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- PDB-6ycm: Crystal structure of GcoA T296S bound to p-vanillin -

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Basic information

Entry
Database: PDB / ID: 6ycm
TitleCrystal structure of GcoA T296S bound to p-vanillin
ComponentsAromatic O-demethylase, cytochrome P450 subunit
KeywordsOXIDOREDUCTASE / Aromatic catabolism / cytochrome P450 / lignin valorization / protein engineering
Function / homology
Function and homology information


Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor / catabolic process / 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-hydroxy-3-methoxybenzaldehyde / Aromatic O-demethylase, cytochrome P450 subunit
Similarity search - Component
Biological speciesAmycolatopsis sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsMallinson, S.J.B. / Hinchen, D.J. / Ellis, E.S. / Beckham, G.T. / DuBois, J.L. / McGeehan, J.E.
Funding support United Kingdom, United States, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P011918/1 United Kingdom
Department of Energy (DOE, United States)DE-AC36-08GO28308 United States
CitationJournal: Jacs Au / Year: 2021
Title: Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.
Authors: Ellis, E.S. / Hinchen, D.J. / Bleem, A. / Bu, L. / Mallinson, S.J.B. / Allen, M.D. / Streit, B.R. / Machovina, M.M. / Doolin, Q.V. / Michener, W.E. / Johnson, C.W. / Knott, B.C. / Beckham, G. ...Authors: Ellis, E.S. / Hinchen, D.J. / Bleem, A. / Bu, L. / Mallinson, S.J.B. / Allen, M.D. / Streit, B.R. / Machovina, M.M. / Doolin, Q.V. / Michener, W.E. / Johnson, C.W. / Knott, B.C. / Beckham, G.T. / McGeehan, J.E. / DuBois, J.L.
History
DepositionMar 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Data collection / Database references
Category: citation / database_2 ...citation / database_2 / diffrn_source / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site
Revision 1.2Jan 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / diffrn_source / pdbx_initial_refinement_model
Item: _diffrn_source.pdbx_synchrotron_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aromatic O-demethylase, cytochrome P450 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,2073
Polymers45,4391
Non-polymers7692
Water8,809489
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Correlates to 5NCB, which was confirmed as monomeric by SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1230 Å2
ΔGint-25 kcal/mol
Surface area15900 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.036, 104.036, 116.842
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-644-

HOH

21A-1013-

HOH

31A-1063-

HOH

41A-1067-

HOH

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Components

#1: Protein Aromatic O-demethylase, cytochrome P450 subunit


Mass: 45438.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Amycolatopsis sp. (strain ATCC 39116 / 75iv2) (bacteria)
Strain: ATCC 39116 / 75iv2 / Gene: gcoA / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): Rosetta II
References: UniProt: P0DPQ7, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one ...References: UniProt: P0DPQ7, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor
#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-V55 / 4-hydroxy-3-methoxybenzaldehyde / p-vanillin


Mass: 152.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H8O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 489 / 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: 3.48 Å3/Da / Density % sol: 64.64 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / Details: Na Malonate, HEPES, p-vanillin

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Aug 3, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.6→52.02 Å / Num. obs: 83956 / % possible obs: 100 % / Redundancy: 2 % / Biso Wilson estimate: 22.2 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.05 / Net I/σ(I): 20.52
Reflection shellResolution: 1.6→1.657 Å / Rmerge(I) obs: 1.118 / Num. unique obs: 8286 / CC1/2: 0.898

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
xia2data reduction
xia2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5NCB

5ncb


Resolution: 1.6→52.02 Å / SU ML: 0.2075 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.8354
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2264 4143 4.93 %
Rwork0.1976 79813 -
obs0.199 84749 99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 30.01 Å2
Refinement stepCycle: LAST / Resolution: 1.6→52.02 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3113 0 54 489 3656
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00583317
X-RAY DIFFRACTIONf_angle_d0.91474560
X-RAY DIFFRACTIONf_chiral_restr0.0504481
X-RAY DIFFRACTIONf_plane_restr0.0059605
X-RAY DIFFRACTIONf_dihedral_angle_d8.1461460
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.620.39461440.38242515X-RAY DIFFRACTION95.48
1.62-1.640.35141380.3532562X-RAY DIFFRACTION97.37
1.64-1.660.32991350.34082570X-RAY DIFFRACTION96.85
1.66-1.680.31081530.33892565X-RAY DIFFRACTION97.42
1.68-1.70.35121380.33872574X-RAY DIFFRACTION97.84
1.7-1.720.36341460.31872614X-RAY DIFFRACTION98.15
1.72-1.750.35041370.30522599X-RAY DIFFRACTION98.59
1.75-1.770.31821400.29762603X-RAY DIFFRACTION98.24
1.77-1.80.34231460.27692618X-RAY DIFFRACTION98.4
1.8-1.830.321430.27262611X-RAY DIFFRACTION98.85
1.83-1.860.33781240.27182661X-RAY DIFFRACTION98.34
1.86-1.90.31781340.25022610X-RAY DIFFRACTION99.06
1.9-1.930.25191310.24142644X-RAY DIFFRACTION99.21
1.93-1.970.25761220.23772678X-RAY DIFFRACTION99.26
1.97-2.020.2651260.22712659X-RAY DIFFRACTION99.5
2.02-2.060.25531510.22532623X-RAY DIFFRACTION99.36
2.06-2.110.2561430.22262686X-RAY DIFFRACTION99.44
2.11-2.170.26921210.21722667X-RAY DIFFRACTION99.54
2.17-2.240.2321210.21012663X-RAY DIFFRACTION99.46
2.24-2.310.27161400.20262682X-RAY DIFFRACTION99.72
2.31-2.390.24751220.2032691X-RAY DIFFRACTION99.5
2.39-2.490.2211500.20212683X-RAY DIFFRACTION99.82
2.49-2.60.22231310.1992697X-RAY DIFFRACTION99.89
2.6-2.740.24481240.21042713X-RAY DIFFRACTION99.86
2.74-2.910.24391360.20792707X-RAY DIFFRACTION99.86
2.91-3.130.21511430.19462725X-RAY DIFFRACTION99.93
3.13-3.450.21071510.1772713X-RAY DIFFRACTION100
3.45-3.940.16571430.14642758X-RAY DIFFRACTION100
3.95-4.970.14051460.132792X-RAY DIFFRACTION100
4.97-52.020.19821640.16412930X-RAY DIFFRACTION99.87

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