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- PDB-6qcv: Crystal structure of influenza B polymerase initiation state with... -

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Basic information

Entry
Database: PDB / ID: 6qcv
TitleCrystal structure of influenza B polymerase initiation state with capped 14-mer RNA primer and CTP
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-D(*(GDM))-R(P*GP*AP*AP*UP*GP*CP*UP*AP*UP*AP*AP*UP*AP*G)-3')
  • RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*UP*CP*UP*GP*CP*UP*AP*UP*U)-3')
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*G)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / RNA dependent RNA polymerase
Function / homology
Function and homology information


cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / ec:2.7.7.48: / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm ...cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / ec:2.7.7.48: / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm / ec:3.1.-.-: / transcription, DNA-templated / host cell nucleus / nucleotide binding / RNA binding / metal ion binding
Influenza RNA-dependent RNA polymerase subunit PA / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / PA/PA-X superfamily / Polymerase acidic protein / PB2, C-terminal / RNA-directed RNA polymerase, negative-strand RNA virus / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PB1
Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Biological speciesInfluenza B virus
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.24 Å
AuthorsCusack, S. / Drncova, P.
Funding supportFrance , 2件
OrganizationGrant numberCountry
European Commission322586France
French National Research AgencyANR-18-CE11-0028France
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2019
Title: Structural snapshots of actively transcribing influenza polymerase.
Authors: Tomas Kouba / Petra Drncová / Stephen Cusack /
Abstract: Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter ...Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter comprising the partially base-paired 3' and 5' extremities of the RNA. A short, capped primer, 'cap-snatched' from a nascent host polymerase II transcript, is directed towards the polymerase active site to initiate RNA synthesis. Here we present structural snapshots, as determined by X-ray crystallography and cryo-electron microscopy, of actively initiating influenza polymerase as it transitions towards processive elongation. Unexpected conformational changes unblock the active site cavity to allow establishment of a nine-base-pair template-product RNA duplex before the strands separate into distinct exit channels. Concomitantly, as the template translocates, the promoter base pairs are broken and the template entry region is remodeled. These structures reveal details of the influenza polymerase active site that will help optimize nucleoside analogs or other compounds that directly inhibit viral RNA synthesis.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 31, 2018 / Release: Jun 5, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 5, 2019Structure modelrepositoryInitial release
1.1Jun 12, 2019Structure modelData collection / Database referencescitation / citation_author / pdbx_database_proc_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
1.2Jun 19, 2019Structure modelData collection / Database referencescitation / citation_author / pdbx_database_proc_citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
M: RNA (5'-D(*(GDM))-R(P*GP*AP*AP*UP*GP*CP*UP*AP*UP*AP*AP*UP*AP*G)-3')
R: RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*UP*CP*UP*GP*CP*UP*AP*UP*U)-3')
V: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)279,4969
Polymers278,8946
Non-polymers6023
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area50270 Å2
ΔGint-316 kcal/mol
Surface area87260 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)200.191, 200.191, 256.208
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

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Protein/peptide , 3 types, 3 molecules ABC

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


Mass: 85822.781 Da / Num. of mol.: 1 / Details: N-terminal His-tag C-terminal linker and TEV site
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8Z9, EC: 3.1.-.-
#2: Protein/peptide RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86207.016 Da / Num. of mol.: 1 / Details: N-terminal linker C-terminal linker and TEV site
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8Y6, EC: 2.7.7.48
#3: Protein/peptide Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 90844.109 Da / Num. of mol.: 1
Details: N-terminal linker C-terminal STREP tag and TEV site
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8X3

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RNA chain , 3 types, 3 molecules MRV

#4: RNA chain RNA (5'-D(*(GDM))-R(P*GP*AP*AP*UP*GP*CP*UP*AP*UP*AP*AP*UP*AP*G)-3')


Mass: 4935.960 Da / Num. of mol.: 1 / Details: Capped RNA / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*UP*CP*UP*GP*CP*UP*AP*UP*U)-3')


Mass: 6525.820 Da / Num. of mol.: 1 / Details: 3' end of vRNA promoter (extended) / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*G)-3')


