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- PDB-6qpg: Influenza A virus Polymerase Heterotrimer A/nt/60/1968(H3N2) in c... -

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Basic information

Entry
Database: PDB / ID: 6qpg
TitleInfluenza A virus Polymerase Heterotrimer A/nt/60/1968(H3N2) in complex with Nanobody NB8205
Components
  • Nanobody NB8205
  • 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 / Nanobody / antiviral inhibitor
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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds ...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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / 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 / 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
Camelidae (mammal)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.34 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/R009945/1 United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 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 / Refinement description / Category: refine_ls_shell / Item: _refine_ls_shell.R_factor_R_free
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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
M: Nanobody NB8205
N: Nanobody NB8205
O: Nanobody NB8205
P: Nanobody NB8205


Theoretical massNumber of molelcules
Total (without water)1,085,67416
Polymers1,085,67416
Non-polymers00
Water0
1
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
M: Nanobody NB8205


Theoretical massNumber of molelcules
Total (without water)271,4194
Polymers271,4194
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
D: Polymerase acidic protein
E: RNA-directed RNA polymerase catalytic subunit
F: Polymerase basic protein 2
N: Nanobody NB8205


Theoretical massNumber of molelcules
Total (without water)271,4194
Polymers271,4194
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
G: Polymerase acidic protein
H: RNA-directed RNA polymerase catalytic subunit
I: Polymerase basic protein 2
O: Nanobody NB8205


Theoretical massNumber of molelcules
Total (without water)271,4194
Polymers271,4194
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
J: Polymerase acidic protein
K: RNA-directed RNA polymerase catalytic subunit
L: Polymerase basic protein 2
P: Nanobody NB8205


Theoretical massNumber of molelcules
Total (without water)271,4194
Polymers271,4194
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)335.181, 192.900, 235.096
Angle α, β, γ (deg.)90.000, 91.781, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
31
41
12
22
32
42
13
23
33
43
14
24
34
44

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain 'A' and (resid 1 through 327 or resid 331 through 716))A1 - 326
121(chain 'A' and (resid 1 through 327 or resid 331 through 716))A331 - 371
131(chain 'A' and (resid 1 through 327 or resid 331 through 716))A398 - 716
241(chain 'D' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))D1 - 326
251(chain 'D' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))D331 - 371
261(chain 'D' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))D398 - 716
371(chain 'G' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))G1 - 326
381(chain 'G' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))G331 - 371
391(chain 'G' and (resid 1 through 327 or resid 331 through 372 or resid 398 through 716))G398 - 716
4101(chain 'J' and (resid 1 through 372 or resid 398 through 716))J1 - 326
4111(chain 'J' and (resid 1 through 372 or resid 398 through 716))J331 - 371
4121(chain 'J' and (resid 1 through 372 or resid 398 through 716))J398 - 716
1132(chain 'C' and (resid 1 through 79 or resid 89 through 524 or resid 537 through 756))C1 - 181
1142(chain 'C' and (resid 1 through 79 or resid 89 through 524 or resid 537 through 756))C208 - 225
1152(chain 'C' and (resid 1 through 79 or resid 89 through 524 or resid 537 through 756))C243 - 350
1162(chain 'C' and (resid 1 through 79 or resid 89 through 524 or resid 537 through 756))C368 - 627
1172(chain 'C' and (resid 1 through 79 or resid 89 through 524 or resid 537 through 756))C687 - 755
2182(chain 'F' and (resid 1 through 177 or resid 183 through 524 or resid 537 through 756))F1 - 181
2192(chain 'F' and (resid 1 through 177 or resid 183 through 524 or resid 537 through 756))F208 - 225
2202(chain 'F' and (resid 1 through 177 or resid 183 through 524 or resid 537 through 756))F243 - 350
2212(chain 'F' and (resid 1 through 177 or resid 183 through 524 or resid 537 through 756))F368 - 627
2222(chain 'F' and (resid 1 through 177 or resid 183 through 524 or resid 537 through 756))F687 - 755
3232(chain 'I' and (resid 1 through 79 or resid 89...I1 - 181
3242(chain 'I' and (resid 1 through 79 or resid 89...I208 - 225
3252(chain 'I' and (resid 1 through 79 or resid 89...I243 - 350
3262(chain 'I' and (resid 1 through 79 or resid 89...I368 - 627
3272(chain 'I' and (resid 1 through 79 or resid 89...I687 - 755
4282(chain 'L' and (resid 1 through 79 or resid 89...L1 - 181
4292(chain 'L' and (resid 1 through 79 or resid 89...L208 - 225
4302(chain 'L' and (resid 1 through 79 or resid 89...L243 - 350
4312(chain 'L' and (resid 1 through 79 or resid 89...L368 - 627
4322(chain 'L' and (resid 1 through 79 or resid 89...L687 - 755
1333(chain 'B' and (resid 1 through 628 or resid 687 through 756))B1 - 78
1343(chain 'B' and (resid 1 through 628 or resid 687 through 756))B89 - 176
1353(chain 'B' and (resid 1 through 628 or resid 687 through 756))B183 - 522
1363(chain 'B' and (resid 1 through 628 or resid 687 through 756))B537 - 674
1373(chain 'B' and (resid 1 through 628 or resid 687 through 756))B693 - 755
2383(chain 'E' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))E1 - 78
2393(chain 'E' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))E89 - 176
2403(chain 'E' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))E183 - 522
2413(chain 'E' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))E537 - 674
2423(chain 'E' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))E693 - 755
3433(chain 'H' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))H1 - 78
3443(chain 'H' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))H89 - 176
3453(chain 'H' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))H183 - 522
3463(chain 'H' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))H537 - 674
3473(chain 'H' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))H693 - 755
4483(chain 'K' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))K1 - 78
4493(chain 'K' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))K89 - 176
4503(chain 'K' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))K183 - 522
4513(chain 'K' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))K537 - 674
4523(chain 'K' and (resid 1 through 351 or resid 368 through 628 or resid 687 through 756))K693 - 755
1534chain 'M'M1 - 120
2544chain 'N'N1 - 120
3554chain 'O'O1 - 120
4564chain 'P'P1 - 120

