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- EMDB-10359: Bat Influenza A polymerase product dissociation complex using 44-... -

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Basic information

Entry
Database: EMDB / ID: EMD-10359
TitleBat Influenza A polymerase product dissociation complex using 44-mer vRNA template with intact oligo(U) sequence
Map data
Sample
  • Complex: Bat Influenza A polymerase product dissociation complex using 44-mer vRNA template with intact oligo(U) sequence
    • Complex: Polymerase
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Nucleic acid
      • RNA: vRNA
KeywordsInfluenza / polymerase / viral transcription / RNA / 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 / host cell mitochondrion / 7-methylguanosine mRNA capping / virion component / host cell cytoplasm / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / virion component / host cell cytoplasm / 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
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 ...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 / 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 basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) / Influenza B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsWandzik JM / Kouba T
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR-18-CE11-0028 France
CitationJournal: Cell / Year: 2020
Title: A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase.
Authors: Joanna M Wandzik / Tomas Kouba / Manikandan Karuppasamy / Alexander Pflug / Petra Drncova / Jan Provaznik / Nayara Azevedo / Stephen Cusack /
Abstract: Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). ...Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
History
DepositionOct 3, 2019-
Header (metadata) releaseApr 15, 2020-
Map releaseApr 15, 2020-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t0u
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10359.png

Downloads & links

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Map

FileDownload / File: emd_10359.map.gz / Format: CCP4 / Size: 115.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 312 pix.
= 252.72 Å		0.81 Å/pix.
x 312 pix.
= 252.72 Å		0.81 Å/pix.
x 312 pix.
= 252.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.07666522 - 0.14892888
Average (Standard dev.)0.00006762049 (±0.0055665234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions312312312
Spacing312312312
CellA=B=C: 252.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z312312312
origin x/y/z0.0000.0000.000
length x/y/z252.720252.720252.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS312312312
D min/max/mean-0.0770.1490.000

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Supplemental data

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Additional map: LocScale map

Fileemd_10359_additional.map
AnnotationLocScale map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10359_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10359_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bat Influenza A polymerase product dissociation complex using 44-...

EntireName: Bat Influenza A polymerase product dissociation complex using 44-mer vRNA template with intact oligo(U) sequence
Components
  • Complex: Bat Influenza A polymerase product dissociation complex using 44-mer vRNA template with intact oligo(U) sequence
    • Complex: Polymerase
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Nucleic acid
      • RNA: vRNA

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Supramolecule #1: Bat Influenza A polymerase product dissociation complex using 44-...

SupramoleculeName: Bat Influenza A polymerase product dissociation complex using 44-mer vRNA template with intact oligo(U) sequence
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Polymerase

SupramoleculeName: Polymerase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))

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Supramolecule #3: Nucleic acid

SupramoleculeName: Nucleic acid / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Influenza B virus

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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: Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Molecular weightTheoretical: 85.432828 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSHHHHHHHH GSGSMENFVR TNFNPMILER AEKTMKEYGE NPQNEGNKFA AISTHMEVCF MYSDFHFIDL EGNTIVKEND DDNAMLKHR FEIIEGQERN IAWTIVNSIC NMTENSKPRF LPDLYDYKTN KFIEIGVTRR KVEDYYYEAA SKLKGENVYI H IFSFDGEE ...String:
GSHHHHHHHH GSGSMENFVR TNFNPMILER AEKTMKEYGE NPQNEGNKFA AISTHMEVCF MYSDFHFIDL EGNTIVKEND DDNAMLKHR FEIIEGQERN IAWTIVNSIC NMTENSKPRF LPDLYDYKTN KFIEIGVTRR KVEDYYYEAA SKLKGENVYI H IFSFDGEE MATDDEYILD EESRARIKTR LFVLRQELAT AGLWDSFRQS EKGEETLEEE FSYPPTFQRL ANQSLPPSFK DY HQFKAYV SSFKANGNIE AKLGAMSEKV NAQIESFDPR TIRELELPEG KFCTQRSKFL LMDAMKLSVL NPAHEGEGIP MKD AKACLD TFWGWKKATI IKKHEKGVNT NYLMIWEQLL ESIKEMEGKF LNLKKTNHLK WGLGEGQAPE KMDFEDCKEV PDLF QYKSE PPEKRKLASW IQSEFNKASE LTNSNWIEFD ELGNDVAPIE HIASRRRNFF TAEVSQCRAS EYIMKAVYIN TALLN SSCT AMEEYQVIPI ITKCRDTSGQ RRTNLYGFII KGRSHLRNDT DVVNFISLEF SLTDPRNEIH KWEKYCVLEI GDMEIR TSI STIMKPVYLY VRTNGTSKIK MKWGMEMRRC LLQSLQQVES MIEAESAVKE KDMTEPFFRN RENDWPIGES PQGIEKG TI GKVCRVLLAK SVFNSIYASA QLEGFSAESR KLLLLIQAFR DNLDPGTFDL KGLYEAIEEC IINDPWVLLN ASWFNSFL K AVQLSMGSGS GENLYFQ

