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- PDB-1w5n: Stepwise introduction of zinc binding site into porphobilinogen s... -

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Basic information

Entry
Database: PDB / ID: 1w5n
TitleStepwise introduction of zinc binding site into porphobilinogen synthase of Pseudomonas aeruginosa (mutations D131C 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: 1.65 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)74,45712
Polymers74,0802
Non-polymers37710
Water11,277626
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)297,82948
Polymers296,3208
Non-polymers1,50940
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)125.322, 125.322, 85.795
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-2002-

HOH

Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (-0.84657, 0.53224, 0.00606), (0.53225, 0.84659, -0.00013), (-0.0052, 0.00311, -0.99998)
Vector: 198.30324, -57.12341, -16.57149)

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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: 37040.008 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 , 6 types, 636 molecules

#2: Chemical ChemComp-FMT / FORMIC ACID


Mass: 46.025 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CH2O2
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / 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: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 626 / 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, ASP 131 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 D131C 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: 2.22 Å3/Da / Density % sol: 44.28 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 7.5
Details: HANGING DROP. DROPS WERE MIXED OF 5 MICROLITER OF PROTEIN SOLUTION (9 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 (9 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 7.5, 40 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: 1.65→20 Å / Num. obs: 81768 / % possible obs: 99.4 % / Redundancy: 5.4 % / Rmerge(I) obs: 0.05 / Net I/σ(I): 20.5
Reflection shellResolution: 1.65→1.71 Å / Redundancy: 5.1 % / Rmerge(I) obs: 0.36 / Mean I/σ(I) obs: 3.1 / % possible all: 99

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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

1b4k


Resolution: 1.65→87.71 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.961 / SU B: 1.699 / SU ML: 0.058 / Cross valid method: THROUGHOUT / ESU R: 0.086 / ESU R Free: 0.089 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.188 4030 5 %RANDOM
Rwork0.151 ---
obs0.152 76946 98.4 %-
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: 19.9 Å2
Baniso -1Baniso -2Baniso -3
1-0.28 Å20 Å20 Å2
2--0.28 Å20 Å2
3----0.55 Å2
Refinement stepCycle: LAST / Resolution: 1.65→87.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5001 0 14 626 5641
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0260.0225588
X-RAY DIFFRACTIONr_bond_other_d0.0030.025171
X-RAY DIFFRACTIONr_angle_refined_deg2.1051.9677643
X-RAY DIFFRACTIONr_angle_other_deg1.146311969
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5355741
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.37223.234269
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.59915947
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.6831561
X-RAY DIFFRACTIONr_chiral_restr0.1650.2836
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.026575
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021194
X-RAY DIFFRACTIONr_nbd_refined0.2390.21232
X-RAY DIFFRACTIONr_nbd_other0.1890.25802
X-RAY DIFFRACTIONr_nbtor_refined0.1810.22900
X-RAY DIFFRACTIONr_nbtor_other0.0860.23507
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1480.2446
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.3020.234
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2930.2190
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1750.253
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.52123524
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it4.6835721
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it5.40722103
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it7.78731922
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: 1.65→1.69 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.262 280
Rwork0.196 5706

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