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- PDB-6qpf: Influenza A virus Polymerase Heterotrimer A/duck/Fujian/01/2002(H5N1) -

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Basic information

Entry
Database: PDB / ID: 6qpf
TitleInfluenza A virus Polymerase Heterotrimer A/duck/Fujian/01/2002(H5N1)
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsRNA BINDING PROTEIN / Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP
Function / homology
Function and homology information


virion component => GO:0044423 / 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 / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase ...virion component => GO:0044423 / 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 / 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 / cytoplasm
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 CAP binding 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 basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.634 Å
AuthorsFan, H.T. / Keown, J.R. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
Authors: Haitian Fan / Alexander P Walker / Loïc Carrique / Jeremy R Keown / Itziar Serna Martin / Dimple Karia / Jane Sharps / Narin Hengrung / Els Pardon / Jan Steyaert / Jonathan M Grimes / Ervin Fodor /
Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented ...Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol) composed of PB1, PB2 and PA subunits. Although the high-resolution crystal structure of FluPol of bat influenza A virus has previously been reported, there are no complete structures available for human and avian FluPol. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol that could be targeted in the development of antiviral drugs.
History
DepositionFeb 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2019Group: Data collection / Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop
Revision 1.2Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Sep 25, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
D: Polymerase acidic protein
E: RNA-directed RNA polymerase catalytic subunit
F: Polymerase basic protein 2
G: Polymerase acidic protein
H: RNA-directed RNA polymerase catalytic subunit
I: Polymerase basic protein 2
J: Polymerase acidic protein
K: RNA-directed RNA polymerase catalytic subunit
L: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)1,024,31412
Polymers1,024,31412
Non-polymers00
Water0
1
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)256,0793
Polymers256,0793
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area33260 Å2
ΔGint-188 kcal/mol
Surface area95620 Å2
MethodPISA
2
D: Polymerase acidic protein
E: RNA-directed RNA polymerase catalytic subunit
F: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)256,0793
Polymers256,0793
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area32480 Å2
ΔGint-192 kcal/mol
Surface area97440 Å2
MethodPISA
3
G: Polymerase acidic protein
H: RNA-directed RNA polymerase catalytic subunit
I: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)256,0793
Polymers256,0793
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area34120 Å2
ΔGint-187 kcal/mol
Surface area95810 Å2
MethodPISA
4
J: Polymerase acidic protein
K: RNA-directed RNA polymerase catalytic subunit
L: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)256,0793
Polymers256,0793
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area32960 Å2
ΔGint-182 kcal/mol
Surface area94100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)337.122, 192.859, 235.688
Angle α, β, γ (deg.)90.000, 91.460, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain C and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))
21(chain F and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))
31(chain I and (resid 1 through 79 or resid 89 through 756))
41(chain L and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))
12(chain B and (resid 1 through 179 or resid 209...
22(chain E and (resid 1 through 348 or resid 374 through 632 or resid 657 through 756))
32(chain H and (resid 1 through 179 or resid 209...
42(chain K and (resid 1 through 179 or resid 209...
13(chain A and (resid 1 through 374 or resid 381 through 716))
23(chain D and (resid 1 through 374 or resid 381 through 716))
33(chain G and (resid 1 through 374 or resid 381 through 716))
43chain J

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111METMETSERSER(chain C and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))CC1 - 791 - 79
121VALVALTRPTRP(chain C and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))CC89 - 53789 - 537
131SERSERMETMET(chain C and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))CC544 - 756544 - 756
211METMETSERSER(chain F and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))FF1 - 791 - 79
221VALVALTRPTRP(chain F and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))FF89 - 53789 - 537
231SERSERMETMET(chain F and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))FF544 - 756544 - 756
311METMETSERSER(chain I and (resid 1 through 79 or resid 89 through 756))II1 - 791 - 79
321VALVALMETMET(chain I and (resid 1 through 79 or resid 89 through 756))II89 - 75689 - 756
411METMETSERSER(chain L and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))LL1 - 791 - 79
421VALVALTRPTRP(chain L and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))LL89 - 53789 - 537
431SERSERMETMET(chain L and (resid 1 through 79 or resid 89 through 537 or resid 544 through 756))LL544 - 756544 - 756
112METMETMETMET(chain B and (resid 1 through 179 or resid 209...BB1 - 1791 - 179
122LYSLYSMETMET(chain B and (resid 1 through 179 or resid 209...BB209 - 348209 - 348
132METMETGLNGLN(chain B and (resid 1 through 179 or resid 209...BB1 - 7561 - 756
142TYRTYRPROPRO(chain B and (resid 1 through 179 or resid 209...BB657 - 668657 - 668
152ASPASPGLNGLN(chain B and (resid 1 through 179 or resid 209...BB685 - 756685 - 756
212METMETMETMET(chain E and (resid 1 through 348 or resid 374 through 632 or resid 657 through 756))EE1 - 3481 - 348
222ALAALAVALVAL(chain E and (resid 1 through 348 or resid 374 through 632 or resid 657 through 756))EE374 - 632374 - 632
232TYRTYRGLNGLN(chain E and (resid 1 through 348 or resid 374 through 632 or resid 657 through 756))EE657 - 756657 - 756
312METMETMETMET(chain H and (resid 1 through 179 or resid 209...HH1 - 1791 - 179
322LYSLYSMETMET(chain H and (resid 1 through 179 or resid 209...HH209 - 348209 - 348
332ALAALAVALVAL(chain H and (resid 1 through 179 or resid 209...HH374 - 632374 - 632
342TYRTYRPROPRO(chain H and (resid 1 through 179 or resid 209...HH657 - 668657 - 668
352ASPASPGLNGLN(chain H and (resid 1 through 179 or resid 209...HH685 - 756685 - 756
412METMETMETMET(chain K and (resid 1 through 179 or resid 209...KK1 - 1791 - 179
422LYSLYSMETMET(chain K and (resid 1 through 179 or resid 209...KK209 - 348209 - 348
432ALAALAPROPRO(chain K and (resid 1 through 179 or resid 209...KK374 - 668374 - 668
442ASPASPGLNGLN(chain K and (resid 1 through 179 or resid 209...KK685 - 756685 - 756
113METMETMETMET(chain A and (resid 1 through 374 or resid 381 through 716))AA1 - 3741 - 374
123PHEPHEARGARG(chain A and (resid 1 through 374 or resid 381 through 716))AA381 - 716381 - 716
213METMETMETMET(chain D and (resid 1 through 374 or resid 381 through 716))DD1 - 3741 - 374
223PHEPHEARGARG(chain D and (resid 1 through 374 or resid 381 through 716))DD381 - 716381 - 716
313METMETMETMET(chain G and (resid 1 through 374 or resid 381 through 716))GG1 - 3741 - 374
323PHEPHEARGARG(chain G and (resid 1 through 374 or resid 381 through 716))GG381 - 716381 - 716
413METMETARGARGchain JJJ1 - 7161 - 716