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/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Molecular weightTheoretical: 87.936312 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSGSGSGSGM DVNPMLIFLK VPVQNAISTT FPYTGDPPYS HGTGTGYTMD TVIRTHDYSS RGIWKTNSET GAQQLNPIDG PLPEDNEPS GYAQTDCVLE LIEGLDRSHP GLFETACQET IDAIQQTRVD KLTQGRQTYD WTLNRNQPAA TALANTIEVF R KNGYKLNE ...String:
GSGSGSGSGM DVNPMLIFLK VPVQNAISTT FPYTGDPPYS HGTGTGYTMD TVIRTHDYSS RGIWKTNSET GAQQLNPIDG PLPEDNEPS GYAQTDCVLE LIEGLDRSHP GLFETACQET IDAIQQTRVD KLTQGRQTYD WTLNRNQPAA TALANTIEVF R KNGYKLNE SGRLIDFLKD VLLSFENDSM EVTTHFQKKK RIRDNHSKKM ITQRTIGKKR VKLTKKNYLI RALTLNTMTK DA ERGKLKR RAIATPGMQI RGFVYFVELL ARNICERLEQ SGLPVGGNEK KAKLANVIKK MMAKSTDEEL SYTITGDNTK WNE NQNPRI FLAMVLRITA GQPEWFRDLL AVAPIMFSNK VARLGRGYMF ESKSMHLRTQ ISAENLSDIN LRYFNEDTKK KIEK IRHLM VEGTASLSPG MMMGMFNMLS TVLGVSVLNL GQREILKRTY WWDGLQSSDD FALIINGHFK EDIQQGVNHF YRTCK LVGI NMSQKKSYIN KTGTFEFTSF FYRYGFVANF SMELPSFGVA GNNESADMSI GTTVIKTNMI NNDLGPATAQ MAIQLF IKD YRYTYRCHRG DTNLETRRTK SIKRLWTETI SKAGLLVADG GPNPYNLRNL HIPEVCLKWS LMDPDYRGRL CNPNNPF VH HMEVESTNLA VVMPAHGPAK SLEYDAVATT HSWTPKRNRS ILNTNQRGIL EDERIYQKCC QVFEKFFPSS TYRRPIGM A SMLDAMLSRA RIDARIDLES GRISSQDFSE ITNTCKAIEA LKRQGSGSGE NLYFQ

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/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Molecular weightTheoretical: 91.027141 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSGSGSGSGM ERIKELMEMV KNSRMREILT TTSVDHMAVI KKYTSGRQEK NPALRMKWMM AMKYPISASS RIREMIPEKD EDGNTLWTN TKDAGSNRVL VSPNAVTWWN RAGPVSDVVH YPRVYKMYFD RLERLTHGTF GPVKFYNQVK VRKRVDINPG H KDLTSREA ...String:
GSGSGSGSGM ERIKELMEMV KNSRMREILT TTSVDHMAVI KKYTSGRQEK NPALRMKWMM AMKYPISASS RIREMIPEKD EDGNTLWTN TKDAGSNRVL VSPNAVTWWN RAGPVSDVVH YPRVYKMYFD RLERLTHGTF GPVKFYNQVK VRKRVDINPG H KDLTSREA QEVIMEVVFP NEVGARTLSS DAQLTITKEK KEELKNCKIS PIMVAYMLER ELVRRTRFLP IAGATSSTYV EV LHLTQGT CWEQQYTPGG EAENDDLDQT LIIASRNIVR RSIVAIDPLA SLLSMCHTTS ISSEPLVEIL RSNPTDEQAV NIC KAALGI RINNSFSFGG YNFKRVKGSS QRTEKAVLTG NLQTLTMTIF EGYEEFNVSG KRASAVLKKG AQRLIQAIIG GRTL EDILN LMITLMVFSQ EEKMLKAVRG DLNFVNRANQ RLNPMYQLLR HFQKDSSTLL KNWGTEEIDP IMGIAGIMPD GTINK TQTL MGVRLSQGGV DEYSFNERIR VNIDKYLRVR NEKGELLISP EEVSEAQGQE KLPINYNSSL MWEVNGPESI LTNTYH WII KNWELLKTQW MTDPTVLYNR IEFEPFQTLI PKGNRAIYSG FTRTLFQQMR DVEGTFDSIQ IIKLLPFSAH PPSLGRT QF SSFTLNIRGA PLRLLIRGNS QVFNYNQMEN VIIVLGKSVG SPERSILTES SSIESAVLRG FLILGKANSK YGPVLTIG E LDKLGRGEKA NVLIGQGDTV LVMKRKRDSS ILTDSQTALK RIRLEESKGW SHPQFEKGGG SGGGSGGSAW SHPQFEKGR SGGENLYFQ

UniProtKB: Polymerase basic protein 2

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Macromolecule #4: vRNA

MacromoleculeName: vRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 13.911169 KDa
SequenceString:
AGUAGUAACA AGAGCAUUUU UUCCGUCUCG CCUCUGCUUC UGCU

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51502
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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