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- PDB-8fie: Crystal Structure of Erwinia tracheiphila CYP114 mutant - A261D -

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Basic information

Entry
Database: PDB / ID: 8fie
TitleCrystal Structure of Erwinia tracheiphila CYP114 mutant - A261D
ComponentsCytochrome P450
KeywordsOXIDOREDUCTASE / cytochrome P450 / gibberellin
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 / Cytochrome P450
Similarity search - Component
Biological speciesErwinia tracheiphila PSU-1 (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.26 Å
AuthorsStewart Jr., C.E. / Alexander, L.E. / Nagel, R.
Funding support United States, Germany, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131885 United States
German Research Foundation (DFG)NA 1261/1-2 Germany
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis.
Authors: Nagel, R. / Alexander, L.E. / Stewart Jr., C.E. / Peters, R.J.
History
DepositionDec 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 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)49,5422
Polymers48,9251
Non-polymers6161
Water4,900272
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)93.211, 97.499, 107.174
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-829-

HOH

21A-840-

HOH

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Components

#1: Protein Cytochrome P450


Mass: 48925.293 Da / Num. of mol.: 1 / Mutation: A261D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Erwinia tracheiphila PSU-1 (bacteria) / Gene: SY86_19565 / Plasmid: pET101 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star / References: UniProt: A0A0M2KDV0
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 272 / 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: 2.49 Å3/Da / Density % sol: 50.57 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 25% PEG 3350, 0.1M Bis-Tris pH 5.5, 0.5% Polyvinylpyrrolidone K15

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54187 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: May 15, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54187 Å / Relative weight: 1
ReflectionResolution: 2.26→67.37 Å / Num. obs: 22010 / % possible obs: 94.8 % / Redundancy: 4.5 % / CC1/2: 0.942 / Rmerge(I) obs: 0.148 / Rpim(I) all: 0.074 / Rrim(I) all: 0.166 / Net I/σ(I): 5.8 / Num. measured all: 99510 / Scaling rejects: 295
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
2.26-2.334.20.355745617830.3270.1940.4092.784.1
9.02-67.374.30.08415813710.9820.0450.0969.392.6

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation6.15 Å57.04 Å
Translation6.15 Å57.04 Å

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Processing

Software
NameVersionClassification
MOSFLM1.18.2data reduction
Aimless0.5.27data scaling
PHASER2.8.2phasing
PHENIX1.14refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7RLP

7rlp
PDB Unreleased entry


Resolution: 2.26→44.37 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 22.04 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.228 1078 4.9 %
Rwork0.184 20904 -
obs0.1862 21982 94.72 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 87.79 Å2 / Biso mean: 28.2832 Å2 / Biso min: 7.38 Å2
Refinement stepCycle: final / Resolution: 2.26→44.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3259 0 73 272 3604
Biso mean--16.1 28.58 -
Num. residues----418
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0033394
X-RAY DIFFRACTIONf_angle_d0.5884629
X-RAY DIFFRACTIONf_dihedral_angle_d19.6451237
X-RAY DIFFRACTIONf_chiral_restr0.038513
X-RAY DIFFRACTIONf_plane_restr0.003605
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 8

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.26-2.360.27211210.19912493261492
2.36-2.490.23471460.18192710285699
2.49-2.640.22421390.17642699283899
2.64-2.850.23931290.19012457258690
2.85-3.130.27451400.19422730287099
3.13-3.590.24331310.19112582271393
3.59-4.520.19471290.16682500262990
4.52-44.370.20081430.1862733287695

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