NCS ensembles :
ID
1
2
3

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Components

#1: Protein
Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 82710.484 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: D3YNB8, Hydrolases; Acting on ester bonds
#2: Protein
RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86546.109 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: D3YNB6, RNA-directed RNA polymerase
#3: Protein
Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 86821.953 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: D3YNB5

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.92 Å3/Da / Density % sol: 68.64 %
Crystal growTemperature: 293 K / Method: evaporation
Details: 1M Phosphate Potassium Sodium pH6.9, 0.07% Dichloromethane

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 30, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.634→235.611 Å / Num. obs: 143100 / % possible obs: 100 % / Redundancy: 3.4 % / Biso Wilson estimate: 76.82 Å2 / CC1/2: 0.88 / Rmerge(I) obs: 0.484 / Rpim(I) all: 0.31 / Rrim(I) all: 0.576 / Net I/σ(I): 1.5
Reflection shell

Diffraction-ID: 1 / % possible all: 100

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all
3.85-4.053.52.539208780.3751.6083.013
12.16-235.613.20.25446590.8820.1630.302

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Processing

Software
NameVersionClassification
PHENIXrefinement
Aimless0.7.3data scaling
PDB_EXTRACT3.24data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5d98

5d98


Resolution: 3.634→235.611 Å / SU ML: 0.58 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 36.15
RfactorNum. reflection% reflection
Rfree0.3229 4059 4.99 %
Rwork0.277 --
obs0.2794 81274 47.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 252.81 Å2 / Biso mean: 101.2285 Å2 / Biso min: 15.26 Å2
Refinement stepCycle: final / Resolution: 3.634→235.611 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms67760 0 0 0 67760
Num. residues----8449
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11C8668X-RAY DIFFRACTION2.831TORSIONAL
12F8668X-RAY DIFFRACTION2.831TORSIONAL
13I8668X-RAY DIFFRACTION2.831TORSIONAL
14L8668X-RAY DIFFRACTION2.831TORSIONAL
21B7744X-RAY DIFFRACTION2.831TORSIONAL
22E7744X-RAY DIFFRACTION2.831TORSIONAL
23H7744X-RAY DIFFRACTION2.831TORSIONAL
24K7744X-RAY DIFFRACTION2.831TORSIONAL
31A8816X-RAY DIFFRACTION2.831TORSIONAL
32D8816X-RAY DIFFRACTION2.831TORSIONAL
33G8816X-RAY DIFFRACTION2.831TORSIONAL
34J8816X-RAY DIFFRACTION2.831TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 29

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.6344-3.67720.457220.50622123
3.6772-3.7220.569230.427686892
3.722-3.76910.224570.3291351422
3.7691-3.81870.4272120.33832212334
3.8187-3.87110.3486150.33552993145
3.8711-3.92640.3297220.32323854077
3.9264-3.9850.4342240.33194314558
3.985-4.04730.3315240.352357359710
4.0473-4.11360.3477410.309174578613
4.1136-4.18460.3307540.3058946100017
4.1846-4.26070.3776630.28541144120721
4.2607-4.34260.3638670.29681387145425
4.3426-4.43130.3241860.2951643172930
4.4313-4.52760.3617930.291920201334
4.5276-4.63290.36151360.27872280241641
4.6329-4.74880.30711390.26072671281048
4.7488-4.87720.31391590.26393037319655
4.8772-5.02080.34451880.27733334352260
5.0208-5.18280.34442010.28613601380265
5.1828-5.36810.322090.29893967417671
5.3681-5.5830.3792300.31394428465880
5.583-5.83710.37772760.30995065534191
5.8371-6.14490.32422860.31575452573898
6.1449-6.52990.36412920.330155665858100
6.5299-7.03410.37622940.317455655859100
7.0341-7.7420.32032930.287555925885100
7.742-8.86240.31142820.236255975879100
8.8624-11.16570.22062810.195456035884100
11.1657-236.10790.31082800.2725521580196

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