NCS ensembles :
ID
1
2
3
4

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Components

#1: Protein
Polymerase acidic protein


Mass: 83100.797 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03434
#2: Protein
RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86524.086 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2 / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03432, RNA-directed RNA polymerase
#3: Protein
Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 86958.242 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2 / Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03429
#4: Antibody
Nanobody NB8205


Mass: 14835.375 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae (mammal) / Production host: Escherichia coli (E. coli)

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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: 3.5 Å3/Da / Density % sol: 64.84 %
Crystal growTemperature: 293 K / Method: evaporation / Details: 0.9-1.2M Phosphate Potassium Sodium, pH6.7

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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: Dec 13, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.34→235 Å / Num. obs: 89646 / % possible obs: 41.5 % / Redundancy: 7 % / Biso Wilson estimate: 78.39 Å2 / CC1/2: 0.99 / Net I/σ(I): 4.8
Reflection shellResolution: 3.34→3.87 Å / Redundancy: 6.9 % / Mean I/σ(I) obs: 1.8 / Num. unique obs: 4718 / CC1/2: 0.7 / % possible all: 6.2

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
Aimlessdata scaling
PDB_EXTRACT3.24data extraction
PHASERphasing
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5d98

5d98


Resolution: 3.34→234.98 Å / SU ML: 0.517 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 34.658
RfactorNum. reflection% reflection
Rfree0.3049 4533 5.06 %
Rwork0.2512 --
obs0.254 89539 41.47 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 78.84 Å2
Refinement stepCycle: LAST / Resolution: 3.34→234.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms70549 0 0 0 70549
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002671910
X-RAY DIFFRACTIONf_angle_d0.660296973
X-RAY DIFFRACTIONf_chiral_restr0.04310664
X-RAY DIFFRACTIONf_plane_restr0.003712491
X-RAY DIFFRACTIONf_dihedral_angle_d8.597343929
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.34-3.3800.212617X-RAY DIFFRACTION0.24
3.38-3.420.391830.350758X-RAY DIFFRACTION0.86
3.42-3.460.41270.3592122X-RAY DIFFRACTION1.83
3.46-3.50.3694100.3412189X-RAY DIFFRACTION2.8
3.5-3.550.4035160.3082297X-RAY DIFFRACTION4.4
3.55-3.60.3208210.3228403X-RAY DIFFRACTION5.95
3.6-3.650.3665260.345518X-RAY DIFFRACTION7.57
3.65-3.710.3324290.353606X-RAY DIFFRACTION8.88
3.71-3.760.4314450.3013727X-RAY DIFFRACTION10.8
3.76-3.830.348360.2894873X-RAY DIFFRACTION12.78
3.83-3.890.3297490.29381029X-RAY DIFFRACTION15
3.89-3.960.3698780.29371184X-RAY DIFFRACTION17.57
3.96-4.040.3314690.27421321X-RAY DIFFRACTION19.37
4.04-4.120.3285820.24691474X-RAY DIFFRACTION21.54
4.12-4.210.3381940.24851709X-RAY DIFFRACTION25.23
4.21-4.310.3532940.24132005X-RAY DIFFRACTION29.34
4.31-4.420.31261290.23652375X-RAY DIFFRACTION34.79
4.42-4.540.30411490.23862779X-RAY DIFFRACTION40.69
4.54-4.670.30971790.23863091X-RAY DIFFRACTION45.64
4.67-4.820.31641760.2333620X-RAY DIFFRACTION52.47
4.82-4.990.32782240.24124141X-RAY DIFFRACTION60.95
4.99-5.190.32782770.25444748X-RAY DIFFRACTION69.8
5.19-5.430.31862720.26575341X-RAY DIFFRACTION77.99
5.43-5.710.33943080.27985758X-RAY DIFFRACTION83.99
5.71-6.070.33083330.29126251X-RAY DIFFRACTION91.22
6.07-6.540.35263610.28316771X-RAY DIFFRACTION98.86
6.54-7.20.32553680.28566871X-RAY DIFFRACTION100
7.2-8.240.28853600.23726866X-RAY DIFFRACTION99.97
8.24-10.380.23233710.19016913X-RAY DIFFRACTION99.93
10.38-235.570.26913670.25446949X-RAY DIFFRACTION98.84

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