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- EMDB-14240: Early transcription elongation state of influenza B polymerase ba... -

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Basic information

Entry
Database: EMDB / ID: EMD-14240
TitleEarly transcription elongation state of influenza B polymerase backtracked due to double incoproation of nucleotide analogue T1106
Map dataLocScale filtered
Sample
  • Complex: B/Mem-BACKTRACKED
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 5' vRNA
    • RNA: 3' vRNA
    • RNA: mRNAMessenger RNA
  • Ligand: MAGNESIUM ION
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE
  • Ligand: water
Function / homology
Function and homology information


symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / host cell mitochondrion / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase ...symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / host cell mitochondrion / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / : / : / : / Influenza RNA polymerase PB2 N-terminal region ...Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / : / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / Influenza RNA polymerase PB2 middle domain / Influenza RNA polymerase PB2 6th domain / Influenza RNA polymerase PB2 C-terminal domain / : / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA polymerase PB2 CAP binding domain / Polymerase acidic protein / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA
Similarity search - Domain/homology
Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza B virus (B/Memphis/13/2003) / Influenza B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.58 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
DepositionFeb 2, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateFeb 15, 2023-
Current statusFeb 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14240.png

Downloads & links

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Map

FileDownload / File: emd_14240.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocScale filtered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 440 pix.
= 361.68 Å		0.82 Å/pix.
x 440 pix.
= 361.68 Å		0.82 Å/pix.
x 440 pix.
= 361.68 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.17675674 - 0.7118502
Average (Standard dev.)0.0010105596 (±0.015078549)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 361.68002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: RELION post-processed

Fileemd_14240_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: #1

Fileemd_14240_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14240_half_map_2.map
Projections & Slices
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Sample components

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Entire : B/Mem-BACKTRACKED

EntireName: B/Mem-BACKTRACKED
Components
  • Complex: B/Mem-BACKTRACKED
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 5' vRNA
    • RNA: 3' vRNA
    • RNA: mRNAMessenger RNA
  • Ligand: MAGNESIUM ION
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: B/Mem-BACKTRACKED

SupramoleculeName: B/Mem-BACKTRACKED / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Influenza B virus (B/Memphis/13/2003)
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 B virus (B/Memphis/13/2003) / Strain: B/Memphis/13/2003
Molecular weightTheoretical: 85.822781 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSHHHHHHHH GSGSMDTFIT RNFQTTIIQK AKNTMAEFSE DPELQPAMLF NICVHLEVCY VISDMNFLDE EGKAYTALEG QGKEQNLRP QYEVIEGMPR TIAWMVQRSL AQEHGIETPK YLADLFDYKT KRFIEVGITK GLADDYFWKK KEKLGNSMEL M IFSYNQDY ...String:
GSHHHHHHHH GSGSMDTFIT RNFQTTIIQK AKNTMAEFSE DPELQPAMLF NICVHLEVCY VISDMNFLDE EGKAYTALEG QGKEQNLRP QYEVIEGMPR TIAWMVQRSL AQEHGIETPK YLADLFDYKT KRFIEVGITK GLADDYFWKK KEKLGNSMEL M IFSYNQDY SLSNESSLDE EGKGRVLSRL TELQAELSLK NLWQVLIGEE DVEKGIDFKL GQTISRLRDI SVPAGFSNFE GM RSYIDNI DPKGAIERNL ARMSPLVSVT PKKLTWEDLR PIGPHIYNHE LPEVPYNAFL LMSDELGLAN MTEGKSKKPK TLA KECLEK YSTLRDQTDP ILIMKSEKAN ENFLWKLWRD CVNTISNEEM SNELQKTNYA KWATGDGLTY QKIMKEVAID DETM CQEEP KIPNKCRVAA WVQTEMNLLS TLTSKRALDL PEIGPDVAPV EHVGSERRKY FVNEINYCKA STVMMKYVLF HTSLL NESN ASMGKYKVIP ITNRVVNEKG ESFDMLYGLA VKGQSHLRGD TDVVTVVTFE FSSTDPRVDS GKWPKYTVFR IGSLFV SGR EKSVYLYCRV NGTNKIQMKW GMEARRCLLQ SMQQMEAIVE QESSIQGYDM TKACFKGDRV NSPKTFSIGT QEGKLVK GS FGKALRVIFT KCLMHYVFGN AQLEGFSAES RRLLLLIQAL KDRKGPWVFD LEGMYSGIEE CISNNPWVIQ SAYWFNEW L GFEKEGSKVL ESVDEIMDEG SGSGENLYFQ

