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- PDB-3a50: Structure of cytochrome P450 Vdh mutant (Vdh-K1) obtained by dire... -

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Basic information

Entry
Database: PDB / ID: 3a50
TitleStructure of cytochrome P450 Vdh mutant (Vdh-K1) obtained by directed evolution with bound vitamin D3
ComponentsVitamin D hydroxylase
KeywordsOXIDOREDUCTASE / Cytochrome P450 / vitamin D3 hydroxylase / hemoprotein / monooxygenase / directed evolution
Function / homology
Function and homology information


cholestanetriol 26-monooxygenase / cholestanetetraol 26-dehydrogenase activity / iron ion binding / heme binding / cytoplasm
Similarity search - Function
Cytochrome P450, B-class / Cytochrome p450 / Cytochrome P450 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
ACETATE ION / PROTOPORPHYRIN IX CONTAINING FE / Chem-VD3 / Vitamin D(3) 25-hydroxylase
Similarity search - Component
Biological speciesPseudonocardia autotrophica (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsYasutake, Y. / Fujii, Y. / Cheon, W.K. / Arisawa, A. / Tamura, T.
CitationJournal: J.Biol.Chem. / Year: 2010
Title: Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase
Authors: Yasutake, Y. / Fujii, Y. / Nishioka, T. / Cheon, W.K. / Arisawa, A. / Tamura, T.
History
DepositionJul 24, 2009Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 28, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 1, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / entity / pdbx_entity_nonpoly / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _chem_comp.name / _database_2.pdbx_DOI ..._chem_comp.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _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.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.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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vitamin D hydroxylase
B: Vitamin D hydroxylase
C: Vitamin D hydroxylase
D: Vitamin D hydroxylase
E: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,72429
Polymers227,9395
Non-polymers5,78524
Water18,5551030
1
A: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,9529
Polymers45,5881
Non-polymers1,3658
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,5893
Polymers45,5881
Non-polymers1,0012
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,7476
Polymers45,5881
Non-polymers1,1595
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
4
D: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,6885
Polymers45,5881
Non-polymers1,1004
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
5
E: Vitamin D hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,7476
Polymers45,5881
Non-polymers1,1595
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)77.384, 172.266, 189.063
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 5 molecules ABCDE

#1: Protein
Vitamin D hydroxylase


Mass: 45587.887 Da / Num. of mol.: 5 / Mutation: T70R, V156L, E216M, E384R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudonocardia autotrophica (bacteria) / Strain: NBRC 12743 / Gene: vdh / Plasmid: pET22 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: C4B644

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Non-polymers , 6 types, 1054 molecules

#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical
ChemComp-VD3 / (1S,3Z)-3-[(2E)-2-[(1R,3AR,7AS)-7A-METHYL-1-[(2R)-6-METHYLHEPTAN-2-YL]-2,3,3A,5,6,7-HEXAHYDRO-1H-INDEN-4-YLIDENE]ETHYLI DENE]-4-METHYLIDENE-CYCLOHEXAN-1-OL / VITAMIN D3


Mass: 384.638 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C27H44O
#4: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H3O2
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#7: 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: 2.76 Å3/Da / Density % sol: 55.5 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1M calcium acetate, 12% PEG3350, pH7.5, VAPOR DIFFUSION, HANGING DROP

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NE3A / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Jun 21, 2009
RadiationMonochromator: Si(111) double crystal monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.05→50 Å / Num. obs: 158815 / % possible obs: 100 % / Redundancy: 7.4 % / Biso Wilson estimate: 23.3 Å2 / Rmerge(I) obs: 0.107 / Net I/σ(I): 26.9
Reflection shellResolution: 2.05→2.09 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.54 / Mean I/σ(I) obs: 2.9 / Num. unique all: 7805 / % possible all: 99.8

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
MOLREPphasing
REFMAC5.2.0019refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3A4G

3a4g


Resolution: 2.05→49.09 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.922 / SU B: 4.278 / SU ML: 0.118 / Cross valid method: THROUGHOUT / ESU R: 0.19 / ESU R Free: 0.168 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23955 7866 5 %RANDOM
Rwork0.20114 ---
obs0.20308 150738 99.91 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 28.916 Å2
Baniso -1Baniso -2Baniso -3
1-0.54 Å20 Å20 Å2
2---0.09 Å20 Å2
3----0.45 Å2
Refinement stepCycle: LAST / Resolution: 2.05→49.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15550 0 397 1030 16977
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.02216308
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.52.03922257
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.6752002
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.94622.937715
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.189152625
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.25115170
X-RAY DIFFRACTIONr_chiral_restr0.1020.22482
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0212487
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.20.27716
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.2990.211157
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1310.21091
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined0.0970.29
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1660.2111
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1880.225
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined0.0340.22
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.0621.510375
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.327216196
X-RAY DIFFRACTIONr_scbond_it2.04836657
X-RAY DIFFRACTIONr_scangle_it3.1614.56051
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.05→2.103 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.293 548 -
Rwork0.243 10966 -
obs--99.17 %

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