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- PDB-2ez9: Pyruvate oxidase variant F479W in complex with reaction intermedi... -

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Basic information

Entry
Database: PDB / ID: 2ez9
TitlePyruvate oxidase variant F479W in complex with reaction intermediate analogue 2-phosphonolactyl-thiamin diphosphate
ComponentsPyruvate oxidase
KeywordsOXIDOREDUCTASE / TPP enzyme / reaction intermediate analogue
Function / homology
Function and homology information


pyruvate oxidase / pyruvate oxidase activity / thiamine pyrophosphate binding / magnesium ion binding
Similarity search - Function
Pyruvate oxidase / : / : / : / TPP-binding enzyme, conserved site / Thiamine pyrophosphate enzymes signature. / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, N-terminal TPP-binding domain / Thiamine pyrophosphate enzyme, N-terminal TPP binding domain ...Pyruvate oxidase / : / : / : / TPP-binding enzyme, conserved site / Thiamine pyrophosphate enzymes signature. / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, central domain / Thiamine pyrophosphate enzyme, N-terminal TPP-binding domain / Thiamine pyrophosphate enzyme, N-terminal TPP binding domain / Thiamine pyrophosphate enzyme, C-terminal TPP-binding / Thiamine pyrophosphate enzyme, C-terminal TPP binding domain / Thiamin diphosphate (ThDP)-binding fold, Pyr/PP domains / TPP-binding domain / Thiamin diphosphate-binding fold / DHS-like NAD/FAD-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Chem-TDK / Pyruvate oxidase
Similarity search - Component
Biological speciesLactobacillus plantarum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsWille, G. / Meyer, D. / Steinmetz, A. / Hinze, E. / Golbik, R. / Tittmann, K.
CitationJournal: Nat.Chem.Biol. / Year: 2006
Title: The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.
Authors: Wille, G. / Meyer, D. / Steinmetz, A. / Hinze, E. / Golbik, R. / Tittmann, K.
History
DepositionNov 10, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 25, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Oct 18, 2017Group: Refinement description / Category: software
Revision 1.4Oct 20, 2021Group: Data collection / Database references / Derived calculations
Category: database_2 / diffrn_source ...database_2 / diffrn_source / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _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.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_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.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_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 / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Aug 23, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Remark 999SEQUENCE Author states that residue 561 is indeed a MET. The THR from the SWS database is an error ...SEQUENCE Author states that residue 561 is indeed a MET. The THR from the SWS database is an error in the database.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyruvate oxidase
B: Pyruvate oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,28410
Polymers132,4922
Non-polymers2,7928
Water18,5551030
1
A: Pyruvate oxidase
B: Pyruvate oxidase
hetero molecules

A: Pyruvate oxidase
B: Pyruvate oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,56820
Polymers264,9834
Non-polymers5,58516
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_565x,-y+1,-z1
Buried area32980 Å2
ΔGint-244 kcal/mol
Surface area69270 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)118.661, 154.517, 165.640
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-1513-

NA

21B-1511-

NA

31A-1684-

HOH

41B-1708-

HOH

DetailsThe biological assembly is a tetramer generated from the dimer in the asymmetric unit by the operation: x, -y+1, -z

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Pyruvate oxidase / Pyruvic oxidase / POX


Mass: 66245.836 Da / Num. of mol.: 2 / Mutation: F479W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus plantarum (bacteria) / Gene: pox5 / Plasmid: pBP200 / Production host: Escherichia coli (E. coli) / Strain (production host): C600 / References: UniProt: P37063, pyruvate oxidase

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Non-polymers , 5 types, 1038 molecules

#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Chemical ChemComp-TDK / 3-[(4-AMINO-2-METHYLPYRIMIDIN-5-YL)METHYL]-2-{(1S)-1-HYDROXY-1-[(R)-HYDROXY(METHOXY)PHOSPHORYL]ETHYL}-5-(2-{[(S)-HYDROXY(PHOSPHONOOXY)PHOSPHORYL]OXY}ETHYL)-4-METHYL-1,3-THIAZOL-3-IUM / 2-PHOSPHONOLACTYLTHIAMIN DIPHOSPHATE


