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- EMDB-16006: Early transcription elongation state of influenza A/H7N9 backtrac... -

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Basic information

Entry
Database: EMDB / ID: EMD-16006
TitleEarly transcription elongation state of influenza A/H7N9 backtracked polymerase with singly incoporated T1106 at the U +1 position
Map dataLoc-Scale filtered
Sample
  • Complex: A/H7N9-Backtracked-T/U+1 complex with only single incorporation
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 3' vRNA
    • RNA: 5' vRNA
  • RNA: mRNA
  • Ligand: MAGNESIUM ION
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE
  • Ligand: water
KeywordsInfluenza / viral RNA-dependent RNA polymerase / antiviral drug / nucleoside analogue / T705 (favipiravir) / T1106 / cap-dependent transcription / backtracking / VIRAL PROTEIN
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding / cytoplasm
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain ...Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Polymerase acidic protein / 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.
Similarity search - Domain/homology
Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9)) / Influenza A virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsCusack S / Kouba T
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research Agency France
CitationJournal: Cell Rep / Year: 2023
Title: Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106.
Authors: Tomas Kouba / Anna Dubankova / Petra Drncova / Elisa Donati / Pietro Vidossich / Valentina Speranzini / Alex Pflug / Johanna Huchting / Chris Meier / Marco De Vivo / Stephen Cusack /
Abstract: The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although ...The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.
History
DepositionOct 21, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16006.png

Downloads & links

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Map

FileDownload / File: emd_16006.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLoc-Scale filtered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 300.096 Å		0.83 Å/pix.
x 360 pix.
= 300.096 Å		0.83 Å/pix.
x 360 pix.
= 300.096 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8336 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.24866463 - 0.7572277
Average (Standard dev.)0.0019061501 (±0.021899123)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 300.096 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Relion post-processed

Fileemd_16006_additional_1.map
AnnotationRelion post-processed
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map

Fileemd_16006_half_map_1.map
AnnotationHalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map

Fileemd_16006_half_map_2.map
AnnotationHalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Sample components

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Entire : A/H7N9-Backtracked-T/U+1 complex with only single incorporation

EntireName: A/H7N9-Backtracked-T/U+1 complex with only single incorporation
Components
  • Complex: A/H7N9-Backtracked-T/U+1 complex with only single incorporation
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 3' vRNA
    • RNA: 5' vRNA
  • RNA: mRNA
  • Ligand: MAGNESIUM ION
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: A/H7N9-Backtracked-T/U+1 complex with only single incorporation

SupramoleculeName: A/H7N9-Backtracked-T/U+1 complex with only single incorporation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #5-#6
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 255 KDa

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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 / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Strain: A/Zhejiang/DTID-ZJU01/2013(H7N9)
Molecular weightTheoretical: 82.930602 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GMEDFVRQCF NPMIVELAEK AMKEYGEDPK IETNKFASIC THLEVCFMYS DFHFIDERGE STIIESGDPN VLLKHRFEII EGRDRTMAW TVVNSICNTT GVEKPKFLPD LYDYKENRFI EIGVTRREVH IYYLEKANKI KSEKTHIHIF SFTGEEMATK A DYTLDEES ...String:
GMEDFVRQCF NPMIVELAEK AMKEYGEDPK IETNKFASIC THLEVCFMYS DFHFIDERGE STIIESGDPN VLLKHRFEII EGRDRTMAW TVVNSICNTT GVEKPKFLPD LYDYKENRFI EIGVTRREVH IYYLEKANKI KSEKTHIHIF SFTGEEMATK A DYTLDEES RARIKTRLFT IRQEMASRGL WDSFRQSERG EETIEERFEI TGTMRRLADQ SLPPNFSSLE NFRAYVDGFE PN GCIEGKL SQMSKEVNAR IEPFLRTTPR PLRLPDGPPC SQRSKFLLMD ALKLSIEDPS HEGEGIPLYD AIKCMKTFFG WKE PNIIKP HEKGINPNYL LTWKQVLAEL QDIENEEKIP RTKNMKKTSQ LKWALGENMA PEKVDFEDCK DVNDLKQYDS DEPE PRSLA CWIQSEFNKA CELTDSSWVE LDEIGEDVAP IEHIASMRRN YFTAEVSHCR ATEYIMKGVY INTALLNASC AAMDD FQLI PMISKCRTKE GRRKTNLYGF IIKGRSHLRN DTDVVNFVSM EFSLTDPRLE PHKWEKYCVL EIGDMLLRTA VGQVSR PMF LYVRTNGTSK IKMKWGMEMR RCLLQSLQQI ESMIEAESSV KEKDLTKEFF ENKSETWPIG ESPKGVEEGS IGKVCRT LL AKSVFNSLYA SPQLEGFSAE SRKLLLIVQA LRDNLEPGTF DLEGLYEAIE ECLINDPWVL LNASWFNSFL THALR

