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

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Basic information

Entry
Database: PDB / ID: 2ezu
TitlePyruvate oxidase variant F479W in complex with reaction intermediate 2-acetyl-thiamin diphosphate
ComponentsPyruvate oxidase
KeywordsOXIDOREDUCTASE / TPP enzyme / reaction intermediate
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 / 2-ACETYL-THIAMINE DIPHOSPHATE / PYRUVIC ACID / Pyruvate oxidase
Similarity search - Component
Biological speciesLactobacillus plantarum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.16 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 ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / database_2 ...chem_comp / database_2 / diffrn_source / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site / _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.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.ptnr1_symmetry / _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_conn.ptnr2_symmetry / _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
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Category: atom_site / chem_comp_atom / chem_comp_bond
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2
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)135,26812
Polymers132,4922
Non-polymers2,77710
Water17,601977
1
A: Pyruvate oxidase
B: Pyruvate oxidase
hetero molecules

A: Pyruvate oxidase
B: Pyruvate oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,53624
Polymers264,9834
Non-polymers5,55320
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_565x,-y+1,-z1
Buried area33810 Å2
ΔGint-193 kcal/mol
Surface area69510 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)118.276, 154.724, 166.003
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
11B-1611-

NA

21A-1688-

HOH

31B-1710-

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 , 6 types, 987 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-HTL / 2-ACETYL-THIAMINE DIPHOSPHATE / 2-ACETYL-3-[(4-AMINO-2-METHYL-5-PYRIMIDINYL)METHYL]-4-METHYL-5-(4,6,6-TRIHYDROXY-3,5-DIOXA-4,6-DIPHOSPHAHEX-1-YL)THIAZO LIUM INNER SALT P,P'-DIOXIDE


Mass: 467.351 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H21N4O8P2S
#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: Chemical ChemComp-PYR / PYRUVIC ACID


Mass: 88.062 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H4O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 977 / 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: 2.16→25 Å / Num. all: 77411 / Num. obs: 77402 / % possible obs: 95.1 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 3.7 / Redundancy: 4 % / Biso Wilson estimate: 15.8 Å2 / Rmerge(I) obs: 0.116 / Χ2: 0.743 / Net I/σ(I): 9.46
Reflection shellResolution: 2.16→2.19 Å / % possible obs: 93.2 % / Redundancy: 2.7 % / Rmerge(I) obs: 0.258 / Mean I/σ(I) obs: 3.2 / Num. measured obs: 2978 / Χ2: 0.716 / % possible all: 0.932

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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: 2.16→25 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.934 / WRfactor Rfree: 0.155 / WRfactor Rwork: 0.124 / SU B: 3.449 / SU ML: 0.091 / Cross valid method: THROUGHOUT / σ(F): 2 / ESU R: 0.171 / ESU R Free: 0.149 / Stereochemistry target values: Engh & Huber / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1839 1543 1.993 %RANDOM
Rwork0.1431 ---
all0.144 77411 --
obs0.144 77402 95.146 %-
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: 12.081 Å2
Baniso -1Baniso -2Baniso -3
1-1.389 Å20 Å20 Å2
2---0.523 Å20 Å2
3----0.865 Å2
Refinement stepCycle: LAST / Resolution: 2.16→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9046 0 180 977 10203
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0229426
X-RAY DIFFRACTIONr_angle_refined_deg1.5211.97512866
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3251168
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.18725.138436
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.496151492
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.8131546
X-RAY DIFFRACTIONr_chiral_restr0.0950.21428
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.027258
X-RAY DIFFRACTIONr_nbd_refined0.1980.24604
X-RAY DIFFRACTIONr_nbtor_refined0.2990.26496
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1340.2842
X-RAY DIFFRACTIONr_metal_ion_refined0.0250.22
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1960.2237
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1480.282
X-RAY DIFFRACTIONr_mcbond_it0.641.55836
X-RAY DIFFRACTIONr_mcangle_it1.19729376
X-RAY DIFFRACTIONr_scbond_it2.34433755
X-RAY DIFFRACTIONr_scangle_it3.7784.53490
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 (%)
2.162-2.2180.212960.15854540.159594193.419
2.218-2.2790.231180.14854440.15580095.897
2.279-2.3450.2261130.14653320.147564096.543
2.345-2.4170.204920.14952390.15551396.699
2.417-2.4960.211070.15250590.153533796.796
2.496-2.5840.184890.15248700.153513796.535
2.584-2.6810.1891000.14547190.146498896.612
2.681-2.7910.181950.1545070.15478896.115
2.791-2.9140.192950.1543410.151461196.205
2.914-3.0570.228790.14741370.148441295.558
3.057-3.2220.174920.14239190.143419495.637
3.222-3.4170.195690.14237030.143396595.132
3.417-3.6520.185690.12535070.126376994.879
3.652-3.9450.128740.12732310.127350294.375
3.945-4.320.143600.12229560.122321393.869
4.32-4.8290.165520.12326980.124295493.094
4.829-5.5740.184580.1523470.151259492.714
5.574-6.820.136410.18220010.181221292.315
6.82-9.620.172250.14415650.144175490.65
9.62-94.0720.155190.1558300.155102782.668

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