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- PDB-7y0u: Crystal structure of the P450 BM3 heme domain mutant F87A in comp... -

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Basic information

Entry
Database: PDB / ID: 7y0u
TitleCrystal structure of the P450 BM3 heme domain mutant F87A in complex with N-imidazolyl-hexanoyl-L-phenylalanyl-L-phenylalanine and hydroxylamine
Components
  • Bifunctional cytochrome P450/NADPH--P450 reductase
  • I7X-PHE-PHE
KeywordsOXIDOREDUCTASE / Dual-functional small molecule / P450 heme domain / Complex
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 / response to hormone / FMN binding / flavin adenine dinucleotide binding / iron ion binding ...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 / response to hormone / 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
PROTOPORPHYRIN IX CONTAINING FE / HYDROXYAMINE / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesPriestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsJiang, Y. / Dong, S. / Feng, Y. / Cong, Z.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21778060 China
National Natural Science Foundation of China (NSFC)21977104 China
CitationJournal: To Be Published
Title: Crystal structure of the P450 BM3 heme domain mutant F87A in complex with N-imidazolyl-hexanoyl-L-phenylalanyl-L-phenylalanine and hydroxylamine
Authors: Jiang, Y. / Dong, S. / Feng, Y. / Cong, Z.
History
DepositionJun 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 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 cytochrome P450/NADPH--P450 reductase
B: Bifunctional cytochrome P450/NADPH--P450 reductase
C: I7X-PHE-PHE
D: I7X-PHE-PHE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,9588
Polymers107,6594
Non-polymers1,2994
Water10,809600
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5980 Å2
ΔGint-59 kcal/mol
Surface area32520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.444, 143.469, 62.344
Angle α, β, γ (deg.)90.000, 98.290, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 167 or (resid 168...
21(chain B and (resid 3 through 157 or (resid 158...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LYSLYSARGARG(chain A and (resid 3 through 167 or (resid 168...AA3 - 1675 - 169
12ASPASPPROPRO(chain A and (resid 3 through 167 or (resid 168...AA168 - 170170 - 172
13LYSLYSLEULEU(chain A and (resid 3 through 167 or (resid 168...AA3 - 4555 - 457
14LYSLYSLEULEU(chain A and (resid 3 through 167 or (resid 168...AA3 - 4555 - 457
15LYSLYSLEULEU(chain A and (resid 3 through 167 or (resid 168...AA3 - 4555 - 457
16LYSLYSLEULEU(chain A and (resid 3 through 167 or (resid 168...AA3 - 4555 - 457
21LYSLYSGLYGLY(chain B and (resid 3 through 157 or (resid 158...BB3 - 1575 - 159
22PHEPHEPHEPHE(chain B and (resid 3 through 157 or (resid 158...BB158160
23LYSLYSLEULEU(chain B and (resid 3 through 157 or (resid 158...BB3 - 4555 - 457
24LYSLYSLEULEU(chain B and (resid 3 through 157 or (resid 158...BB3 - 4555 - 457
25LYSLYSLEULEU(chain B and (resid 3 through 157 or (resid 158...BB3 - 4555 - 457
26LYSLYSLEULEU(chain B and (resid 3 through 157 or (resid 158...BB3 - 4555 - 457

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Components

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


Mass: 53352.758 Da / Num. of mol.: 2 / Mutation: F87A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
Strain: ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / VKM B-512
Gene: cyp102A1, cyp102, BG04_163 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase
#2: Protein/peptide I7X-PHE-PHE


Mass: 476.568 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: 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
#4: Chemical ChemComp-HOA / HYDROXYAMINE


Mass: 33.030 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: H3NO / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 600 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.35 Å3/Da / Density % sol: 47.7 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.38 M MgCl2, 0.1 M Tris 8.5, 12-18%PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17B1 / Wavelength: 0.97857 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Jul 16, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97857 Å / Relative weight: 1
ReflectionResolution: 2→50 Å / Num. obs: 59348 / % possible obs: 88.6 % / Redundancy: 2.9 % / Rmerge(I) obs: 0.118 / Rpim(I) all: 0.08 / Rrim(I) all: 0.144 / Χ2: 0.795 / Net I/σ(I): 4.8 / Num. measured all: 172214
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.032.60.51126800.5840.3710.6350.52181.1
2.03-2.072.90.48828550.6820.3350.5940.53184.9
2.07-2.1130.42228900.7480.2820.510.55486.4
2.11-2.1530.40228510.7550.2640.4830.59585.8
2.15-2.230.37228990.7830.2450.4470.65786.7
2.2-2.2530.35228750.7940.2310.4220.67386.7
2.25-2.3130.31329140.8390.2050.3760.7386
2.31-2.3730.28828810.840.1910.3470.75287
2.37-2.4430.2629270.8630.1720.3130.79687.8
2.44-2.522.90.22429530.880.1490.270.80488.6
2.52-2.612.90.21429920.8830.1430.2580.87288.5
2.61-2.712.80.18129520.8960.1230.220.95388.4
2.71-2.842.60.16128650.8960.1150.1991.02186.2
2.84-2.992.70.1428770.9220.0970.1711.01386
2.99-3.1730.11830250.9450.0790.1431.07990.7
3.17-3.422.90.0930910.960.0610.111.03891.9
3.42-3.762.90.06931080.9750.0480.0850.92692.9
3.76-4.312.80.05832240.9790.0410.0710.82695.9
4.31-5.432.70.05532090.9830.0390.0670.78195.4
5.43-503.20.05732800.9890.0380.0690.76195.6

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7EGN

7egn


Resolution: 2→45.17 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.39 / Phase error: 22.8 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2261 1990 3.55 %
Rwork0.1992 54093 -
obs0.2001 56083 83.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 73.4 Å2 / Biso mean: 29.367 Å2 / Biso min: 11.63 Å2
Refinement stepCycle: final / Resolution: 2→45.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6950 0 160 600 7710
Biso mean--22.16 32.07 -
Num. residues----884
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4228X-RAY DIFFRACTION6.615TORSIONAL
12B4228X-RAY DIFFRACTION6.615TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2-2.050.25911120.21683018313065
2.05-2.110.24941280.2083437356575
2.11-2.170.2671340.20933533366777
2.17-2.240.25741260.20813576370277
2.24-2.320.22581330.20323571370478
2.32-2.410.25061330.20383727386081
2.41-2.520.25031460.21113866401284
2.52-2.660.26221440.2154008415287
2.66-2.820.24991510.21913986413786
2.82-3.040.2661400.22153987412787
3.04-3.350.21921610.20374194435591
3.35-3.830.23021440.18964321446593
3.83-4.820.18031730.17434453462696
4.82-45.170.20391650.19294416458195

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