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- PDB-1w5m: Stepwise introduction of zinc binding site into porphobilinogen s... -

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Basic information

Entry
Database: PDB / ID: 1w5m
TitleStepwise introduction of zinc binding site into porphobilinogen synthase of Pseudomonas aeruginosa (mutations A129C and D139C)
ComponentsDELTA-AMINOLEVULINIC ACID DEHYDRATASE
KeywordsSYNTHASE / EVOLUTION / METALLOENZYME / PORPHOBILINOGEN SYNTHASE / PSEUDOMONAS AERUGINOSA / PROTEIN ENGINEERING
Function / homology
Function and homology information


porphobilinogen synthase / porphobilinogen synthase activity / porphyrin-containing compound biosynthetic process / protoporphyrinogen IX biosynthetic process / heme biosynthetic process / zinc ion binding / cytosol
Similarity search - Function
Delta-aminolevulinic acid dehydratase / Delta-aminolevulinic acid dehydratase, active site / Delta-aminolevulinic acid dehydratase / Delta-aminolevulinic acid dehydratase active site. / Delta-aminolevulinic acid dehydratase / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
FORMIC ACID / Delta-aminolevulinic acid dehydratase
Similarity search - Component
Biological speciesPSEUDOMONAS AERUGINOSA (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsFrere, F. / Reents, H. / Schubert, W.-D. / Heinz, D.W. / Jahn, D.
CitationJournal: J.Mol.Biol. / Year: 2005
Title: Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro
Authors: Frere, F. / Reents, H. / Schubert, W.-D. / Heinz, D.W. / Jahn, D.
History
DepositionAug 9, 2004Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2005Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3May 8, 2019Group: Data collection / Experimental preparation / Other
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.method / _pdbx_database_status.recvd_author_approval
Revision 1.4Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / 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_database_status.status_code_sf / _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.ptnr1_symmetry / _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.ptnr3_symmetry / _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
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA BA" IN EACH CHAIN ON SHEET RECORDS ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA BA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 11-STRANDED BARREL THIS IS REPRESENTED BY A 12-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
B: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,63215
Polymers74,1682
Non-polymers46413
Water15,979887
1
A: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
B: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
hetero molecules

A: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
B: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
hetero molecules

A: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
B: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
hetero molecules

A: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
B: DELTA-AMINOLEVULINIC ACID DEHYDRATASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)298,52860
Polymers296,6728
Non-polymers1,85652
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_645-y+3/2,x-1/2,z1
crystal symmetry operation4_565y+1/2,-x+3/2,z1
crystal symmetry operation2_765-x+2,-y+1,z1
MethodPQS
Unit cell
Length a, b, c (Å)126.307, 126.307, 85.156
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number90
Space group name H-MP4212
Components on special symmetry positions
IDModelComponents
11A-2027-

HOH

Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (-0.83857, 0.54479, 0.00358), (0.54479, 0.83857, -0.00096), (-0.00352, 0.00115, -0.99999)
Vector: 198.00226, -58.65075, -16.38513)

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein DELTA-AMINOLEVULINIC ACID DEHYDRATASE / / PORPHOBILINOGEN SYNTHASE / ALAD / ALADHPORPHOBILINOGEN SYNTHASE


Mass: 37084.020 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PSEUDOMONAS AERUGINOSA (bacteria) / Strain: PAO1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q59643, porphobilinogen synthase

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Non-polymers , 5 types, 900 molecules

#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-FMT / FORMIC ACID / Formic acid


Mass: 46.025 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CH2O2
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 887 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsCATALYTIC ACTIVITY: 2 5-AMINOLEVULINATE = PORPHOBILINOGEN + 2 H(2)O. ENGINEERED MUTATION IN CHAINS ...CATALYTIC ACTIVITY: 2 5-AMINOLEVULINATE = PORPHOBILINOGEN + 2 H(2)O. ENGINEERED MUTATION IN CHAINS A AND B, ALA 129 TO CYS ENGINEERED MUTATION IN CHAINS A AND B, ASP 139 TO CYS ACCIDENTALLY INTRODUCED MUTATION IN CHAINS A AND B, ILE 199 TO VAL
Sequence detailsACCIDENTAL POINT MUTATION IN THE ORIGINAL GENE CAUSES I199V, TWO ADDITIONAL ENGINEERED MUTATIONS ...ACCIDENTAL POINT MUTATION IN THE ORIGINAL GENE CAUSES I199V, TWO ADDITIONAL ENGINEERED MUTATIONS A129C AND D139C

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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: 2.24 Å3/Da / Density % sol: 44.75 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 8.5
Details: HANGING DROP. DROPS WERE MIXED OF 5 MICROLITER OF PROTEIN SOLUTION (7.5 MG/ML PROTEIN, 50 MM NA-HEPES PH 7.5, 10MM MGCL2, 10MM ZNCL2, 10 MM BETA-MERCAPTOETHANOLE) PLUS 5 MICROLITER OF ...Details: HANGING DROP. DROPS WERE MIXED OF 5 MICROLITER OF PROTEIN SOLUTION (7.5 MG/ML PROTEIN, 50 MM NA-HEPES PH 7.5, 10MM MGCL2, 10MM ZNCL2, 10 MM BETA-MERCAPTOETHANOLE) PLUS 5 MICROLITER OF RESERVOIR SOLUTION (30.0 % (W/V) PEG 400, 100MM NA-HEPES PH 8.5, 200 MM MGCL2, 20MM BETA-MERCAPTOETHANOLE) ON GLASS COVER SLIDES, HANGING ABOVE 500 MICROLITER OF RESERVOIR SOLUTION.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: MPG/DESY, HAMBURG / Beamline: BW6 / Wavelength: 1.05
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Aug 21, 2002
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.05 Å / Relative weight: 1
ReflectionResolution: 1.6→20 Å / Num. obs: 90595 / % possible obs: 99.9 % / Redundancy: 6.4 % / Rmerge(I) obs: 0.05 / Net I/σ(I): 32.7
Reflection shellResolution: 1.6→1.63 Å / Redundancy: 6.1 % / Rmerge(I) obs: 0.28 / Mean I/σ(I) obs: 5.5 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.2.0005refinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1B4K
Resolution: 1.6→87.71 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.964 / SU B: 1.21 / SU ML: 0.044 / Cross valid method: THROUGHOUT / ESU R: 0.069 / ESU R Free: 0.074 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.165 4488 5 %RANDOM
Rwork0.129 ---
obs0.131 85549 99 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 14.2 Å2
Baniso -1Baniso -2Baniso -3
1-0.24 Å20 Å20 Å2
2--0.24 Å20 Å2
3----0.48 Å2
Refinement stepCycle: LAST / Resolution: 1.6→87.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5051 0 17 887 5955
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0230.0225693
X-RAY DIFFRACTIONr_bond_other_d0.0020.025248
X-RAY DIFFRACTIONr_angle_refined_deg2.0611.9677793
X-RAY DIFFRACTIONr_angle_other_deg1.034312164
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.7675760
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.21423.333276
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.49315970
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.1211562
X-RAY DIFFRACTIONr_chiral_restr0.1570.2848
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.026737
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021217
X-RAY DIFFRACTIONr_nbd_refined0.2330.21308
X-RAY DIFFRACTIONr_nbd_other0.2020.25999
X-RAY DIFFRACTIONr_nbtor_refined0.1820.22987
X-RAY DIFFRACTIONr_nbtor_other0.0860.23570
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.120.2552
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1760.231
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2780.2181
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1210.251
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.74423666
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it3.47935835
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it4.67922239
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it6.72731958
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.6→1.64 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.199 332
Rwork0.153 6327

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