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- PDB-5jtd: Crystal structure of the Ru(bpy)2PhenA functionalized P450 BM3 L4... -

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Basic information

Entry
Database: PDB / ID: 5jtd
TitleCrystal structure of the Ru(bpy)2PhenA functionalized P450 BM3 L407C heme domain mutant in complex with DMSO.
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / hybrid P450 BM3 enzymes / electron transfer / photosensitizer / photocatalytic activity
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 / FMN binding / iron ion binding / heme binding / identical protein binding / cytoplasm
Similarity search - Function
Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Cytochrome p450 / Cytochrome P450 / 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. ...Bifunctional cytochrome P450/NADPH--cytochrome P450 reductase / Cytochrome p450 / Cytochrome P450 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Chem-RU8 / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.5 Å
AuthorsKloos, M.
CitationJournal: Biochim.Biophys.Acta / Year: 2016
Title: Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies.
Authors: Spradlin, J. / Lee, D. / Mahadevan, S. / Mahomed, M. / Tang, L. / Lam, Q. / Colbert, A. / Shafaat, O.S. / Goodin, D. / Kloos, M. / Kato, M. / Cheruzel, L.E.
History
DepositionMay 9, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 12, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_initial_refinement_model / pdbx_struct_oper_list / struct_conn / struct_conn_type
Item: _citation.journal_id_CSD / _database_2.pdbx_DOI ..._citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.symmetry_operation / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,46910
Polymers107,3702
Non-polymers3,0998
Water12,574698
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5680 Å2
ΔGint-38 kcal/mol
Surface area37230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.690, 145.390, 62.900
Angle α, β, γ (deg.)90.000, 97.080, 90.000
Int Tables number4
Space group name H-MP1211

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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: 53685.062 Da / Num. of mol.: 2 / Mutation: L407C
Source method: isolated from a genetically manipulated source
Details: A Ru(bpy)2)PhenA was covalently attached to a non-native single cysteine (L407C) mutant of P450 BM3 mutant (post translational modification)
Source: (gene. exp.) Bacillus megaterium (bacteria)
Strain: ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / VKM B-512
Gene: cyp102A1, cyp102, BG04_163 / Plasmid: pC-Wori / Production host: Escherichia coli (E. coli)
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical
ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#4: Chemical ChemComp-RU8 / bis(2,2'-bipyridine-kappa~2~N~1~,N~1'~)[2-iodo-N-(1,10-phenanthrolin-5-yl-kappa~2~N~1~,N~10~)acetamide]ruthenium(2+)


Mass: 776.591 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H26IN7ORu
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 698 / Source method: isolated from a natural source / Formula: H2O

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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: 2.54 Å3/Da / Density % sol: 51.61 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 150 mM MgCl2, 125 mM Na-MOPS, 20% PEG 3350, cryo: 30% glycerol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 15, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.4→47.36 Å / Num. obs: 183724 / % possible obs: 79.5 % / Observed criterion σ(I): -3 / Redundancy: 3.2 % / Biso Wilson estimate: 24.898 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.044 / Net I/σ(I): 9.61
Reflection shell
Resolution (Å)Rmerge(I) obsMean I/σ(I) obsDiffraction-ID% possible all
1.4-1.50.8290.96148.1
1.5-1.60.51.81180.6
1.6-1.70.3172.87185.6
1.7-1.80.1974.23185.7
1.8-1.90.1376.2187.9
1.9-20.0948.21188
2-2.30.05812.02188.4
2.3-2.70.0416.62189.2
2.7-3.10.0321.58191
3.1-3.50.02725.13189.6
3.5-40.02626.48188.1
4-60.0233.88191
6-100.01735.8192.5
10-47.360.01144.13195.8

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Phasing

PhasingMethod: molecular replacement
Phasing MRPacking: 2

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Processing

Software
NameVersionClassification
REFMACrefinement
XSCALEv.Oct-2015data scaling
PHASER2,5,7phasing
PDB_EXTRACT3.2data extraction
XDSv.Oct-2015data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1JPZ

1jpz


Resolution: 1.5→47.36 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.957 / SU B: 1.554 / SU ML: 0.055 / SU R Cruickshank DPI: 0.0705 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.07 / ESU R Free: 0.071
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY ALTERNATIVE CONFORMATION OF HEM 460 AND RES 267-269 WERE DETECTED IN BOTH CHAINS BUT NOT MODELLED. WE ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY ALTERNATIVE CONFORMATION OF HEM 460 AND RES 267-269 WERE DETECTED IN BOTH CHAINS BUT NOT MODELLED. WE OBSERVED DIFFERENCE DENSITY CLOSE TO THE RU8-LIGANDS WHICH MIGHT EITHER RESULT FROM THEIR STATIC DISORDER, OR ALTERNATIV CONFORMATION DUE TO A PHOTOSENSITIZER-ISOMER WHICH WAS COVALENTLY ATTACHED.
RfactorNum. reflection% reflectionSelection details
Rfree0.1977 8058 5 %RANDOM
Rwork0.1733 ---
obs0.1745 153091 96.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 74.9 Å2 / Biso mean: 22.68 Å2 / Biso min: 11.22 Å2
Baniso -1Baniso -2Baniso -3
1--0.9 Å20 Å2-0.26 Å2
2---0.65 Å20 Å2
3---1.57 Å2
Refinement stepCycle: final / Resolution: 1.5→47.36 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7368 0 188 699 8255
Biso mean--19.36 31.42 -
Num. residues----916
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0197842
X-RAY DIFFRACTIONr_bond_other_d0.0020.027468
X-RAY DIFFRACTIONr_angle_refined_deg1.5612.00210670
X-RAY DIFFRACTIONr_angle_other_deg0.961317229
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0745938
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.7824.814376
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.79151392
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.8651542
X-RAY DIFFRACTIONr_chiral_restr0.0910.21119
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0218830
X-RAY DIFFRACTIONr_gen_planes_other0.0060.021812
X-RAY DIFFRACTIONr_mcbond_it1.3612.0873686
X-RAY DIFFRACTIONr_mcbond_other1.362.0873685
X-RAY DIFFRACTIONr_mcangle_it2.1173.1254610
LS refinement shellResolution: 1.5→1.539 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.305 540 -
Rwork0.293 10259 -
all-10799 -
obs--87.78 %

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