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- PDB-9f2r: Influenza A/H17N10 polymerase with bound promoter and 3' end of t... -

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Basic information

Entry
Database: PDB / ID: 9f2r
TitleInfluenza A/H17N10 polymerase with bound promoter and 3' end of template in active site
Components
  • 3' end of the promoter vRNA
  • 5' end of the promoter vRNA
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / RNA-dependent RNA polymerase
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / GTP binding / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
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 / : ...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 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / 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
7N-METHYL-8-HYDROGUANOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.959 Å
AuthorsCusack, S. / Drncova, P.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE11-0028 France
CitationJournal: To Be Published
Title: High-resolution structure of a replication-initiation like configuration of influenza polymerase active site visualises the essential role of a conserved dibasic motif in the PA subunit
Authors: Cusack, S. / Drncova, P. / Krischuns, T. / Naffakh, N.
History
DepositionApr 23, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release

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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
R: 3' end of the promoter vRNA
V: 5' end of the promoter vRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)277,06810
Polymers276,2285
Non-polymers8405
Water19,6901093
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area44380 Å2
ΔGint-256 kcal/mol
Surface area85310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.080, 119.267, 251.555
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Polymerase acidic protein


Mass: 85490.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal his-tag and C-terminal linker and TEV cleavage site
Source: (gene. exp.) Influenza A virus / Strain: A/little-yellow-shouldered-bat/H17N10 / Gene: PA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM92
#2: Protein RNA-directed RNA polymerase catalytic subunit


Mass: 87936.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal linker and C-terminal linker and TEV cleavage site
Source: (gene. exp.) Influenza A virus / Strain: A/little-yellow-shouldered-bat/H17N10 / Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM91, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2


Mass: 91027.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal linker and C-terminal linker and double STREP tag
Source: (gene. exp.) Influenza A virus / Strain: A/little-yellow-shouldered-bat/H17N10 / Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM90

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RNA chain , 2 types, 2 molecules RV

#4: RNA chain 3' end of the promoter vRNA


Mass: 6525.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus
#5: RNA chain 5' end of the promoter vRNA


Mass: 5248.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus

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Non-polymers , 4 types, 1098 molecules

#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-MGT / 7N-METHYL-8-HYDROGUANOSINE-5'-TRIPHOSPHATE


Mass: 539.223 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H20N5O14P3 / Feature type: SUBJECT OF INVESTIGATION
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1093 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.47 Å3/Da / Density % sol: 50.27 % / Description: long prisms
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 5 mg/ml polymerase with 5.1 mg/ml (19.5 microM) with 1.14 x molar excess of each RNA (v5' 1-16, v3' 1-18+3 and 15-mer capped primer), mixed in 1:1 ratio of 100 mM amino acids, 100 mM ...Details: 5 mg/ml polymerase with 5.1 mg/ml (19.5 microM) with 1.14 x molar excess of each RNA (v5' 1-16, v3' 1-18+3 and 15-mer capped primer), mixed in 1:1 ratio of 100 mM amino acids, 100 mM Tris/Bicine pH8.5, 8% ethylene glycol (v/v), 4% PEG 8000 (w/v) by hanging drop at room temperature

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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 / Detector: PIXEL / Date: Dec 13, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.959→125.799 Å / Num. obs: 135837 / % possible obs: 69.7 % / Redundancy: 13.3 % / CC1/2: 0.997 / Rmerge(I) obs: 0.159 / Rrim(I) all: 0.166 / Net I/σ(I): 9.7
Reflection shellResolution: 1.965→2.189 Å / Redundancy: 10.7 % / Rmerge(I) obs: 1.227 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 345 / CC1/2: 0.736 / Rrim(I) all: 1.29 / % possible all: 68.4

