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

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Entry
Database: PDB / ID: 6qcx
TitleCrystal structure of influenza B polymerase initiation state with capped 15-mer RNA primer
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*GP*C)-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: 3.08 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

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
M: RNA (5'-D(*(GDM))-R(P*GP*AP*AP*UP*GP*CP*UP*AP*UP*AP*AP*UP*AP*GP*C)-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)279,4949
Polymers279,1996
Non-polymers2953
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area50350 Å2
ΔGint-317 kcal/mol
Surface area87670 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)200.407, 200.407, 256.372
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*GP*C)-3')


Mass: 5241.142 Da / Num. of mol.: 1 / Details: Capped 15-mer 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 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 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-POP / PYROPHOSPHATE 2-


Mass: 175.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H2O7P2 / Pyrophosphate
#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: 5.32 Å3/Da / Density % sol: 76.88 %
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 15-mer capped RNA. The mother liquor contained 200 mM di-ammonium phosphate and 100 mM sodium acetate between pH 4.0 and 4.4.
PH range: 4.0-4.4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.966 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: May 29, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.966 Å / Relative weight: 1
ReflectionResolution: 3.08→50 Å / Num. obs: 83143 / % possible obs: 75.2 % / Redundancy: 4.8 % / CC1/2: 0.999 / Rrim(I) all: 0.086 / Rsym value: 0.076 / Net I/σ(I): 14.3
Reflection shellResolution: 3.08→3.27 Å / Mean I/σ(I) obs: 1.7 / CC1/2: 0.54 / Rrim(I) all: 0.888 / Rsym value: 0.756 / % possible all: 61

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Processing

Software
NameVersionClassification
REFMAC5.8.0232refinement
XDSdata reduction
AutoPROCdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5MSG
Resolution: 3.08→173.56 Å / Cor.coef. Fo:Fc: 0.865 / Cor.coef. Fo:Fc free: 0.93 / SU B: 45.732 / SU ML: 0.324 / Cross valid method: THROUGHOUT / ESU R: 1.37 / ESU R Free: 0.376 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22582 4024 4.8 %RANDOM
Rwork0.20243 ---
Obs0.20358 79129 75.17 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 114.342 Å2
Baniso -1Baniso -2Baniso -3
1-0.33 Å20.16 Å20 Å2
2--0.33 Å2-0 Å2
3----1.05 Å2
Refinement stepCycle: 1 / Resolution: 3.08→173.56 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17483 1084 15 0 18582
Refine LS restraints

