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- PDB-9kt5: Synchrotron X-ray crystal structure of oxygen-bound F87A/F393H P4... -

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Basic information

Entry
Database: PDB / ID: 9kt5
TitleSynchrotron X-ray crystal structure of oxygen-bound F87A/F393H P450BM3 with decoy C7ProPhe (N-enanthyl-L-prolyl-L-phenylalanine) and substrate styrene at 2 MGy X-ray dose
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / Cytochrome P450 / heme enzyme
Function / homology
Function and homology information


aromatase activity / 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 / 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 / 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 ...Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / 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
Similarity search - Domain/homology
Chem-D0L / PROTOPORPHYRIN IX CONTAINING FE / OXYGEN MOLECULE / ethenylbenzene / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsNagao, S. / Kuwano, W. / Tosha, T. / Yamashita, K. / Stanfield, J.K. / Kasai, C. / Ariyasu, S. / Shoji, O. / Sugimoto, H. / Kubo, M.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR15P3 Japan
Japan Society for the Promotion of Science (JSPS)JP19H05784 Japan
Japan Society for the Promotion of Science (JSPS)JP18KK0397 Japan
Japan Society for the Promotion of Science (JSPS)JP19K22403 Japan
CitationJournal: Commun Chem / Year: 2025
Title: XFEL crystallography reveals catalytic cycle dynamics during non-native substrate oxidation by cytochrome P450BM3.
Authors: Nagao, S. / Kuwano, W. / Tosha, T. / Yamashita, K. / Stanfield, J.K. / Kasai, C. / Ariyasu, S. / Hirata, K. / Ueno, G. / Murakami, H. / Ago, H. / Yamamoto, M. / Shoji, O. / Sugimoto, H. / Kubo, M.
History
DepositionDec 1, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 2, 2025Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)107,02614
Polymers104,4312
Non-polymers2,59512
Water15,853880
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A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,4216
Polymers52,2161
Non-polymers1,2055
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area19840 Å2
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,6058
Polymers52,2161
Non-polymers1,3897
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area230 Å2
ΔGint0 kcal/mol
Surface area19770 Å2
Unit cell
Length a, b, c (Å)59.130, 129.340, 150.150
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, 2 molecules AB

#1: Protein Bifunctional cytochrome P450/NADPH--P450 reductase / Cytochrome P450(BM-3) / Cytochrome P450BM-3 / Fatty acid monooxygenase / Flavocytochrome P450 BM3


Mass: 52215.535 Da / Num. of mol.: 2 / Mutation: F87A,F393H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
Strain: ATCC 14581/NBRC 15308 / Gene: cyp102A1, cyp102, BG04_163 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase

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Non-polymers , 6 types, 892 molecules

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


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-SYN / ethenylbenzene / styrene


Mass: 104.149 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H8 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-OXY / OXYGEN MOLECULE


Mass: 31.999 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-D0L / (2S)-2-[[(2S)-1-heptylpyrrolidin-2-yl]carbonylamino]-3-phenyl-propanoic acid


Mass: 360.490 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H32N2O3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 880 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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 growTemperature: 277 K / Method: batch mode / pH: 7.4
Details: 50 mM Tris-HCl buffer, 120 mM MgCl2, 16-18% PEG 8000, 0.2 mM N-Enanthyl-L-Prolyl-L-Phenylalanine, 1%(v/v) styrene

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL32XU / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jul 20, 2022
RadiationMonochromator: Si / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.6→43.64 Å / Num. obs: 152182 / % possible obs: 100 % / Redundancy: 19.5 % / CC1/2: 0.987 / Rmerge(I) obs: 0.303 / Rrim(I) all: 0.311 / Net I/σ(I): 8.85
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2% possible allRrim(I) all
1.6-1.718.22.8831.13249820.564100
1.7-1.8118.71.4612.15215600.814100
1.81-1.9620.60.8663.89221910.9221000.887
1.96-2.1520.490.5226.71199590.9611000.535
2.15-2.420.20.34610.14175970.9731000.355
2.4-2.7719.40.26213.43158030.9811000.269
2.77-3.3917.90.20217.29134610.9861000.208
3.39-4.8210.16725.69105980.9811000.171
4.8-43.6420.20.15325.8960310.98499.80.158

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XSCALEJan 10, 2022 BUILT=20220220data scaling
PHENIX1.20.1_4487phasing
Coot0.9.8.7model building
XDSJan 10, 2022 BUILT=20220220data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→43.64 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 18.12 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2003 7420 5.02 %Random selection
Rwork0.149 ---
obs0.1515 147863 97.75 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.6→43.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7328 0 182 880 8390
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0067775
X-RAY DIFFRACTIONf_angle_d0.8210552
X-RAY DIFFRACTIONf_dihedral_angle_d13.3322981
X-RAY DIFFRACTIONf_chiral_restr0.0491115
X-RAY DIFFRACTIONf_plane_restr0.0091385
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.620.407930.33081849X-RAY DIFFRACTION39
1.62-1.640.38782360.30634418X-RAY DIFFRACTION93
1.64-1.660.35322450.27244710X-RAY DIFFRACTION100
1.66-1.680.30392660.26114756X-RAY DIFFRACTION100
1.68-1.70.31322490.22594705X-RAY DIFFRACTION100
1.7-1.730.28722490.19244746X-RAY DIFFRACTION100
1.73-1.750.24422790.17594742X-RAY DIFFRACTION100
1.75-1.780.24652280.16894734X-RAY DIFFRACTION100
1.78-1.810.23072730.15024752X-RAY DIFFRACTION100
1.81-1.840.20532440.13444753X-RAY DIFFRACTION100
1.84-1.870.22452620.13444718X-RAY DIFFRACTION100
1.87-1.90.20382680.14424754X-RAY DIFFRACTION100
1.9-1.940.2212610.14564718X-RAY DIFFRACTION100
1.94-1.980.22352430.15524798X-RAY DIFFRACTION100
1.98-2.020.20532300.164779X-RAY DIFFRACTION100
2.02-2.070.19752640.13164739X-RAY DIFFRACTION100
2.07-2.120.18372690.12634775X-RAY DIFFRACTION100
2.12-2.180.18852570.12714766X-RAY DIFFRACTION100
2.18-2.240.18552650.1274744X-RAY DIFFRACTION100
2.24-2.310.19152320.12794787X-RAY DIFFRACTION100
2.31-2.40.19042210.1264823X-RAY DIFFRACTION100
2.4-2.490.17082700.1264793X-RAY DIFFRACTION100
2.49-2.60.17812540.13134776X-RAY DIFFRACTION100
2.6-2.740.1952190.14424860X-RAY DIFFRACTION100
2.74-2.910.19692790.14464780X-RAY DIFFRACTION100
2.91-3.140.18812440.15244849X-RAY DIFFRACTION100
3.14-3.450.19992520.14684865X-RAY DIFFRACTION100
3.45-3.950.16992410.13744905X-RAY DIFFRACTION100
3.95-4.980.15992450.13514934X-RAY DIFFRACTION100
4.98-43.640.20072820.17385115X-RAY DIFFRACTION100

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