Mass: 4557.820 Da / Num. of mol.: 1 / Details: 5' end of vRNA promoter / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Phosphate
#8: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3 / Cytidine triphosphate
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Magnesium

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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: 5.31 Å3/Da / Density % sol: 76.85 % / Description: marquise shaped
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 4.4
Details: Influenza B polymerase at 9 mg per ml in was mixed with 40 microM vRNA 5 prime end 14-mer, 40 microM vRNA 3 prime end 21-mer and 80 microM 14-mer capped RNA. The mother liquor contained 200 mM di-ammonium phosphate and 100 mM sodium acetate between pH 4.0 and 4.4. CTP was soaked for 18 hours into grown crystals at a final concentration of 5 mM in the drop.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.977 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Aug 24, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 3.24→50 Å / Num. obs: 64019 / % possible obs: 67.9 % / Redundancy: 6.9 % / CC1/2: 0.996 / Rmerge(I) obs: 0.175 / Rrim(I) all: 0.189 / Net I/σ(I): 7.6
Reflection shellResolution: 3.24→3.48 Å / Redundancy: 7.3 % / Rmerge(I) obs: 1.19 / Mean I/σ(I) obs: 1.5 / CC1/2: 0.65 / Rrim(I) all: 1.28 / % possible all: 60.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
XDSdata reduction
AutoPROCdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5MSG
Resolution: 3.24→50 Å / Cor.coef. Fo:Fc: 0.815 / Cor.coef. Fo:Fc free: 0.905 / SU B: 55.338 / SU ML: 0.381 / Cross valid method: THROUGHOUT / ESU R Free: 0.486 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23448 3069 4.8 %RANDOM
Rwork0.20868 ---
Obs0.20995 60950 67.65 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 116.801 Å2
Baniso -1Baniso -2Baniso -3
1--2.36 Å2-1.18 Å2-0 Å2
2---2.36 Å20 Å2
3---7.65 Å2
Refinement stepCycle: 1 / Resolution: 3.24→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17470 1064 35 0 18569
Refine LS restraints

Refinement-ID: X-RAY DIFFRACTION

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0020.01319022
r_bond_other_d0.0010.01717353
r_angle_refined_deg1.1331.62225874
r_angle_other_deg1.0621.61940432
r_dihedral_angle_1_deg5.31252190
r_dihedral_angle_2_deg31.27122.53921
r_dihedral_angle_3_deg15.284153368
r_dihedral_angle_4_deg13.93715118
r_chiral_restr0.0390.22518
r_gen_planes_refined0.0020.0220246
r_gen_planes_other0.0010.023910
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_it0.5475.7538778
r_mcbond_other0.5475.7538777
r_mcangle_it0.9778.6310962
r_mcangle_other0.9778.62910963
r_scbond_it0.4825.82910244
r_scbond_other0.4825.82910243
r_scangle_it
r_scangle_other0.8868.72514913
r_long_range_B_refined2.14969.28521980
r_long_range_B_other2.14969.28321981
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
LS refinement shellResolution: 3.24→3.324 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.3 33 -
Rwork0.334 382 -
Obs--5.98 %
Refinement TLS params.

