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- PDB-7tnu: The crystal structure of F298V CYP199A4 bound to 4-cyclohexylbenz... -

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Basic information

Entry
Database: PDB / ID: 7tnu
TitleThe crystal structure of F298V CYP199A4 bound to 4-cyclohexylbenzoic acid
ComponentsCytochrome P450
KeywordsOXIDOREDUCTASE / Cytochrome P450 / F298V mutant / CYP199A4 / 4-cyclohexylbenzoic acid
Function / homology
Function and homology information


oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen / monooxygenase activity / iron ion binding / heme binding
Similarity search - Function
Cytochrome P450, B-class / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450
Similarity search - Domain/homology
4-cyclohexylbenzoic acid / PROTOPORPHYRIN IX CONTAINING FE / Cytochrome P450
Similarity search - Component
Biological speciesRhodopseudomonas palustris HaA2 (phototrophic)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.584 Å
AuthorsPodgorski, M. / Bell, S.G.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC) Australia
CitationJournal: Chemistry / Year: 2022
Title: Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering.
Authors: Coleman, T. / Lee, J.Z.H. / Kirk, A.M. / Doherty, D.Z. / Podgorski, M.N. / Pinidiya, D.K. / Bruning, J.B. / De Voss, J.J. / Krenske, E.H. / Bell, S.G.
History
DepositionJan 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 4, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 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

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Assembly

Deposited unit
A: Cytochrome P450
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,3964
Polymers44,5391
Non-polymers8563
Water10,701594
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1370 Å2
ΔGint-37 kcal/mol
Surface area14920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.656, 51.297, 78.532
Angle α, β, γ (deg.)90.000, 92.610, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Cytochrome P450 / CYP199A4


Mass: 44539.387 Da / Num. of mol.: 1 / Mutation: F298V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodopseudomonas palustris HaA2 (phototrophic)
Strain: HaA2 / Gene: RPB_3613 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q2IU02
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-EH1 / 4-cyclohexylbenzoic acid


Mass: 204.265 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H16O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 594 / 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: 2.02 Å3/Da / Density % sol: 39.03 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: 100 mM Bis-Tris (adjusted to pH 5.0-5.75 with acetic acid), 0.2 M magnesium acetate, 20-32% PEG3350
PH range: 5.00-5.75

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX1 / Wavelength: 0.95372 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Apr 29, 2021
RadiationMonochromator: Double-crystal Si(111) water-cooled / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95372 Å / Relative weight: 1
ReflectionResolution: 1.58→44.61 Å / Num. obs: 47014 / % possible obs: 97.1 % / Redundancy: 6.8 % / Biso Wilson estimate: 18.65 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.106 / Rpim(I) all: 0.044 / Rrim(I) all: 0.115 / Net I/σ(I): 8.6 / Num. measured all: 319434 / Scaling rejects: 15
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.58-1.615.71.395960416950.5060.6071.5270.868.6
8.53-44.616.80.02822683350.9990.0110.0335.799.3

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation5.84 Å44.61 Å
Translation5.84 Å44.61 Å

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimless0.7.4data scaling
PHASER2.8.2phasing
PHENIX1.11.1-2575refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5UVB

5uvb


Resolution: 1.584→39.562 Å / SU ML: 0.23 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 23.75 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2113 2014 4.29 %
Rwork0.1797 44927 -
obs0.1811 46941 96.85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 68.14 Å2 / Biso mean: 22.0203 Å2 / Biso min: 11.26 Å2
Refinement stepCycle: final / Resolution: 1.584→39.562 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3017 0 74 594 3685
Biso mean--16.1 31.16 -
Num. residues----393
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023234
X-RAY DIFFRACTIONf_angle_d0.7054428
X-RAY DIFFRACTIONf_chiral_restr0.041480
X-RAY DIFFRACTIONf_plane_restr0.004591
X-RAY DIFFRACTIONf_dihedral_angle_d22.7351202
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.584-1.62340.3711270.3289246576
1.6234-1.66730.31591330.2974325098
1.6673-1.71640.30071530.2686325998
1.7164-1.77180.28441380.2473325599
1.7718-1.83510.28111440.2342327799
1.8351-1.90860.26381500.218322699
1.9086-1.99540.25471320.2133320597
1.9954-2.10060.24081510.1906317097
2.1006-2.23220.2171390.1819332999
2.2322-2.40450.19761510.17463260100
2.4045-2.64650.22431470.1761331499
2.6465-3.02930.20941440.1675330999
3.0293-3.81610.16431520.155320296
3.8161-39.5620.17161530.1388340699

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