNCKTHEE MFKQAETLRL TLKLVGINMS PSKCILISPA GIGEFNSKFH HRDFV GNVA TELPALVPNG TNPMTDLAMG LNVIKHSVNT GQMNLCTGAL AMRIFNHAYK YAYMALGVTR RTRFMEENAI TPLLTN QGA SPVHSFSTMH LDEVALRRHL GLLDEETLRR ILNPNNPVTQ KGDPSMFFRI ENKMPQIMED YSVPSCFKYT LSRNRTI QD KPHKALLNKE ERYQRVTSII NKLFPEVLIQ EASAPGTVRE SLKRRLELVV ERSDLDEERK KRILSRIF

UniProtKB: RNA-directed RNA polymerase catalytic subunit

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Macromolecule #3: Polymerase basic protein 2

MacromoleculeName: Polymerase basic protein 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Molecular weightTheoretical: 94.418578 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDREEPAESE CTLRALVEEY NGACKEAPKE MSKQFTDYNT FKRYTTSKKD HAPQMRLVYS VRKPWPISMT PSKEIPLVFN GTKLKDTIL DLGESKRTRA NIVVPDYWSK YGSQTSLEVV NAILYAEDLK VQRFFSTEWG EIRYGRMLPF RKPVQACPTI E EVNPASIP ...String:
MDREEPAESE CTLRALVEEY NGACKEAPKE MSKQFTDYNT FKRYTTSKKD HAPQMRLVYS VRKPWPISMT PSKEIPLVFN GTKLKDTIL DLGESKRTRA NIVVPDYWSK YGSQTSLEVV NAILYAEDLK VQRFFSTEWG EIRYGRMLPF RKPVQACPTI E EVNPASIP HTLLQVFCPQ YTTLDSKRKA HMGAVEKLKR VMEPICKVQT QESAVHIARS LIDSNKKWLP TVVDHTPRTA EM AHFLCSK YHYVHTNTQD LSDTRSIDNL CGELVKRSLK CRCPKETLVA NLDKITIQGR PMREVLADHD GELPYLGICR VAM GLSTHH TMKIRSTKFS ILNSDHPRIE VKKVFSLSPD VQVTIPYRRF KGKAKVYFQN DQIQGYFSCT DRQIDEIKIS APKN APLLE PLLDICYYGS FIEPGFEQTF GFYPAGKREF VDSFFMHHSK DHKAFLIHMG LDKDLSLPLS PELNWKEPAL SKVCR VTEL DSTVQPYTSA TREFVLGETL NVYTQHENGL ELLICPTEIR STRGPLPPGT NLSGSEFIDI YQDPFSRAKS LLKSTI LHA ERCKEFVGNM LEEYQDPAET TVQSLVPINT WGKSAKRKLQ EEITSDPDWH QCPRKRAKMS YLAIIAGSIQ DRDKKQT NV PRAFMLRGSQ IEYDMKATRG LVVDTTNRII VGGETVLREG KGGPEGYVQT GVFEEQPRCY LVDTPDHGLS MGLSRFCV H SQGRYFQYEK KISIWEETDN IKATIDSQRD LKRRRDIEEM VSKRARIVLE VLFQGPGHHH HHHHHSADYK DDDDKGGWS HPQFEKGGGS GGGGSGGSAW SHPQFEK

UniProtKB: Polymerase basic protein 2

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Macromolecule #4: RNA (5'-R(*AP*GP*AP*GP*AP*AP*AP*UP*CP*AP*AP*GP*GP*CP*AP*GP*UP*U)-3')

MacromoleculeName: RNA (5'-R(*AP*GP*AP*GP*AP*AP*AP*UP*CP*AP*AP*GP*GP*CP*AP*GP*UP*U)-3')
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Molecular weightTheoretical: 5.84358 KDa
SequenceString:
AGAGAAAUCA AGGCAGUU

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Macromolecule #5: RNA (5'-R(*GP*AP*CP*UP*GP*CP*CP*UP*GP*UP*UP*UP*UP*UP*GP*CP*U)-3')

MacromoleculeName: RNA (5'-R(*GP*AP*CP*UP*GP*CP*CP*UP*GP*UP*UP*UP*UP*UP*GP*CP*U)-3')
type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Thogoto virus (isolate SiAr 126)
Molecular weightTheoretical: 5.335125 KDa
SequenceString:
GACUGCCUGU UUUUGCU

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Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 132762
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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