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- PDB-6fhh: Crystal structure of bat influenza A/H17N10 polymerase with viral... -

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Basic information

Entry
Database: PDB / ID: 6fhh
TitleCrystal structure of bat influenza A/H17N10 polymerase with viral RNA promoter bound to a 22-mer modified Pol II CTD peptide with serine 5 thiophosphorylated.
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*U)-3')
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*GP*GP*G)-3')
  • RNA-directed RNA polymerase catalytic subunit
  • TYR-SER-PRO-THR-TPS-PRO-SER
KeywordsVIRAL PROTEIN / Influenza virus / RNA-dependent RNA polymerase / modified Pol II CTD peptide
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 / host cell cytoplasm / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity ...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 / host cell cytoplasm / 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 / cytoplasm
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / : / : / : ...Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / 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 / 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. / 3-Layer(aba) Sandwich / Alpha Beta
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 A virus
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsLukarska, M. / Cusack, S.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council322586 France
CitationJournal: To Be Published
Title: Towards New Anti-Influenza Therapeutics: Structure-Activity Relationships in the Interaction between Heterotrimeric Influenza Polymerase and Pol II C-terminal domain
Authors: Claron, M. / Lukarska, M. / Uhrig, U. / Sehr, P. / Drncova, P. / Lewis, J.D. / Will, D.W. / Cusack, S.
History
DepositionJan 14, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 30, 2019Provider: repository / Type: Initial release
Revision 2.0Feb 1, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Other / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / cell ...atom_site / cell / chem_comp / database_2 / entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_entity_nonpoly / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / struct_asym / struct_ref / struct_ref_seq
Item: _atom_site.label_entity_id / _cell.Z_PDB ..._atom_site.label_entity_id / _cell.Z_PDB / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_entity_nonpoly.entity_id / _pdbx_nonpoly_scheme.entity_id / _pdbx_poly_seq_scheme.entity_id / _pdbx_poly_seq_scheme.mon_id / _pdbx_unobs_or_zero_occ_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id / _struct_asym.entity_id / _struct_ref_seq.ref_id
Revision 2.1Feb 7, 2024Group: Data collection / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_conn
Item: _struct_conn.pdbx_leaving_atom_flag

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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: RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*U)-3')
V: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*GP*GP*G)-3')
X: TYR-SER-PRO-THR-TPS-PRO-SER
Y: TYR-SER-PRO-THR-TPS-PRO-SER
hetero molecules


Theoretical massNumber of molelcules
Total (without water)282,03423
Polymers280,5447
Non-polymers1,49016
Water2,468137
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area47290 Å2
ΔGint-402 kcal/mol
Surface area90110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)267.770, 147.810, 88.410
Angle α, β, γ (deg.)90.00, 97.08, 90.00
Int Tables number5
Space group name H-MC121

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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 C-terminal linker plus TEV protease site
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(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 C-terminal linker and TEV protease site
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(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
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(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 RNA (5'-R(*UP*AP*UP*AP*CP*CP*UP*CP*UP*GP*CP*UP*U)-3')


Mass: 5584.283 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: vRNA promoter 3' end / Source: (synth.) Influenza A virus
#5: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*AP*CP*AP*AP*GP*AP*GP*GP*G)-3')


Mass: 5248.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: vRNA promoter 5' end / Source: (synth.) Influenza A virus

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Protein/peptide , 1 types, 2 molecules XY

#6: Protein/peptide TYR-SER-PRO-THR-TPS-PRO-SER


Mass: 2628.546 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Mimic of Pol II CTD with thiophospho-serine at position 5
Source: (synth.) Homo sapiens (human)

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Non-polymers , 3 types, 153 molecules

#7: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: PO4
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 137 / Source method: isolated from a natural source / Formula: H2O

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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: 3.09 Å3/Da / Density % sol: 60.2 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: Bat FluA polymerase bound to the ends of the RNA promotor (5 prime end nts 1-16, 3 prime end nts 1-18) was co-crystallized in a 1:8 ratio with the thiophospho-serine-5 22-mer Pol II CTD ...Details: Bat FluA polymerase bound to the ends of the RNA promotor (5 prime end nts 1-16, 3 prime end nts 1-18) was co-crystallized in a 1:8 ratio with the thiophospho-serine-5 22-mer Pol II CTD peptide mimic in 0.8 M sodium-potassium phosphate at pH 5.0. Crystals were set at 4 C using seeding.
PH range: 5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.97242 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 9, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97242 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 92066 / % possible obs: 98.6 % / Redundancy: 2.92 % / CC1/2: 0.996 / Rsym value: 0.049 / Net I/σ(I): 14.34
Reflection shellResolution: 2.7→2.77 Å / Redundancy: 2.69 % / Mean I/σ(I) obs: 1.87 / CC1/2: 0.761 / Rsym value: 0.5 / % possible all: 96.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5m3h

5m3h


Resolution: 2.7→50 Å / Cor.coef. Fo:Fc: 0.946 / Cor.coef. Fo:Fc free: 0.92 / SU B: 14.635 / SU ML: 0.286 / Cross valid method: THROUGHOUT / ESU R: 0.685 / ESU R Free: 0.327 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25763 4534 4.9 %RANDOM
Rwork0.2129 ---
obs0.21514 87532 98.6 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 90.198 Å2
Baniso -1Baniso -2Baniso -3
1-3.28 Å2-0 Å23.39 Å2
2---3.86 Å2-0 Å2
3----0.25 Å2
Refinement stepCycle: 1 / Resolution: 2.7→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17644 600 76 137 18457
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.01918724
X-RAY DIFFRACTIONr_bond_other_d0.0010.0217114
X-RAY DIFFRACTIONr_angle_refined_deg1.0851.93725402
X-RAY DIFFRACTIONr_angle_other_deg0.874339805
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.74152191
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.50524.097842
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.455153343
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.66915134
X-RAY DIFFRACTIONr_chiral_restr0.0620.22789
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.02120079
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023791
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_it2.9239.1378797
X-RAY DIFFRACTIONr_mcbond_other2.9239.1378796
X-RAY DIFFRACTIONr_mcangle_it4.88713.69910977
X-RAY DIFFRACTIONr_mcangle_other4.88713.69810978
X-RAY DIFFRACTIONr_scbond_it2.7789.3099927
X-RAY DIFFRACTIONr_scbond_other2.7519.2989868
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.75813.82514336
X-RAY DIFFRACTIONr_long_range_B_refined7.86420626
X-RAY DIFFRACTIONr_long_range_B_other7.86120617
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.703→2.773 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.361 340 -
Rwork0.328 6312 -
obs--96.41 %

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