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- PDB-8yaz: XFEL crystal structure of the oxidized form of F87A/F393H P450BM3... -

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Basic information

Entry
Database: PDB / ID: 8yaz
TitleXFEL crystal structure of the oxidized form of F87A/F393H P450BM3 with N-enanthyl-L-prolyl-L-phenylalanine in complex with styrene
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / Cytochrome P450
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 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / 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 / Flavoprotein-like superfamily / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450
Similarity search - Domain/homology
Chem-D0L / PROTOPORPHYRIN IX CONTAINING FE / ethenylbenzene / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
MethodX-RAY DIFFRACTION / FREE ELECTRON LASER / MOLECULAR REPLACEMENT / Resolution: 1.85 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
DepositionFeb 11, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Apr 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

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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)106,60711
Polymers104,1692
Non-polymers2,4399
Water9,008500
1
A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,3506
Polymers52,0841
Non-polymers1,2655
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1310 Å2
ΔGint-22 kcal/mol
Surface area18950 Å2
MethodPISA
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2585
Polymers52,0841
Non-polymers1,1734
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1320 Å2
ΔGint-22 kcal/mol
Surface area19110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.900, 128.440, 149.050
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: 52084.340 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: NBRC 15308 = ATCC 14581 / 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 , 5 types, 509 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-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
#4: Chemical ChemComp-SYN / ethenylbenzene / styrene


Mass: 104.149 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H8 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 500 / 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: 2.71 Å3/Da / Density % sol: 54.55 %
Crystal growTemperature: 277 K / Method: batch mode / pH: 7.4
Details: 50 mM Tris-HCl buffer, 120 mM MgCl2, 16-18% PEG 8000, 200 uM N-Enanthyl-L-Prolyl-L-Phenylalanine, 1%(v/v) styrene

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: Y
Diffraction sourceSource: FREE ELECTRON LASER / Site: SACLA / Beamline: BL2 / Wavelength: 0.9539 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Dec 9, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9539 Å / Relative weight: 1
ReflectionResolution: 1.85→10 Å / Num. obs: 96618 / % possible obs: 100 % / Redundancy: 92.2 % / CC1/2: 0.972 / Net I/σ(I): 6.53
Reflection shellResolution: 1.85→1.87 Å / Redundancy: 34.1 % / Mean I/σ(I) obs: 1.95 / Num. unique obs: 4723 / CC1/2: 0.608
Serial crystallography measurementCollection time total: 4.1 hours / Focal spot size: 19.313 µm2 / Pulse duration: 10 fsec. / Pulse photon energy: 13 keV / XFEL pulse repetition rate: 30 Hz
Serial crystallography sample deliveryDescription: Fixed-target / Method: fixed target
Serial crystallography sample delivery fixed targetCrystals per unit: 50
Description: microcrystals were mounted on mesh loop and flash-frozen by liquid nitrogen
Motion control: stepper motors / Sample dehydration prevention: none / Sample holding: polyimide film
Sample solvent: 125 mM Tris-HCl (pH 8.3), 14% w/v PEG 8000, 60 mM MgCl2, saturated styrene, and 30% glycerol
Sample unit size: 36 µm / Support base: SPINE magnet base
Serial crystallography data reductionCrystal hits: 18419 / Frames indexed: 11660

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
CrystFELdata reduction
CrystFELdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.85→9.98 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.69 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2199 4809 4.98 %
Rwork0.1859 --
obs0.1876 96521 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.85→9.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7328 0 172 500 8000
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0087710
X-RAY DIFFRACTIONf_angle_d1.06610446
X-RAY DIFFRACTIONf_dihedral_angle_d8.9831055
X-RAY DIFFRACTIONf_chiral_restr0.0571109
X-RAY DIFFRACTIONf_plane_restr0.0091368
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.85-1.870.35771610.30362970X-RAY DIFFRACTION100
1.87-1.890.3291820.29313027X-RAY DIFFRACTION100
1.89-1.920.36241720.28763003X-RAY DIFFRACTION100
1.92-1.940.32581510.28233008X-RAY DIFFRACTION100
1.94-1.970.32641530.26133054X-RAY DIFFRACTION100
1.97-1.990.2971460.25623018X-RAY DIFFRACTION100
1.99-2.020.29641590.22663050X-RAY DIFFRACTION100
2.02-2.050.27821630.22753034X-RAY DIFFRACTION100
2.05-2.080.27191690.22082988X-RAY DIFFRACTION100
2.08-2.120.25271810.20793026X-RAY DIFFRACTION100
2.12-2.150.24461480.19463013X-RAY DIFFRACTION100
2.15-2.190.23431850.19493030X-RAY DIFFRACTION100
2.19-2.230.24041580.18473020X-RAY DIFFRACTION100
2.23-2.280.22661520.18973055X-RAY DIFFRACTION100
2.28-2.330.19041330.18213050X-RAY DIFFRACTION100
2.33-2.380.23481440.18533055X-RAY DIFFRACTION100
2.38-2.440.2321620.18713068X-RAY DIFFRACTION100
2.44-2.50.23981810.1873005X-RAY DIFFRACTION100
2.5-2.580.22821490.18823091X-RAY DIFFRACTION100
2.58-2.660.23481500.19133057X-RAY DIFFRACTION100
2.66-2.750.24611560.19123045X-RAY DIFFRACTION100
2.75-2.860.23371790.1913055X-RAY DIFFRACTION100
2.86-2.980.24381450.20113090X-RAY DIFFRACTION100
2.98-3.140.23881660.19353069X-RAY DIFFRACTION100
3.14-3.330.22481580.19273082X-RAY DIFFRACTION100
3.33-3.570.19491580.18083098X-RAY DIFFRACTION100
3.57-3.910.18661530.17213120X-RAY DIFFRACTION100
3.91-4.430.16571440.15443140X-RAY DIFFRACTION100
4.43-5.430.18211760.15383146X-RAY DIFFRACTION100
5.43-9.980.20191750.17353245X-RAY DIFFRACTION100

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