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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 B virus (B/Memphis/13/2003)
Molecular weightTheoretical: 86.207016 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSGSGSGSGM NINPYFLFID VPIQAAISTT FPYTGVPPYS HGTGTGYTID TVIRTHEYSN KGKQYISDVT GCTMVDPTNG PLPEDNEPS AYAQLDCVLE ALDRMDEEHP GLFQAASQNA METLMVTTVD KLTQGRQTFD WTVCRNQPAA TALNTTITSF R LNDLNGAD ...String:
GSGSGSGSGM NINPYFLFID VPIQAAISTT FPYTGVPPYS HGTGTGYTID TVIRTHEYSN KGKQYISDVT GCTMVDPTNG PLPEDNEPS AYAQLDCVLE ALDRMDEEHP GLFQAASQNA METLMVTTVD KLTQGRQTFD WTVCRNQPAA TALNTTITSF R LNDLNGAD KGGLIPFCQD IIDSLDRPEM TFFSVKNIKK KLPAKNRKGF LIKRIPMKVK DKITKVEYIK RALSLNTMTK DA ERGKLKR RAIATAGIQI RGFVLVVENL AKNICENLEQ SGLPVGGNEK KAKLSNAVAK MLSNCPPGGI SMTVTGDNTK WNE CLNPRI FLAMTERITR DSPIWFRDFC SIAPVLFSNK IARLGKGFMI TSKTKRLKAQ IPCPDLFSIP LERYNEETRA KLKK LKPFF NEEGTASLSP GMMMGMFNML STVLGVAALG IKNIGNKEYL WDGLQSSDDF ALFVNAKDEE TCMEGINDFY RTCKL LGIN MSKKKSYCNE TGMFEFTSMF YRDGFVSNFA MELPSFGVAG VNESADMAIG MTIIKNNMIN NGMGPATAQT AIQLFI ADY RYTYKCHRGD SKVEGKRMKI IKELWENTKG RDGLLVADGG PNIYNLRNLH IPEIVLKYNL MDPEYKGRLL HPQNPFV GH LSIEGIKEAD ITPAHGPVKK MDYDAVSGTH SWRTKRNRSI LNTDQRNMIL EEQCYAKCCN LFEACFNSAS YRKPVGQH S MLEAMAHRLR MDARLDYESG RMSKDDFEKA MAHLGEIGYI GSGSGENLYF Q

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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 B virus (B/Memphis/13/2003) / Strain: B/Memphis/13/2003
Molecular weightTheoretical: 90.844109 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSGSGSGSGM TLAKIELLKQ LLRDNEAKTV LKQTTVDQYN IIRKFNTSRI EKNPSLRMKW AMCSNFPLAL TKGDMANRIP LEYKGIQLK TNAEDIGTKG QMCSIAAVTW WNTYGPIGDT EGFERVYESF FLRKMRLDNA TWGRITFGPV ERVRKRVLLN P LTKEMPPD ...String:
GSGSGSGSGM TLAKIELLKQ LLRDNEAKTV LKQTTVDQYN IIRKFNTSRI EKNPSLRMKW AMCSNFPLAL TKGDMANRIP LEYKGIQLK TNAEDIGTKG QMCSIAAVTW WNTYGPIGDT EGFERVYESF FLRKMRLDNA TWGRITFGPV ERVRKRVLLN P LTKEMPPD EASNVIMEIL FPKEAGIPRE STWIHRELIK EKREKLKGTM ITPIVLAYML ERELVARRRF LPVAGATSAE FI EMLHCLQ GENWRQIYHP GGNKLTESRS QSMIVACRKI IRRSIVASNP LELAVEIANK TVIDTEPLKS CLAAIDGGDV ACD IIRAAL GLKIRQRQRF GRLELKRISG RGFKNDEEIL IGNGTIQKIG IWDGEEEFHV RCGECRGILK KSKMKLEKLL INSA KKEDM RDLIILCMVF SQDTRMFQGV RGEINFLNRA GQLLSPMYQL QRYFLNRSND LFDQWGYEES PKASELHGIN ESMNA SDYT LKGVVVTRNV IDDFSSTETE KVSITKNLSL IKRTGEVIMG ANDVSELESQ AQLMITYDTP KMWEMGTTKE LVQNTY QWV LKNLVTLKAQ FLLGKEDMFQ WDAFEAFESI IPQKMAGQYS GFARAVLKQM RDQEVMKTDQ FIKLLPFCFS PPKLRSN GE PYQFLKLVLK GGGENFIEVR KGSPLFSYNP QTEVLTICGR MMSLKGKIED EERNRSMGNA VLAGFLVSGK YDPDLGDF K TIEELEKLKP GEKANILLYQ GKPVKVVKRK RYSALSNDIS QGIKRQRMTV ESMGWALSGW SHPQFEKGSG SENLYFQ

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Macromolecule #4: 5' vRNA

MacromoleculeName: 5' vRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 4.55782 KDa
SequenceString:
AGUAGUAACA AGAG

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Macromolecule #5: 3' vRNA

MacromoleculeName: 3' vRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 6.706053 KDa
SequenceString:
UAUACAACUG AGAAAGCUAU U

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Macromolecule #6: mRNA

MacromoleculeName: mRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 6.282715 KDa
SequenceString:
AUCUAUAAUA GCUUUCUC(K1F)(K1F)

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 3 / 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: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 33.4 e/Å2
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

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 328180
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7r1f:
Early transcription elongation state of influenza B polymerase backtracked due to double incoproation of nucleotide analogue T1106

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