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- PDB-3dgi: Crystal structure of F87A/T268A mutant of CYP BM3 -

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Basic information

Entry
Database: PDB / ID: 3dgi
TitleCrystal structure of F87A/T268A mutant of CYP BM3
ComponentsBifunctional P-450/NADPH-P450 reductase
KeywordsOXIDOREDUCTASE / cytochrome P450 / BM3 / DMSO / Cytoplasm / Electron transport / FAD / Flavoprotein / FMN / Heme / Iron / Metal-binding / Monooxygenase / Multifunctional enzyme / NADP / Transport
Function / homology
Function and homology information


NADPH-hemoprotein reductase / NADPH-hemoprotein reductase activity / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen / unspecific monooxygenase / aromatase activity / FMN binding / flavin adenine dinucleotide binding / iron ion binding / heme binding / identical protein binding / cytosol
Similarity search - Function
Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Cytochrome p450 / Cytochrome P450 / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. ...Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Cytochrome p450 / Cytochrome P450 / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Flavoprotein-like superfamily / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsLe Trong, I. / Katayama, J.H. / Totah, R.A. / Stenkamp, R.E. / Fox, E.P.
CitationJournal: To be Published
Title: Homolytic versus heterolytic dioxygen bond cleavage in cytochrome P450 BM3.
Authors: Katayama, J.H. / Roberts, A. / Kaspera, R. / Le Trong, I. / Stenkamp, R.E. / Thompson, J. / Totah, R.A.
History
DepositionJun 13, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 16, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / 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 / _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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional P-450/NADPH-P450 reductase
B: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,1746
Polymers105,7842
Non-polymers1,3894
Water9,152508
1
A: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,5873
Polymers52,8921
Non-polymers6952
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,5873
Polymers52,8921
Non-polymers6952
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)58.934, 146.625, 63.515
Angle α, β, γ (deg.)90.00, 97.36, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Bifunctional P-450/NADPH-P450 reductase / Cytochrome P450(BM-3) / P450BM-3 [Full length protein includes: Cytochrome P450 102 (EC 1.14.14.1) ...Cytochrome P450(BM-3) / P450BM-3 [Full length protein includes: Cytochrome P450 102 (EC 1.14.14.1) and NADPH-cytochrome P450 reductase (EC 1.6.2.4)]


Mass: 52892.215 Da / Num. of mol.: 2 / Fragment: Cytochrome P450 102, heme domain: Residues 2-456 / Mutation: F87A, T268A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (bacteria) / Gene: CYP102A1, cyp102 / Plasmid: pET21d / Production host: Escherichia coli (E. coli) / Strain (production host): DH5alpha / References: UniProt: P14779, unspecific monooxygenase
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 508 / 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: 2.57 Å3/Da / Density % sol: 52.19 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 12% PEG 8000, 100mM Tris-HCl pH 8.5, 200mM MgCl2, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.54178 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: May 3, 2007 / Details: Mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 1.95→50 Å / Num. all: 69973 / Num. obs: 69973 / % possible obs: 89.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.9 % / Biso Wilson estimate: 29.2 Å2 / Rmerge(I) obs: 0.081 / Net I/σ(I): 15.1
Reflection shellResolution: 1.95→2.02 Å / Redundancy: 2.5 % / Rmerge(I) obs: 0.637 / Mean I/σ(I) obs: 1.4 / % possible all: 48.2

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Processing

Software
NameVersionClassification
REFMAC5.4.0066refinement
CrystalCleardata collection
HKL-2000data reduction
HKL-2000data scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1BU7

1bu7


Resolution: 1.95→40.36 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.937 / SU B: 3.801 / SU ML: 0.109 / Cross valid method: THROUGHOUT / ESU R: 0.17 / ESU R Free: 0.155 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22043 3507 5 %RANDOM
Rwork0.17697 ---
obs0.17914 66413 89.76 %-
all-66414 --
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: 32.888 Å2
Baniso -1Baniso -2Baniso -3
1--0.58 Å20 Å2-0.25 Å2
2---0.66 Å20 Å2
3---1.18 Å2
Refinement stepCycle: LAST / Resolution: 1.95→40.36 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7139 0 94 508 7741
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0227407
X-RAY DIFFRACTIONr_bond_other_d0.0010.025116
X-RAY DIFFRACTIONr_angle_refined_deg1.4552.00510037
X-RAY DIFFRACTIONr_angle_other_deg0.951312512
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0545869
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.22924.755368
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.598151342
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.6121543
X-RAY DIFFRACTIONr_chiral_restr0.0930.21076
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0218084
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021425
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_it0.7811.54389
X-RAY DIFFRACTIONr_mcbond_other0.1821.51737
X-RAY DIFFRACTIONr_mcangle_it1.42527114
X-RAY DIFFRACTIONr_scbond_it2.17433018
X-RAY DIFFRACTIONr_scangle_it3.5114.52923
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.95→2 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.303 152 -
Rwork0.275 2467 -
obs--45.49 %

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