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- PDB-5msg: Influenza B polymerase bound to vRNA promoter and capped RNA primer -

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Basic information

Entry
Database: PDB / ID: 5msg
TitleInfluenza B polymerase bound to vRNA promoter and capped RNA primer
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-D(*(GDM))-R(P*AP*AP*UP*C)-3')
  • RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*UP*CP*UP*GP*CP*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 / influenza B virus RNA-dependent RNA polymerase / vRNA promoter / capped RNA primer
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 / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase ...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 / 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 PB2 / 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-dependent RNA polymerase subunit PB2 / 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 6th domain / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical 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
PHOSPHATE ION / RNA / RNA (> 10) / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza B virus
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.8 Å
AuthorsCusack, S. / Guilligay, D.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council322586 France
CitationJournal: Nucleic Acids Res. / Year: 2017
Title: An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
Authors: Reich, S. / Guilligay, D. / Cusack, S.
History
DepositionJan 4, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 8, 2017Provider: repository / Type: Initial release
Revision 1.1May 3, 2017Group: Database references
Revision 1.2Jan 17, 2024Group: Advisory / Author supporting evidence ...Advisory / Author supporting evidence / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_audit_support / pdbx_initial_refinement_model / pdbx_validate_polymer_linkage
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 2.0Mar 6, 2024Group: Data collection / Non-polymer description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / entity
Item: _chem_comp.formula / _chem_comp.formula_weight / _entity.formula_weight

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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*AP*AP*UP*C)-3')
R: RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*UP*CP*UP*GP*CP*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)277,7578
Polymers277,5676
Non-polymers1902
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area45710 Å2
ΔGint-277 kcal/mol
Surface area88300 Å2
MethodPISA
Unit cell
Length a, b, c (Å)200.410, 200.410, 254.610
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 , 3 types, 3 molecules ABC

#1: Protein Polymerase acidic protein


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


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


Mass: 90844.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: PB2 subunit with N-terminal linker and C-terminal STREP tag + TEV site
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*AP*AP*UP*C)-3')


Mass: 4550.729 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: capped 13-mer primer / 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*U)-3')


Mass: 5584.283 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: vRNA promoter 3' end nucleotides 1-18 / Source: (synth.) Influenza B virus
#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 / Source method: obtained synthetically / Details: vRNA promoter 5' end nucleotides 1-14 / Source: (synth.) Influenza B virus

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Non-polymers , 1 types, 2 molecules

#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4

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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: 76.87 % / Description: diamond
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 4
Details: 35 microM of FluB polymerase was mixed with 40 microM of the vRNA promoter and 40 microM 13-mer capped RNA primer in a buffer containing 500 mM NaCl, 50 mM HEPES pH 7.5, 5% glycerol and 2 mM ...Details: 35 microM of FluB polymerase was mixed with 40 microM of the vRNA promoter and 40 microM 13-mer capped RNA primer in a buffer containing 500 mM NaCl, 50 mM HEPES pH 7.5, 5% glycerol and 2 mM TCEP. The best diffracting crystals appeared in 100 mM sodium acetate pH 3.8 - 4.0 and 150 mM di-ammonium phosphate
PH range: 3.8-4.0

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 1.2724 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 2, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2724 Å / Relative weight: 1
ReflectionResolution: 3.8→173.56 Å / Num. obs: 58685 / % possible obs: 99.9 % / Redundancy: 46.4 % / CC1/2: 0.999 / Rmerge(I) obs: 0.411 / Net I/σ(I): 9.76
Reflection shellResolution: 3.8→4 Å / Redundancy: 4.1 % / Rmerge(I) obs: 2.743 / Mean I/σ(I) obs: 1.2 / CC1/2: 0.72 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4WRT

4wrt


Resolution: 3.8→173.56 Å / Cor.coef. Fo:Fc: 0.947 / Cor.coef. Fo:Fc free: 0.927 / SU B: 46.916 / SU ML: 0.563 / Cross valid method: THROUGHOUT / ESU R Free: 0.59 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26745 2785 4.7 %RANDOM
Rwork0.23471 ---
obs0.23631 55900 99.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 172.682 Å2
Baniso -1Baniso -2Baniso -3
1--6.58 Å2-3.29 Å2-0 Å2
2---6.58 Å20 Å2
3---21.34 Å2
Refinement stepCycle: 1 / Resolution: 3.8→173.56 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17457 769 10 0 18236
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.01918650
X-RAY DIFFRACTIONr_bond_other_d0.0010.0217203
X-RAY DIFFRACTIONr_angle_refined_deg0.9961.93525294
X-RAY DIFFRACTIONr_angle_other_deg0.8692.99940060
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.12152188
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.49324.377802
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.967153370
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.17815118
X-RAY DIFFRACTIONr_chiral_restr0.0540.22774
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.02119930
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023720
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.00217.5538770
X-RAY DIFFRACTIONr_mcbond_other2.99717.5548769
X-RAY DIFFRACTIONr_mcangle_it5.21226.32410952
X-RAY DIFFRACTIONr_mcangle_other5.21326.32410953
X-RAY DIFFRACTIONr_scbond_it2.37317.769880
X-RAY DIFFRACTIONr_scbond_other2.37317.769879
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.38926.54214343
X-RAY DIFFRACTIONr_long_range_B_refined8.17221371
X-RAY DIFFRACTIONr_long_range_B_other8.17221372
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.397 199 -
Rwork0.424 4074 -
obs--99.98 %

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