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: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 86.496156 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDVNPTLLFL KVPVQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHKYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEIVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK ...String:
MDVNPTLLFL KVPVQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHKYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEIVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK DVMDSMDKEE MEITTHFQRK RRVRDNMTKK MVTQRTIGKK KQRLNKRSYL IRALTLNTMT KDAERGKLKR RA IATPGMQ IRGFVYFVEA LARSICEKLE QSGLPVGGNE KKAKLANVVR KMMTNSQDTE LSFTITGDNT KWNENQNPRM FLA MITYIT RNQPEWFRNV LSIAPIMFSN KMARLGKGYM FESKSMKLRT QVPAEMLANI DLKYFNKSTR EKIEKIRPLL IDGT ASLSP GMMMGMFNML STVLGVSILN LGQKKYTKTT YWWDGLQSSD DFALIVNAPN HEGIQAGVDR FYRTCKLVGI NMSKK KSYI NRTGTFEFTS FFYRYGFVAN FSMELPSFGV SGINESADMS VGVTVIKNNM INNDLGPATA QMALQLFIKD YRYTYR CHR GDTQIQTRRA FELKKLWEQT RSKAGLLVSD GGPNLYNIRN LHIPEVCLKW ELMDEDYQGR LCNPMNPFVS HKEIDSV NN AVVMPAHGPA KSMEYDAVAT THSWIPKRNR SILNTSQRGI LEDEQMYQKC CNLFEKFFPS SSYRRPVGIS SMVEAMVS R ARIDARIDFE SGRIKKEEFA EIMKICSTIE ELRRQK

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: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Strain: A/Zhejiang/DTID-ZJU01/2013(H7N9)
Molecular weightTheoretical: 86.00732 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF ...String:
MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF PNEVGARILT SESQLTITKE KKKELQDCKI APLMVAYMLE RELVRKTRFL PVAGGTSSVY IEVLHLTQGT CW EQMYTPG GEVRNDDVDQ SLIIAARNIV RRATVSADPL ASLLEMCHST QIGGVRMVDI LRQNPTEEQA VDICKAAMGL RIS SSFSFG GFTFKRTSGS SVKREEEVLT GNLQTLKIRV HEGYEEFTMV GRRATAILRK ATRRLIQLIV SGKDEQSIAE AIIV AMVFS QEDCMIKAVR GDLNFVNRAN QRLNPMHQLL RHFQKDAKVL FQNWGIEPID NVMGMIGILP DMTPSTEMSL RGVRV SKMG VDEYSSTERV VVSIDRFLRV RDQRGNVLLS PEEVSETQGT EKLTITYSSS MMWEINGPES VLVNTYQWII RNWENV KIQ WSQDPTMLYN KMEFEPFQSL VPKAARGQYS GFVRVLFQQM RDVLGTFDTV QIIKLLPFAA APPEQSRMQF SSLTVNV RG SGMRIVVRGN SPVFNYNKAT KRLTVLGKDA GALMEDPDEG TAGVESAVLR GFLILGKENK RYGPALSINE LSNLAKGE K ANVLIGQGDV VLVMKRKRDS SILTDSQTAT KRIRMAIN

UniProtKB: Polymerase basic protein 2

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Macromolecule #4: mRNA

MacromoleculeName: mRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 5.340144 KDa
SequenceString:
AUCUAUAAUA GUUU(K1F)UU

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Macromolecule #5: 3' vRNA

MacromoleculeName: 3' vRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 6.603949 KDa
SequenceString:
UAUACCUCUG AAUAAACUAU U

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Macromolecule #6: 5' vRNA

MacromoleculeName: 5' vRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 4.55782 KDa
SequenceString:
AGUAGUAACA AGAG

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE

MacromoleculeName: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: GTA
Molecular weightTheoretical: 787.441 Da
Chemical component information

ChemComp-GTA:
P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 102 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6t0v

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99386
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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