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- PDB-4wsa: Crystal structure of Influenza B polymerase bound to the vRNA pro... -

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Basic information

Entry
Database: PDB / ID: 4wsa
TitleCrystal structure of Influenza B polymerase bound to the vRNA promoter (FluB1 form)
Components
  • Influenza B vRNA promoter 3' end
  • Influenza B vRNA promoter 5' end
  • PA
  • PB2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsTRANSFERASE/RNA / TRANSFERASE-RNA COMPLEX
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 / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase ...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 / 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
RNA / RNA (> 10) / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza B virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / Resolution: 3.4 Å
AuthorsReich, S. / Guilligay, D. / Pflug, A. / Cusack, S.
CitationJournal: Nature / Year: 2014
Title: Structural insight into cap-snatching and RNA synthesis by influenza polymerase.
Authors: Reich, S. / Guilligay, D. / Pflug, A. / Malet, H. / Berger, I. / Crepin, T. / Hart, D. / Lunardi, T. / Nanao, M. / Ruigrok, R.W. / Cusack, S.
History
DepositionOct 26, 2014Deposition site: RCSB / Processing site: PDBE
Revision 1.0Nov 19, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 3, 2014Group: Database references
Revision 1.2Dec 24, 2014Group: Database references
Revision 1.3Apr 3, 2019Group: Data collection / Source and taxonomy / Category: entity_src_gen / Item: _entity_src_gen.pdbx_host_org_cell_line

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Influenza B vRNA promoter 3' end
V: Influenza B vRNA promoter 5' end
A: PA
B: RNA-directed RNA polymerase catalytic subunit
C: PB2


Theoretical massNumber of molelcules
Total (without water)273,0165
Polymers273,0165
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area40320 Å2
ΔGint-246 kcal/mol
Surface area90160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)199.700, 199.700, 252.680
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: RNA chain Influenza B vRNA promoter 3' end


Mass: 5584.283 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus
#2: RNA chain Influenza B vRNA promoter 5' end


Mass: 4557.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus
#3: Protein PA


Mass: 85822.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal extension: his-tag and linker C-terminal extension: linker and TEV site
Source: (gene. exp.) Influenza B virus / Strain: B/Memphis/13/03 / Gene: PA / Plasmid: pKL-PBac / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8Z9
#4: Protein RNA-directed RNA polymerase catalytic subunit


Mass: 86207.016 Da / Num. of mol.: 1 / Mutation: Trichoplusia ni
Source method: isolated from a genetically manipulated source
Details: N-terminal extension: linker C-terminal extension: linker and TEV site
Source: (gene. exp.) Influenza B virus / Strain: B/Memphis/13/03 / Gene: PB1 / Plasmid: pKL-PBac / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8Y6, RNA-directed RNA polymerase
#5: Protein PB2


Mass: 90844.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal extension: linker C-terminal extension: Strep-tag and TEV site
Source: (gene. exp.) Influenza B virus / Strain: B/Memphis/13/03 / Gene: PB2 / Plasmid: pKL-PBac / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8X3

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 5.33 Å3/Da / Density % sol: 75 % / Description: bipyramids
Crystal growTemperature: 281 K / Method: vapor diffusion, sitting drop / pH: 9
Details: FluB polymerase was concentrated to 9 mg per ml (37 microM) in a buffer containing 500 mM NaCl, 50 mM Hepes pH 7.5, 5% glycerol and 2mM Tris(2-carboxyethyl)phosphine (TCEP), and mixed with ...Details: FluB polymerase was concentrated to 9 mg per ml (37 microM) in a buffer containing 500 mM NaCl, 50 mM Hepes pH 7.5, 5% glycerol and 2mM Tris(2-carboxyethyl)phosphine (TCEP), and mixed with 40 microM vRNA for crystallization in hanging drops at 4 C. A trigonal crystal form (FluB1) was obtained by mixing polymerase with nucleotides 5-18 of the 3 prime end and 1-14 of the 5 prime end of the vRNA in a condition containing 0.1 M bicine pH 9.0, 10% MPD.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.973 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Apr 4, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.973 Å / Relative weight: 1
ReflectionResolution: 3.4→50 Å / Num. obs: 79266 / % possible obs: 98.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 11.8 % / Rmerge(I) obs: 0.121 / Net I/σ(I): 12.6
Reflection shellResolution: 3.4→3.52 Å / Redundancy: 5.8 % / Rmerge(I) obs: 1.3 / Mean I/σ(I) obs: 1.5 / % possible all: 89.9

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Processing

Software
NameVersionClassification
PHENIX(phenix.refine: dev_1760)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
SHARPphasing
RefinementMethod to determine structure: SIRAS / Resolution: 3.4→48.978 Å / SU ML: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 32.94 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2653 3820 4.82 %
Rwork0.2286 --
obs0.2304 79233 98.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: 1 / Resolution: 3.4→48.978 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17351 589 0 0 17940
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00318361
X-RAY DIFFRACTIONf_angle_d0.65724866
X-RAY DIFFRACTIONf_dihedral_angle_d12.2047104
X-RAY DIFFRACTIONf_chiral_restr0.0282756
X-RAY DIFFRACTIONf_plane_restr0.0033088
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.4001-3.44310.44591170.35662159X-RAY DIFFRACTION78
3.4431-3.48840.35641490.31952672X-RAY DIFFRACTION96
3.4884-3.53620.34431250.32992776X-RAY DIFFRACTION99
3.5362-3.58670.35181550.29842725X-RAY DIFFRACTION98
3.5867-3.64020.35821440.29442788X-RAY DIFFRACTION98
3.6402-3.69710.31071740.29512742X-RAY DIFFRACTION98
3.6971-3.75760.36651160.28252796X-RAY DIFFRACTION98
3.7576-3.82240.35361480.29032717X-RAY DIFFRACTION98
3.8224-3.89190.3071280.29092749X-RAY DIFFRACTION97
3.8919-3.96670.31291430.26642788X-RAY DIFFRACTION100
3.9667-4.04760.28461430.26522796X-RAY DIFFRACTION100
4.0476-4.13560.30391270.26732815X-RAY DIFFRACTION100
4.1356-4.23180.28091460.24232839X-RAY DIFFRACTION100
4.2318-4.33750.29551600.24442836X-RAY DIFFRACTION100
4.3375-4.45470.2531350.23492805X-RAY DIFFRACTION100
4.4547-4.58570.2851440.23382826X-RAY DIFFRACTION100
4.5857-4.73360.23671530.23082799X-RAY DIFFRACTION100
4.7336-4.90270.26691370.22462824X-RAY DIFFRACTION100
4.9027-5.09880.27541240.22642874X-RAY DIFFRACTION100
5.0988-5.33050.25871180.21042862X-RAY DIFFRACTION100
5.3305-5.61120.26121560.21952816X-RAY DIFFRACTION100
5.6112-5.96220.22611320.2132878X-RAY DIFFRACTION100
5.9622-6.42160.2641320.22762874X-RAY DIFFRACTION100
6.4216-7.06610.28221440.21452864X-RAY DIFFRACTION100
7.0661-8.08470.24681370.20642893X-RAY DIFFRACTION100
8.0847-10.17080.21181610.16812899X-RAY DIFFRACTION100
10.1708-48.98320.24091720.21953001X-RAY DIFFRACTION100

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