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- PDB-4rsn: Crystal structure of the E267V mutant of cytochrome P450 BM3 -

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Basic information

Entry
Database: PDB / ID: 4rsn
TitleCrystal structure of the E267V mutant of cytochrome P450 BM3
ComponentsBifunctional P-450/NADPH-P450 reductase
KeywordsOXIDOREDUCTASE / Bifunctional P-450/NADPH-P450 reductase / heme domain
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 / MOLECULAR REPLACEMENT / Resolution: 2.701 Å
AuthorsZhou, W.H. / Zhang, A.L. / Zhang, T. / Ren, X.K. / Yorke, J.A. / Taylor, A. / Wong, L.-L.
CitationJournal: To be Published
Title: Expanding the drug metabolism function of P450BM3
Authors: Ren, X.K. / Yorke, J.A. / Taylor, A. / Zhang, A.L. / Zhang, T. / Zhou, W.H. / Wong, L.-L.
History
DepositionNov 9, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Dec 2, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / 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_label_asym_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_label_asym_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_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: Bifunctional P-450/NADPH-P450 reductase
B: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,5214
Polymers105,2882
Non-polymers1,2332
Water19811
1
A: Bifunctional P-450/NADPH-P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2612
Polymers52,6441
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)53,2612
Polymers52,6441
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)78.299, 78.299, 203.190
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection details
111CHAIN A AND (RESSEQ -4:142 OR RESSEQ 144:455 )
211CHAIN B AND (RESSEQ -4:142 OR RESSEQ 144:455 )

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Components

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


Mass: 52644.121 Da / Num. of mol.: 2 / Fragment: heme domain, UNP residues 1-456 / Mutation: R48L, Y52F, I402P, E268V, F88V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (bacteria) / Gene: cyp102, cyp102A1, p450bm-3 / Plasmid: pGEX-6P-1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / 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.: 11 / 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: 3.42 Å3/Da / Density % sol: 63.99 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: 2.0M Sodium Chloride 10% w/v PEG 6000, pH 7.4, VAPOR DIFFUSION, HANGING DROP, temperature 289K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: May 25, 2014 / Details: mirrors
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. all: 40848 / Num. obs: 37989 / % possible obs: 93 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 5.3 % / Biso Wilson estimate: 65.1 Å2 / Rmerge(I) obs: 0.097 / Rsym value: 0.069 / Net I/σ(I): 44.6
Reflection shellResolution: 2.7→2.75 Å / Redundancy: 5.7 % / Rmerge(I) obs: 0.456 / Mean I/σ(I) obs: 9.8 / Num. unique all: 1868 / Rsym value: 0.474 / % possible all: 99.7

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Processing

Software
NameVersionClassification
HKL-2000data collection
PHASESphasing
PHENIX(phenix.refine: 1.6.2_432)refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3M4V

3m4v


Resolution: 2.701→40.655 Å / SU ML: 0.32 / Cross valid method: THROUGHOUT / σ(F): 0.09 / Phase error: 21.64 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2068 1818 4.97 %RANDOM
Rwork0.1755 ---
obs0.1771 36578 95.71 %-
all-38217 --
Solvent computationShrinkage radii: 0.83 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 32.987 Å2 / ksol: 0.314 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-1.749 Å2-0 Å2-0 Å2
2--1.749 Å20 Å2
3----3.4979 Å2
Refinement stepCycle: LAST / Resolution: 2.701→40.655 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7358 0 86 11 7455
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0097628
X-RAY DIFFRACTIONf_angle_d1.30110350
X-RAY DIFFRACTIONf_dihedral_angle_d15.4932864
X-RAY DIFFRACTIONf_chiral_restr0.0881114
X-RAY DIFFRACTIONf_plane_restr0.0051344
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A3666X-RAY DIFFRACTIONPOSITIONAL
12B3666X-RAY DIFFRACTIONPOSITIONAL0.018
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.7006-2.77360.34261370.2413244489
2.7736-2.85520.261450.2361252791
2.8552-2.94730.28151230.2238259091
2.9473-3.05260.25761230.2088261093
3.0526-3.17480.21651380.1914267395
3.1748-3.31920.23441290.1784272498
3.3192-3.49410.20761480.179272498
3.4941-3.71290.21911470.1758277299
3.7129-3.99940.20471310.1557276399
3.9994-4.40140.19911380.1509277999
4.4014-5.03730.18061580.1508273599
5.0373-6.34260.19591550.18242778100
6.3426-40.65960.18461460.1787264194

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