Refinement-ID: X-RAY DIFFRACTION

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0020.01319035
r_bond_other_d0.0010.01717371
r_angle_refined_deg1.1411.62225892
r_angle_other_deg1.0711.61940471
r_dihedral_angle_1_deg5.32352192
r_dihedral_angle_2_deg31.2422.53921
r_dihedral_angle_3_deg15.769153370
r_dihedral_angle_4_deg15.19115118
r_chiral_restr0.0390.22519
r_gen_planes_refined0.0020.0220262
r_gen_planes_other0.0010.023916
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_it0.4654.6798786
r_mcbond_other0.4654.6798785
r_mcangle_it0.8337.01910972
r_mcangle_other0.8337.01910973
r_scbond_it0.4244.72710249
r_scbond_other0.4244.72710248
r_scangle_it
r_scangle_other0.7757.07114921
r_long_range_B_refined2.30155.99221774
r_long_range_B_other2.30155.99121775
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
LS refinement shellResolution: 3.076→3.156 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.31 48 -
Rwork0.312 917 -
Obs--11.95 %
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)
12.5687-0.41071.25950.4141-0.21310.81090.46511.1789-0.8474-0.7461-0.21750.30490.63370.1704-0.24762.2118-0.35380.04671.8132-0.37381.3704-72.514323.5931-68.2612
21.01560.215-0.33383.19380.11791.63390.2689-0.05020.09950.462-0.28130.0719-0.08280.18090.01240.6184-0.44560.05940.3634-0.04540.4707-51.141160.3379-6.5253
31.79450.67930.03711.7865-0.71331.1840.2496-0.29020.31930.7173-0.20370.1474-0.1035-0.0681-0.04591.0238-0.49470.28220.2885-0.16870.4326-66.178157.50062.8958
40.74380.5264-0.27491.2614-0.91671.88580.3703-0.1806-0.02130.1908-0.3158-0.37980.31440.4165-0.05450.7829-0.37130.02420.3973-0.08760.5408-49.777138.5179-10.1644
51.11760.1587-0.09772.0858-1.61441.4036-0.05630.3864-0.2414-0.2029-0.0858-0.26490.4829-0.00360.14210.8839-0.16720.19280.6385-0.17420.6236-52.067936.4208-39.4592
61.52820.2180.01731.2876-0.55460.78640.02570.1831-0.3110.02580.0058-0.34180.57060.021-0.03141.0172-0.26080.14980.3562-0.10340.6516-54.723728.9512-24.8782
71.30942.42380.71366.94811.65241.67220.2155-0.1697-0.2580.0766-0.2349-0.47950.34370.2280.01940.6539-0.16130.12210.5850.04770.6058-34.318346.3574-22.1903
83.021-1.0782-1.94233.0889-3.109910.2684-0.0655-0.157-0.44730.4297-0.2773-0.29910.4287-0.09040.34280.9621-0.30630.00610.2696-0.0390.73-58.889317.2858-8.7205
96.3522-3.1599-1.23554.0143-1.55563.2660.0257-0.3830.05720.2651-0.2783-0.60380.46120.4470.25260.9898-0.3520.01430.2775-0.06730.6339-56.46423.8724-2.6967
102.02980.43250.09691.4895-0.81943.80430.10320.21290.55420.25670.04280.2816-0.1122-0.4768-0.14610.5956-0.32360.32420.3034-0.03490.6186-82.535259.3095-19.1586
111.2426-2.034-2.74213.58684.72886.29340.21210.4167-0.2796-0.0465-0.60930.1002-0.0464-0.90930.39731.0445-0.3657-0.05590.94210.07140.888-69.431556.6737-42.2348
125.2014-0.2727-0.22842.7535-0.23174.33210.34931.21390.5048-1.0279-0.14120.46050.1679-0.6205-0.20821.1381-0.27930.02351.15770.03960.6808-76.326945.6892-62.909
131.21050.4209-0.42651.4986-0.47381.9308-0.02240.65820.0966-0.36280.08880.48480.4735-0.2998-0.06640.7576-0.32020.11510.6030.020.6755-81.408248.4226-35.072
144.13860.075-0.57033.10020.55542.91730.05971.1118-0.7046-0.68680.0656-0.20610.4792-0.1217-0.12531.2357-0.55230.12010.7461-0.11310.8872-98.247711.5345-22.6559
153.7436-1.5631.03674.6289-1.48223.92950.1038-0.8006-0.29630.7532-0.03820.010.1296-0.0555-0.06561.1343-0.60650.14560.49460.02220.4937-82.595917.110316.9204
160.6560.18270.80881.74692.23123.431-0.14060.51610.2157-0.42610.2239-0.374-0.46730.5883-0.08331.4388-0.33280.02161.07080.01781.2758-81.335228.9354-25.7299
179.06174.05970.72862.1644-0.13745.2547-0.23070.46030.342-0.00230.0321-0.2211-0.11070.17180.19860.3642-0.25090.16740.49780.08650.6974-46.44577.4712-36.6328
180.1329-0.40120.09186.7086-1.03590.19450.15540.12710.02490.8997-0.3029-0.9804-0.12550.21470.14740.7792-0.11360.11050.8615-0.05110.6983-51.685654.4586-28.6496
195.52911.8002-5.3468.3442-3.31455.501-0.5022-0.0318-0.32220.80840.2910.69190.30630.03710.21130.9019-0.17730.2390.4714-0.00010.7376-62.825438.0959-20.3107
204.47153.1305-2.17432.7058-1.61711.08210.16530.06370.22890.2125-0.1161-0.0306-0.0147-0.0359-0.04930.8881-0.42890.17950.24830.01860.4508-44.810364.0008-17.4303
Refinement TLS group

Refinement-ID: X-RAY DIFFRACTION

IDRefine TLS-IDAuth asym-IDAuth seq-ID
11A0 - 222
22A223 - 392
33A393 - 724
44B0 - 72
55B73 - 203
66B204 - 346
77B347 - 413
88B414 - 454
99B455 - 497
1010B498 - 623
1111B624 - 698
1212B699 - 749
1313C0 - 253
1414C254 - 542
1515C543 - 740
1616M0 - 10
1717R1 - 10
1818R11 - 15
1919R16 - 21
2019M11 - 15
2120V1 - 14

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