Mass: 563.373 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H26N4O11P3S
#5: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1030 / 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: 2.9 Å3/Da / Density % sol: 56.8 %
Crystal growTemperature: 280 K / Method: vapor diffusion, hanging drop / pH: 5.7
Details: ammonium sulfate, potassium phosphate, pH 5.7, hanging drop, temperature 280K, VAPOR DIFFUSION, HANGING DROP

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: BW7B / Wavelength: 0.84
DetectorType: MAR scanner 345 mm plate / Detector: IMAGE PLATE / Date: Mar 16, 2005
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.84 Å / Relative weight: 1
ReflectionResolution: 1.6→19.62 Å / Num. all: 165388 / Num. obs: 165340 / % possible obs: 83 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 3.7 / Redundancy: 4.6 % / Biso Wilson estimate: 12.9 Å2 / Rmerge(I) obs: 0.089 / Χ2: 0.766 / Net I/σ(I): 12.14
Reflection shellResolution: 1.6→1.62 Å / % possible obs: 75.9 % / Redundancy: 3.1 % / Rmerge(I) obs: 0.343 / Mean I/σ(I) obs: 2.4 / Num. measured obs: 5969 / Χ2: 0.533 / % possible all: 0.759

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
REFMACrefmac_5.2.0005refinement
PDB_EXTRACT1.7data extraction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 1POW

1pow


Resolution: 1.6→19.62 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.954 / WRfactor Rfree: 0.161 / WRfactor Rwork: 0.142 / SU B: 1.147 / SU ML: 0.041 / Cross valid method: THROUGHOUT / σ(F): 2 / ESU R: 0.079 / ESU R Free: 0.078 / Stereochemistry target values: Engh & Huber / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1804 3328 2.013 %RANDOM
Rwork0.1583 ---
all0.159 165388 --
obs0.159 165339 82.942 %-
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: 13.157 Å2
Baniso -1Baniso -2Baniso -3
1-1.141 Å20 Å20 Å2
2---0.663 Å20 Å2
3----0.477 Å2
Refinement stepCycle: LAST / Resolution: 1.6→19.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9046 0 178 1030 10254
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0229426
X-RAY DIFFRACTIONr_angle_refined_deg1.3181.97612874
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.86751168
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.84225.138436
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.261151492
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9461546
X-RAY DIFFRACTIONr_chiral_restr0.0820.21430
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.027234
X-RAY DIFFRACTIONr_nbd_refined0.1980.24613
X-RAY DIFFRACTIONr_nbtor_refined0.3030.26566
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.0950.2771
X-RAY DIFFRACTIONr_metal_ion_refined0.0250.21
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1920.2237
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1020.266
X-RAY DIFFRACTIONr_mcbond_it0.471.55836
X-RAY DIFFRACTIONr_mcangle_it0.91829376
X-RAY DIFFRACTIONr_scbond_it1.73633756
X-RAY DIFFRACTIONr_scangle_it2.8914.53498
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 all% reflection obs (%)
1.599-1.640.2342370.187107770.1881458075.542
1.64-1.6850.2112280.186109790.1861431178.31
1.685-1.7340.2162350.182121640.1831386189.452
1.734-1.7880.22140.172116370.1721346788
1.788-1.8460.192200.17107070.1711310883.361
1.846-1.9110.1752120.161106640.1611266485.881
1.911-1.9830.1812130.151105410.1511225087.788
1.983-2.0640.1682080.15697370.1561177184.487
2.064-2.1560.1771890.15495090.1541130185.815
2.156-2.2610.1881720.15589490.1561080984.383
2.261-2.3830.1781800.15383590.1531029282.967
2.383-2.5280.1761500.15779640.158974583.263
2.528-2.7020.1911650.1674450.161918382.871
2.702-2.9180.1951210.16268910.163853782.137
2.918-3.1960.1811560.15862360.159790880.83
3.196-3.5730.1641310.14755720.148714979.773
3.573-4.1250.1531020.13549020.136634078.927
4.125-5.050.133790.13641390.136539778.155
5.05-7.1310.178740.18631750.186423776.682
7.131-94.0720.211420.16416640.166243370.119

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