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Entry
Database: PDB / ID: 3mfl
TitleAxial Ligand Swapping In Double Mutant Maintains Intradiol-cleavage Chemistry in Protocatechuate 3,4-Dioxygenase
Components(Protocatechuate 3,4-dioxygenase ...) x 2
KeywordsOXIDOREDUCTASE / dioxygenase / non-heme / iron / homoprotocatechuate / intradiol / substrate analogue
Function / homology
Function and homology information


protocatechuate 3,4-dioxygenase / protocatechuate 3,4-dioxygenase activity / 3,4-dihydroxybenzoate catabolic process / beta-ketoadipate pathway / ferric iron binding
Similarity search - Function
Protocatechuate 3,4-dioxygenase, beta subunit / Protocatechuate 3,4-dioxygenase, alpha subunit / Protocatechuate 3,4-dioxygenase beta subunit, N-terminal / Protocatechuate 3,4-dioxygenase beta subunit N terminal / Intradiol ring-cleavage dioxygenases signature. / Protocatechuate 3,4-Dioxygenase, subunit A / Aromatic compound dioxygenase / Intradiol ring-cleavage dioxygenase, C-terminal / Intradiol ring-cleavage dioxygenase, core / Dioxygenase ...Protocatechuate 3,4-dioxygenase, beta subunit / Protocatechuate 3,4-dioxygenase, alpha subunit / Protocatechuate 3,4-dioxygenase beta subunit, N-terminal / Protocatechuate 3,4-dioxygenase beta subunit N terminal / Intradiol ring-cleavage dioxygenases signature. / Protocatechuate 3,4-Dioxygenase, subunit A / Aromatic compound dioxygenase / Intradiol ring-cleavage dioxygenase, C-terminal / Intradiol ring-cleavage dioxygenase, core / Dioxygenase / Sandwich / Mainly Beta
Similarity search - Domain/homology
BETA-MERCAPTOETHANOL / CARBONATE ION / 2-(3,4-DIHYDROXYPHENYL)ACETIC ACID / : / Protocatechuate 3,4-dioxygenase alpha chain / Protocatechuate 3,4-dioxygenase beta chain
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.78 Å
AuthorsPurpero, V.M. / Lipscomb, J.D.
CitationJournal: To be Published
Title: Axial Ligand Swapping In Double Mutant Maintains Intradiol-cleavage Chemistry in Protocatechuate 3,4-Dioxygenase
Authors: Purpero, V.M. / Lipscomb, J.D.
History
DepositionApr 2, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 13, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 8, 2017Group: Refinement description / Category: software
Revision 1.3Oct 6, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / 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 / _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_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.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_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_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.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_label_alt_id / _struct_conn.pdbx_ptnr2_label_alt_id / _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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protocatechuate 3,4-dioxygenase alpha chain
M: Protocatechuate 3,4-dioxygenase beta chain
B: Protocatechuate 3,4-dioxygenase alpha chain
N: Protocatechuate 3,4-dioxygenase beta chain
C: Protocatechuate 3,4-dioxygenase alpha chain
O: Protocatechuate 3,4-dioxygenase beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,86427
Polymers146,9256
Non-polymers1,93921
Water18,8801048
1
A: Protocatechuate 3,4-dioxygenase alpha chain
M: Protocatechuate 3,4-dioxygenase beta chain
B: Protocatechuate 3,4-dioxygenase alpha chain
N: Protocatechuate 3,4-dioxygenase beta chain
C: Protocatechuate 3,4-dioxygenase alpha chain
O: Protocatechuate 3,4-dioxygenase beta chain
hetero molecules

A: Protocatechuate 3,4-dioxygenase alpha chain
M: Protocatechuate 3,4-dioxygenase beta chain
B: Protocatechuate 3,4-dioxygenase alpha chain
N: Protocatechuate 3,4-dioxygenase beta chain
C: Protocatechuate 3,4-dioxygenase alpha chain
O: Protocatechuate 3,4-dioxygenase beta chain
hetero molecules

A: Protocatechuate 3,4-dioxygenase alpha chain
M: Protocatechuate 3,4-dioxygenase beta chain
B: Protocatechuate 3,4-dioxygenase alpha chain
N: Protocatechuate 3,4-dioxygenase beta chain
C: Protocatechuate 3,4-dioxygenase alpha chain
O: Protocatechuate 3,4-dioxygenase beta chain
hetero molecules

