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- PDB-5vqn: E119D mutant of 2009 H1N1 PA Endonuclease in complex with RO-7 -

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Basic information

Entry
Database: PDB / ID: 5vqn
TitleE119D mutant of 2009 H1N1 PA Endonuclease in complex with RO-7
ComponentsPolymerase acidic protein
KeywordsHYDROLASE/HYDROLASE INHIBITOR / Nuclease Influenza Resistance Inhibitor / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


cap snatching / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / viral RNA genome replication / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / 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 / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
: / Chem-R07 / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.002 Å
AuthorsKumar, G. / White, S.W.
Funding support United States, Switzerland, 3items
OrganizationGrant numberCountry
Department of Health & Human Services (HHS)HHSN272201400006C United States
Roche Innovation Center Switzerland
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: MBio / Year: 2018
Title: Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors.
Authors: Jones, J.C. / Kumar, G. / Barman, S. / Najera, I. / White, S.W. / Webby, R.J. / Govorkova, E.A.
History
DepositionMay 9, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 11, 2018Provider: repository / Type: Initial release
Revision 1.1May 9, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_DOI ..._citation.journal_abbrev / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.3Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polymerase acidic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,9246
Polymers23,1341
Non-polymers7895
Water37821
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Protein elutes as a monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area520 Å2
ΔGint-35 kcal/mol
Surface area9020 Å2
Unit cell
Length a, b, c (Å)89.848, 89.848, 133.261
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number97
Space group name H-MI422

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Components

#1: Protein Polymerase acidic protein


Mass: 23134.316 Da / Num. of mol.: 1 / Mutation: E119D, Loop replaced with GGS linker
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: swl A/California/04/2009 H1N1 / Gene: PA / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: C3W5S0
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-R07 / 1-[(11S)-6,11-dihydrodibenzo[b,e]thiepin-11-yl]-5-hydroxy-3-[(2R)-1,1,1-trifluoropropan-2-yl]-2,3-dihydro-1H-pyrido[2,1-f][1,2,4]triazine-4,6-dione / RO-7


Mass: 487.494 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H20F3N3O3S
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / 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: 2.91 Å3/Da / Density % sol: 57.68 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 9.5
Details: 0.1 M CAPSO pH 9.5, 1 M Ammonium Sulfate, 50 mM MgCl2, 50 mM MnCl2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Dec 17, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2→50 Å / Num. obs: 17177 / % possible obs: 91.6 % / Redundancy: 8.5 % / Biso Wilson estimate: 42.82 Å2 / Rmerge(I) obs: 0.089 / Rpim(I) all: 0.031 / Rrim(I) all: 0.095 / Χ2: 1.208 / Net I/σ(I): 9.3
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.074.60.8560.7010.4070.9540.64453.4
2.07-2.155.40.6360.8860.2730.6950.67968.8
2.15-2.2560.6170.8870.2520.6690.73392.5
2.25-2.377.80.6060.9560.2240.6470.73999.7
2.37-2.529.20.5010.9740.1710.530.77599.9
2.52-2.719.90.3680.9850.1210.3880.84799.9
2.71-2.99100.2050.9940.0670.2161.03100
2.99-3.429.90.1140.9970.0380.121.434100
3.42-4.319.80.0710.9980.0240.0751.99699.9
4.31-509.10.0560.9980.0190.062.01699.6

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation5.37 Å45.98 Å
Translation5.37 Å45.98 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.7.16phasing
PHENIXrefinement
PDB_EXTRACT3.22data extraction
HKL-2000data reduction
SERGUIdata collection
HKLdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5CZN

5czn


Resolution: 2.002→38.47 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.71
RfactorNum. reflection% reflection
Rfree0.2416 900 5.25 %
Rwork0.1972 --
obs0.1993 17151 91.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 176.32 Å2 / Biso mean: 70.0541 Å2 / Biso min: 38.49 Å2
Refinement stepCycle: final / Resolution: 2.002→38.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1436 0 66 21 1523
Biso mean--68.45 59.74 -
Num. residues----179
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0051513
X-RAY DIFFRACTIONf_angle_d0.9092045
X-RAY DIFFRACTIONf_chiral_restr0.048214
X-RAY DIFFRACTIONf_plane_restr0.004258
X-RAY DIFFRACTIONf_dihedral_angle_d15.36886
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.0023-2.12770.3664770.29051702177958
2.1277-2.2920.30161470.252624277190
2.292-2.52260.25961950.222728683063100
2.5226-2.88750.25641750.210629363111100
2.8875-3.63760.2541630.211529643127100
3.6376-38.47760.21531430.17531573300100
Refinement TLS params.Method: refined / Origin x: 37.8108 Å / Origin y: 110.6125 Å / Origin z: 284.6096 Å
111213212223313233
T0.5894 Å2-0.1082 Å20.1392 Å2-0.3427 Å20.0409 Å2--0.431 Å2
L3.5293 °2-0.5308 °20.6468 °2-1.3301 °2-0.3676 °2--1.8007 °2
S0.2141 Å °-0.0859 Å °-0.178 Å °0.0726 Å °-0.2597 Å °0.0563 Å °0.6151 Å °-0.1064 Å °0.0003 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA-2 - 176
2X-RAY DIFFRACTION1allB201 - 202
3X-RAY DIFFRACTION1allC203 - 204
4X-RAY DIFFRACTION1allS1 - 28
5X-RAY DIFFRACTION1allD1

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