Method: refined / Refinement-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
10.7147-0.39450.1560.8692-0.08140.10270.26010.8592-0.6437-0.8432-0.09250.2490.32780.0869-0.16762.0346-0.26730.0231.9072-0.41450.9495-72.394423.6055-68.248
20.97530.2862-0.24783.11080.08761.42890.2919-0.04540.10130.4643-0.27590.0907-0.1360.1739-0.0160.6454-0.46640.07520.383-0.05110.1232-51.119760.357-6.5611
31.82970.39520.27961.7647-0.85081.29290.2015-0.33230.2920.7116-0.16560.1185-0.1603-0.037-0.03591.0432-0.50030.28390.3113-0.19430.1328-66.133857.56922.7782
40.86540.49690.34411.2212-0.62190.94380.3878-0.0657-0.1610.1733-0.3732-0.25990.15310.3665-0.01460.8208-0.38510.0250.4065-0.05380.1497-49.744238.5364-10.2345
50.9887-0.06350.01921.5203-1.23341.2448-0.01060.3731-0.1336-0.1856-0.0959-0.26840.42920.0040.10650.9173-0.17580.15580.6576-0.14380.2439-52.031836.4823-39.4373
61.33140.1130.21871.5586-0.40510.3410.06610.2069-0.35730.1151-0.0691-0.40090.39150.01270.0031.0989-0.26190.04670.3799-0.11560.3078-54.679928.9937-24.8934
71.47630.936-0.5723.2251-0.43150.47260.2732-0.0208-0.21050.4487-0.4098-0.32830.19130.22090.13660.9667-0.20430.04660.54040.01930.1319-43.129235.9742-17.4098
87.5148-2.2605-2.04683.2165-1.82623.11550.2152-0.61930.28790.2272-0.4266-0.42230.12070.70710.21141.0852-0.2947-0.00830.3501-0.12320.3519-56.411523.8985-2.8105
91.19760.4994-0.02393.1169-0.9073.41560.01990.14710.55390.17820.1391-0.0466-0.1566-0.6227-0.1590.5831-0.3210.29680.3456-0.07590.4537-82.466159.2637-19.1521
101.4567-2.4122-2.42454.11134.12444.14380.23040.4472-0.1462-0.1208-0.53260.2421-0.0698-0.63920.30221.0454-0.27310.03150.97180.18390.5521-69.182556.6648-42.0459
112.8594-0.1435-0.05041.67290.67734.42320.22181.07060.1135-0.90370.00730.25610.2497-1.0121-0.22911.1257-0.2950.03231.22780.07240.3548-76.245945.6749-62.8143
120.95250.1805-0.41861.2779-0.05341.70280.02680.64870.1177-0.31650.04680.45060.4135-0.3422-0.07360.8082-0.3230.12420.6906-0.00570.3937-81.349548.3887-35.0703
133.2473-0.0363-0.18062.98590.45922.33810.17241.0871-0.547-0.6902-0.0022-0.16910.49290.056-0.17021.3052-0.49530.15070.8672-0.10430.5072-98.127611.4763-22.6255
143.3746-1.71391.29233.7491-1.22693.98590.055-0.6717-0.30020.766-0.05340.10690.0548-0.0382-0.00161.1178-0.59710.14640.5223-0.00690.1889-82.512117.147116.8668
1513.41889.29410.18486.50680.30860.7308-0.78331.4330.2881-0.70020.86260.2604-0.0543-0.4322-0.07931.353-0.15980.01340.8564-0.04680.9851-74.917132.3089-22.5467
1610.63484.9581-1.27532.42970.02775.3956-0.02860.3251-0.18330.00480.074-0.21110.00760.2402-0.04550.329-0.21190.12830.45890.14670.4216-46.449977.5446-36.5892
172.03861.6263-0.226.7946-1.90940.58040.24920.3196-0.22480.8035-0.5215-0.9793-0.23160.28040.27230.6534-0.13730.11730.6819-0.02060.5097-51.669954.4809-28.6488
1814.02599.4903-3.85148.7759-1.37681.715-0.20830.55280.14640.65830.25390.21930.4101-0.1812-0.04560.6748-0.16430.110.4295-0.03610.3668-63.684736.7735-24.0019
194.41142.1657-2.96811.6932-1.30742.03810.3171-0.10540.14030.351-0.23520.1191-0.16580.0548-0.08190.666-0.32470.14570.3172-0.05270.2042-44.823764.0479-17.4342
Refinement TLS group

Refinement-ID: X-RAY DIFFRACTION

IDRefine TLS-IDAuth asym-IDAuth seq-ID
11A0 - 222
22A223 - 392
33A393 - 727
44B0 - 72
55B73 - 203
66B204 - 346
77B347 - 454
88B455 - 497
99B498 - 623
1010B624 - 698
1111B699 - 749
1212C0 - 253
1313C254 - 542
1414C543 - 740
1515M0 - 14
1615N1
1716R1 - 10
1817R11 - 15
1918R16 - 21
2019V1 - 14

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