A: Protocatechuate 3,4-dioxygenase alpha chain
M: Protocatechuate 3,4-dioxygenase beta chain
B: Protocatechuate 3,4-dioxygenase alpha chain
N: Protocatechuate 3,4-dioxygenase beta chain
C: Protocatechuate 3,4-dioxygenase alpha chain
O: Protocatechuate 3,4-dioxygenase beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)595,456108
Polymers587,70124
Non-polymers7,75584
Water43224
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-x,y,-z1
crystal symmetry operation4_555x,-y,-z1
Buried area157940 Å2
ΔGint-927 kcal/mol
Surface area168540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)128.319, 140.769, 168.183
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222
Detailsexists as a dodecamer (12) of dimer in solution. This space group (I222) shows 3 dimers per asymmetric unit (x,y,z), therefore applying the symmetry operators (-x,y,-z), (x,-y,-z) and (-x,-y,z) yields the biological assembly

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Components

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Protocatechuate 3,4-dioxygenase ... , 2 types, 6 molecules ABCMNO

#1: Protein Protocatechuate 3,4-dioxygenase alpha chain / 3 / 4-PCD


Mass: 22278.812 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: pcaG / Plasmid: pCE vector, pT7-7 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3)
References: UniProt: P00436, protocatechuate 3,4-dioxygenase
#2: Protein Protocatechuate 3,4-dioxygenase beta chain / 3 / 4-PCD


Mass: 26696.287 Da / Num. of mol.: 3 / Mutation: Y148H, H163Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: pcaH / Plasmid: pCE vector, pT7-7 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3)
References: UniProt: P00437, protocatechuate 3,4-dioxygenase

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Non-polymers , 8 types, 1069 molecules

#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-BME / BETA-MERCAPTOETHANOL / 2-Mercaptoethanol


Mass: 78.133 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6OS
#6: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe
#7: Chemical ChemComp-CO3 / CARBONATE ION / Carbonate


Mass: 60.009 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: CO3
#8: Chemical ChemComp-DHY / 2-(3,4-DIHYDROXYPHENYL)ACETIC ACID / 3,4-Dihydroxyphenylacetic acid


Mass: 168.147 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H8O4 / Comment: neurotransmitter*YM
#9: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#10: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1048 / 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: 2.58 Å3/Da / Density % sol: 52.41 %
Crystal growTemperature: 277 K / pH: 8.5
Details: 1.5-1.8 M ammonium sulfate, 40-60 mM Tris buffer pH 8.5, 5 mM BME, with varying ML to enzyme ratios 1:2 to 4:1, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 0.98
DetectorType: SBC-3 / Detector: CCD / Date: Nov 24, 2008 / Details: MIRRORS
RadiationMonochromator: SAGITALLY FOCUSING DOUBLE CRYSTA / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.78→50 Å / Num. obs: 145553 / % possible obs: 99.8 % / Observed criterion σ(I): 3 / Redundancy: 6.3 % / Biso Wilson estimate: 17.9 Å2 / Rmerge(I) obs: 0.066 / Net I/σ(I): 10.6
Reflection shellResolution: 1.78→1.81 Å / Redundancy: 6.1 % / Rmerge(I) obs: 0.408 / Mean I/σ(I) obs: 3.96 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
REFMACrefinement
PDB_EXTRACT3.1data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.78→23.87 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.95 / Occupancy max: 1 / Occupancy min: 0.1 / SU B: 1.902 / SU ML: 0.061 / SU R Cruickshank DPI: 0.102 / Cross valid method: THROUGHOUT / σ(F): 1 / ESU R: 0.102 / ESU R Free: 0.098 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING
RfactorNum. reflection% reflectionSelection details
Rfree0.185 7292 5 %RANDOM
Rwork0.157 ---
obs0.159 138259 99.8 %-
all-145551 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 20.78 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20 Å20 Å2
2--0.02 Å20 Å2
3---0.01 Å2
Refinement stepCycle: LAST / Resolution: 1.78→23.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10374 0 112 1048 11534
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.02211053
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.4041.95515090
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.28351381
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.44323.845567
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.125151687
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.9671587
X-RAY DIFFRACTIONr_chiral_restr0.1060.21576
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0218813
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.7471.56679
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.342210821
X-RAY DIFFRACTIONr_scbond_it2.07834374
X-RAY DIFFRACTIONr_scangle_it3.3394.54232
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.78→1.82 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.236 525 -
Rwork0.186 9947 -
obs--98.99 %

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