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Processing

Software
NameVersionClassification
REFMAC5.8.0425refinement
autoPROCdata reduction
autoPROCdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.959→125.799 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.92 / SU B: 5.052 / SU ML: 0.137 / Cross valid method: FREE R-VALUE / ESU R: 0.262 / ESU R Free: 0.214
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2455 6693 4.927 %
Rwork0.1892 129144 -
all0.192 --
obs-135837 69.07 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 45.222 Å2
Baniso -1Baniso -2Baniso -3
1--0.06 Å20 Å20 Å2
2--0.204 Å2-0 Å2
3----0.144 Å2
Refinement stepCycle: LAST / Resolution: 1.959→125.799 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17427 631 52 1093 19203
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.01218732
X-RAY DIFFRACTIONr_bond_other_d0.0010.01617541
X-RAY DIFFRACTIONr_angle_refined_deg1.4071.83925454
X-RAY DIFFRACTIONr_angle_other_deg0.4871.77240449
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.77852213
X-RAY DIFFRACTIONr_dihedral_angle_2_deg6.6355138
X-RAY DIFFRACTIONr_dihedral_angle_other_2_deg0.257523
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.967103329
X-RAY DIFFRACTIONr_dihedral_angle_6_deg13.80110851
X-RAY DIFFRACTIONr_chiral_restr0.0690.22827
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0221740
X-RAY DIFFRACTIONr_gen_planes_other0.0010.024362
X-RAY DIFFRACTIONr_nbd_refined0.2170.24041
X-RAY DIFFRACTIONr_symmetry_nbd_other0.190.217138
X-RAY DIFFRACTIONr_nbtor_refined0.1830.29040
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0780.29935
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.160.21083
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0530.23
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1430.28
X-RAY DIFFRACTIONr_nbd_other0.1520.271
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1530.214
X-RAY DIFFRACTIONr_mcbond_it3.274.6368810
X-RAY DIFFRACTIONr_mcbond_other3.274.6368810
X-RAY DIFFRACTIONr_mcangle_it5.1478.30611037
X-RAY DIFFRACTIONr_mcangle_other5.1478.30611038
X-RAY DIFFRACTIONr_scbond_it3.4534.9419922
X-RAY DIFFRACTIONr_scbond_other3.4534.9419923
X-RAY DIFFRACTIONr_scangle_it5.6358.91614417
X-RAY DIFFRACTIONr_scangle_other5.6358.91614418
X-RAY DIFFRACTIONr_lrange_it8.10842.82421706
X-RAY DIFFRACTIONr_lrange_other8.08642.69221499
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.959-2.010.353150.3173300.318143770.8850.9282.39970.314
2.01-2.0650.319480.2579980.259140540.9430.9587.44270.256
2.065-2.1250.2581000.25618680.256136460.9570.95914.42180.254
2.125-2.1910.31740.25133870.254132870.9440.96126.80060.245
2.191-2.2620.3113040.24258690.245129060.9390.96347.83050.233
2.262-2.3420.2664230.23382210.235125090.9530.96669.10220.221
2.342-2.430.2765410.22199830.223119920.9510.96987.75850.204
2.43-2.5290.2815400.209108100.213116400.9520.97297.50860.191
2.529-2.6420.2625610.207105220.209110930.9580.97399.90990.188
2.642-2.7710.2625610.194101110.198106720.9570.9761000.178
2.771-2.920.2615180.18996320.193101500.9550.9781000.177
2.92-3.0970.2374730.18991380.19196110.9630.9781000.178
3.097-3.3110.2454250.19286440.19590690.9630.9781000.186
3.311-3.5760.2453860.19880750.20184610.9650.9791000.196
3.576-3.9170.2433860.18974020.19177900.9660.98199.97430.192
3.917-4.3790.223320.15967650.16270980.9720.98599.98590.171
4.379-5.0560.1993110.14959760.15262890.9780.98799.96820.169
5.056-6.1890.2482490.1851080.18353600.9720.98499.9440.202
6.189-8.7420.2362150.17239930.17542090.9740.98599.97620.203
8.742-125.7990.2611310.21223140.21424560.9510.9699.55210.373

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