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- PDB-5we9: Crystal structure of the influenza virus PA endonuclease in compl... -

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Basic information

Entry
Database: PDB / ID: 5we9
TitleCrystal structure of the influenza virus PA endonuclease in complex with inhibitor 7b (SRI-29731)
ComponentsPolymerase acidic protein
KeywordsHYDROLASE/HYDROLASE INHIBITOR / Virus / Nuclease / Transcription / Cap-snatching / HYDROLASE / 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 translational frameshifting / 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-GY7 / : / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.804 Å
AuthorsKumar, G. / White, S.W.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI098757 United States
St. Jude Children's Research Hospital (ALSAC) United States
St. Jude Children's Research Hospital (ALSAC) United States
CitationJournal: Sci Rep / Year: 2017
Title: Protein-Structure Assisted Optimization of 4,5-Dihydroxypyrimidine-6-Carboxamide Inhibitors of Influenza Virus Endonuclease.
Authors: Beylkin, D. / Kumar, G. / Zhou, W. / Park, J. / Jeevan, T. / Lagisetti, C. / Harfoot, R. / Webby, R.J. / White, S.W. / Webb, T.R.
History
DepositionJul 7, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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_alt_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_alt_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.pdbx_ptnr2_label_alt_id / _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)22,5805
Polymers21,9701
Non-polymers6104
Water2,270126
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Protein elutes as a monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area340 Å2
ΔGint-7 kcal/mol
Surface area9380 Å2
2
A: Polymerase acidic protein
hetero molecules

A: Polymerase acidic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,16010
Polymers43,9402
Non-polymers1,2208
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_465y-1,x+1,-z+1/31
Buried area1980 Å2
ΔGint-27 kcal/mol
Surface area17460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.081, 74.081, 127.694
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number181
Space group name H-MP6422

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Polymerase acidic protein


Mass: 21970.160 Da / Num. of mol.: 1
Mutation: Loop 51-72 is replaced a GGS linker, C-terminal has residual His-tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: swl A/California/04/2009 H1N1 / Gene: PA / Plasmid: pET52b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: C3W5S0

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Non-polymers , 5 types, 130 molecules

#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-GY7 / 2-[(2S)-1-(2,6-difluorobenzene-1-carbonyl)pyrrolidin-2-yl]-5-hydroxy-6-oxo-N-(2-phenylethyl)-1,6-dihydropyrimidine-4-carboxamide / SRI-29731


Mass: 468.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H22F2N4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 126 / 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: 2.3 Å3/Da / Density % sol: 46.57 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 0.1 M Tris pH 8.5, 30% PEG 4000, 0.2 M MgCl2, 2 mM MnCl2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 0.9789 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Apr 5, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9789 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. obs: 19618 / % possible obs: 99.5 % / Redundancy: 17.7 % / Biso Wilson estimate: 19.02 Å2 / Rmerge(I) obs: 0.056 / Rpim(I) all: 0.013 / Rrim(I) all: 0.057 / Χ2: 0.938 / Net I/σ(I): 11.5 / Num. measured all: 346860
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.8-1.867.70.9050.6880.3330.9680.76395.4
1.86-1.94140.890.9040.2410.9230.785100
1.94-2.0318.10.630.9630.150.6480.852100
2.03-2.1319.60.3710.9870.0850.3810.928100
2.13-2.2719.90.2330.9950.0530.2390.991100
2.27-2.4419.80.1640.9970.0370.1681.045100
2.44-2.6919.80.110.9980.0250.1131.177100
2.69-3.0819.70.0660.9990.0150.0671.057100
3.08-3.8819.40.0450.9990.010.0461.082100
3.88-5017.90.0250.9990.0060.0260.52399.5

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIXrefinement
PDB_EXTRACT3.22data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.804→35.574 Å / SU ML: 0.17 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 22.17
RfactorNum. reflection% reflection
Rfree0.2258 917 4.92 %
Rwork0.1895 --
obs0.1914 18640 94.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 77.66 Å2 / Biso mean: 30.4329 Å2 / Biso min: 7.35 Å2
Refinement stepCycle: final / Resolution: 1.804→35.574 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1445 0 74 126 1645
Biso mean--23.58 38.24 -
Num. residues----178
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0081552
X-RAY DIFFRACTIONf_angle_d0.9142091
X-RAY DIFFRACTIONf_chiral_restr0.056216
X-RAY DIFFRACTIONf_plane_restr0.006267
X-RAY DIFFRACTIONf_dihedral_angle_d5.152955
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 7

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8038-1.89890.2957760.24051683175964
1.8989-2.01780.23391260.20422481260795
2.0178-2.17360.22221250.187226542779100
2.1736-2.39230.24351400.186526362776100
2.3923-2.73840.26851570.193526612818100
2.7384-3.44960.25541280.191527242852100
3.4496-35.5810.18421650.179928843049100
Refinement TLS params.Method: refined / Origin x: -14.425 Å / Origin y: 75.618 Å / Origin z: 14.074 Å
111213212223313233
T0.2375 Å20.0173 Å20.1065 Å2-0.047 Å2-0.0817 Å2--0.2071 Å2
L2.1207 °2-0.138 °20.0953 °2-3.0081 °20.8169 °2--2.5165 °2
S-0.1001 Å °0.1097 Å °-0.48 Å °-0.1138 Å °-0.1192 Å °-0.1298 Å °0.4554 Å °0.1217 Å °0.1105 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND ( RESID 1:178 OR RESID 477:477 ) )A1 - 178
2X-RAY DIFFRACTION1( CHAIN A AND ( RESID 1:178 OR RESID 477:477 ) )A477

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