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- PDB-3kx4: Crystal structure of Bacillus megaterium BM3 heme domain mutant I401E -

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Basic information

Entry
Database: PDB / ID: 3kx4
TitleCrystal structure of Bacillus megaterium BM3 heme domain mutant I401E
ComponentsBifunctional P-450/NADPH-P450 reductase
KeywordsOXIDOREDUCTASE / cytochrome P450 / I401E mutant / Heme domain / 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 / metabolic process / FMN binding / iron ion binding / heme binding / identical protein binding / cytoplasm
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 / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.95 Å
AuthorsGirvan, H.M. / Levy, C.W. / Leys, D. / Munro, A.W.
CitationJournal: Biochem.J. / Year: 2010
Title: Glutamate-haem ester bond formation is disfavoured in flavocytochrome P450 BM3: characterization of glutamate substitution mutants at the haem site of P450 BM3.
Authors: Girvan, H.M. / Levy, C.W. / Williams, P. / Fisher, K. / Cheesman, M.R. / Rigby, S.E. / Leys, D. / Munro, A.W.
History
DepositionDec 2, 2009Deposition site: RCSB / Processing site: PDBJ
Revision 1.0May 19, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 10, 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.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.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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)108,5434
Polymers107,3102
Non-polymers1,2332
Water15,835879
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A: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,2722
Polymers53,6551
Non-polymers6161
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)54,2722
Polymers53,6551
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)58.540, 152.830, 59.710
Angle α, β, γ (deg.)90.00, 94.64, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Bifunctional P-450/NADPH-P450 reductase / flavocytochrome P450 BM3 / Cytochrome P450(BM-3) / P450BM-3 / Cytochrome P450 102 / NADPH-- ...flavocytochrome P450 BM3 / Cytochrome P450(BM-3) / P450BM-3 / Cytochrome P450 102 / NADPH--cytochrome P450 reductase


Mass: 53655.055 Da / Num. of mol.: 2 / Fragment: Heme domain (UNP residues 2-471) / Mutation: I401E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (bacteria) / Gene: CYP102A1, cyp102 / Plasmid: TG1 / Production host: Escherichia coli (E. coli) / References: UniProt: P14779, unspecific monooxygenase
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 879 / 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: 2.48 Å3/Da / Density % sol: 50.42 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.4
Details: 100mM cacodylic acid, pH 6.0, containing 18% PEG 3350, 140mM MgCl2, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 1.0698 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Apr 6, 2009
RadiationMonochromator: SAGITALLY FOCUSED Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0698 Å / Relative weight: 1
ReflectionResolution: 1.95→30 Å / Num. all: 75924 / Num. obs: 74558 / % possible obs: 98.2 % / Observed criterion σ(F): 1 / Observed criterion σ(I): 1 / Rmerge(I) obs: 0.117 / Net I/σ(I): 10.3
Reflection shellResolution: 1.95→1.97 Å / Rmerge(I) obs: 0.319 / Mean I/σ(I) obs: 4.08 / % possible all: 93.9

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Processing

Software
NameVersionClassification
DNAdata collection
PHENIXmodel building
PHENIX(phenix.refine)refinement
XDSdata reduction
XSCALEdata scaling
PHENIXphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.95→29.757 Å / SU ML: 0.23 / σ(F): 2.03 / Phase error: 20.18 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2101 3717 4.99 %
Rwork0.1625 --
obs0.1649 74551 98.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 49.68 Å2 / ksol: 0.446 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-0.9844 Å20 Å21.0509 Å2
2---7.1557 Å20 Å2
3----5.1603 Å2
Refinement stepCycle: LAST / Resolution: 1.95→29.757 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7302 0 86 879 8267
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00914824
X-RAY DIFFRACTIONf_angle_d0.99826887
X-RAY DIFFRACTIONf_dihedral_angle_d16.093692
X-RAY DIFFRACTIONf_chiral_restr0.0821092
X-RAY DIFFRACTIONf_plane_restr0.0052272
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 27

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.9501-1.97480.23311150.1938207579
1.9748-2.00070.24381690.1817259698
2.0007-2.02810.23841270.1919263398
2.0281-2.05710.26061290.1897262798
2.0571-2.08780.23931220.1759264498
2.0878-2.12040.24491440.1727262298
2.1204-2.15520.21461310.1692264199
2.1552-2.19230.24581100.1663263799
2.1923-2.23220.23881360.1617265099
2.2322-2.27510.2111360.1507262499
2.2751-2.32150.20411540.1454263399
2.3215-2.3720.22831340.1472264599
2.372-2.42710.22231490.1481264599
2.4271-2.48780.21371310.1479267199
2.4878-2.5550.20371460.15262499
2.555-2.63020.20581300.1482648100
2.6302-2.7150.21681470.15552649100
2.715-2.8120.23741400.1582264199
2.812-2.92450.19561690.1658266199
2.9245-3.05740.24051530.1737261099
3.0574-3.21850.1991580.16892682100
3.2185-3.41980.18311350.15882643100
3.4198-3.68340.19631290.1502268499
3.6834-4.05330.17371450.1406264299
4.0533-4.63790.15851150.13692714100
4.6379-5.8360.21921350.162702100
5.836-29.76060.20461280.2007259195

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