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- PDB-8tnk: The crystal structure of the T252E mutant of CYP199A4 bound to 4-... -

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Basic information

Entry
Database: PDB / ID: 8tnk
TitleThe crystal structure of the T252E mutant of CYP199A4 bound to 4-benzylbenzoic acid
ComponentsCytochrome P450
KeywordsOXIDOREDUCTASE / Cytochrome P450 / peroxygenase mutant
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
PROTOPORPHYRIN IX CONTAINING FE / 4-benzylbenzoic acid / Cytochrome P450
Similarity search - Component
Biological speciesRhodopseudomonas palustris HaA2 (phototrophic)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.79 Å
AuthorsPodgorski, M.N. / Bell, S.G.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC) Australia
CitationJournal: J.Inorg.Biochem. / Year: 2023
Title: Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant.
Authors: Podgorski, M.N. / Lee, J.H.Z. / Harbort, J.S. / Nguyen, G.T.H. / Doherty, D.Z. / Donald, W.A. / Harmer, J.R. / Bruning, J.B. / Bell, S.G.
History
DepositionAug 2, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release

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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)45,4804
Polymers44,6151
Non-polymers8643
Water8,125451
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)44.193, 51.590, 79.208
Angle α, β, γ (deg.)90.00, 92.44, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Cytochrome P450


Mass: 44615.438 Da / Num. of mol.: 1 / Mutation: T252E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodopseudomonas palustris HaA2 (phototrophic)
Gene: RPB_3613 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q2IU02
#2: Chemical ChemComp-IRJ / 4-benzylbenzoic acid


Mass: 212.244 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H12O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#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.: 451 / 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: 39.17 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: The crystallization conditions used were those optimized for CYP199A4 previously: 100 mM Bis-Tris buffer (adjusted to pH 5.0-5.75 with acetic acid), 0.2 M magnesium acetate and 20-32% PEG 3350.
PH range: 5.0-5.75

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95373 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 15, 2018
RadiationMonochromator: Double-crystal Si(111) liquid nitrogen cooled (DC) or channel-cut Si(111) liquid nitrogen cooled (CC)
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95373 Å / Relative weight: 1
ReflectionResolution: 1.79→44.153 Å / Num. obs: 33558 / % possible obs: 99.3 % / Redundancy: 6.2 % / CC1/2: 0.991 / Rmerge(I) obs: 0.167 / Rpim(I) all: 0.074 / Rrim(I) all: 0.183 / Χ2: 0.93 / Net I/σ(I): 10.5
Reflection shellResolution: 1.79→1.83 Å / % possible obs: 91.8 % / Redundancy: 5.7 % / Rmerge(I) obs: 1.294 / Num. measured all: 10347 / Num. unique obs: 1818 / CC1/2: 0.312 / Rpim(I) all: 0.597 / Rrim(I) all: 1.431 / Χ2: 0.82 / Net I/σ(I) obs: 2.7

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.79→44.153 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.42 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2077 1724 5.15 %
Rwork0.183 --
obs0.1844 33446 99.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.79→44.153 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3023 0 60 451 3534
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0033233
X-RAY DIFFRACTIONf_angle_d0.6174425
X-RAY DIFFRACTIONf_dihedral_angle_d23.3541194
X-RAY DIFFRACTIONf_chiral_restr0.042477
X-RAY DIFFRACTIONf_plane_restr0.004593
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7902-1.84290.28861170.23982466X-RAY DIFFRACTION92
1.8429-1.90240.2431270.22562635X-RAY DIFFRACTION100
1.9024-1.97040.29251390.21242658X-RAY DIFFRACTION100
1.9704-2.04930.23491490.20262641X-RAY DIFFRACTION100
2.0493-2.14250.22691310.19262662X-RAY DIFFRACTION100
2.1425-2.25550.22941570.19072644X-RAY DIFFRACTION100
2.2555-2.39680.22711350.19072653X-RAY DIFFRACTION100
2.3968-2.58190.20161470.18582683X-RAY DIFFRACTION100
2.5819-2.84160.21681430.19542627X-RAY DIFFRACTION99
2.8416-3.25270.20461340.18822665X-RAY DIFFRACTION99
3.2527-4.09760.18751560.15642682X-RAY DIFFRACTION100
4.0976-44.1530.17251890.15862706X-RAY DIFFRACTION100

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