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- PDB-5jqu: Crystal structure of Cytochrome P450 BM3 heme domain G265F/T269V/... -

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Basic information

Entry
Database: PDB / ID: 5jqu
TitleCrystal structure of Cytochrome P450 BM3 heme domain G265F/T269V/L272W/L322I/F405M/A406S (WIVS-FM) variant with iron(III) deuteroporphyrin IX bound
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / P450 BM3 / iron(III) deuteroporphyrin IX
Function / homology
Function and homology information


aromatase activity / 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 / FMN binding / flavin adenine dinucleotide binding / iron ion binding / heme binding / identical protein binding / cytosol
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 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / 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 / Flavoprotein-like superfamily / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
FE(III) DEUTEROPORPHYRIN IX / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.162 Å
AuthorsReynolds, E.W. / McHenry, M.W. / Cannac, F. / Gober, J.G. / Snow, C.D. / Brustad, E.M.
Funding support United States, 2items
OrganizationGrant numberCountry
Defense Advanced Research Projects AgencyD13AP00024 United States
National Science Foundation (NSF, United States)DGE1144081 United States
CitationJournal: J.Am.Chem.Soc. / Year: 2016
Title: An Evolved Orthogonal Enzyme/Cofactor Pair.
Authors: Reynolds, E.W. / McHenry, M.W. / Cannac, F. / Gober, J.G. / Snow, C.D. / Brustad, E.M.
History
DepositionMay 5, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2016Group: Database references
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Sep 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 / database_2 / diffrn_radiation_wavelength / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _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

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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
C: Bifunctional cytochrome P450/NADPH--P450 reductase
D: Bifunctional cytochrome P450/NADPH--P450 reductase
E: Bifunctional cytochrome P450/NADPH--P450 reductase
F: Bifunctional cytochrome P450/NADPH--P450 reductase
G: Bifunctional cytochrome P450/NADPH--P450 reductase
H: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)437,30416
Polymers432,7898
Non-polymers4,5158
Water21,5461196
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19510 Å2
ΔGint-181 kcal/mol
Surface area140530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)106.057, 166.299, 229.331
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and ((resid 3 and (name N or name...
21(chain B and (resseq 3 or (resid 4 and (name...
31(chain C and ((resid 3 and (name N or name...
41(chain D and ((resid 3 and (name N or name...
51(chain E and ((resid 3 and (name N or name...
61(chain F and ((resid 3 and (name N or name...
71(chain G and ((resid 3 and (name N or name...
81(chain H and (resseq 3 or (resid 4 and (name...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and ((resid 3 and (name N or name...A0
211(chain B and (resseq 3 or (resid 4 and (name...B0
311(chain C and ((resid 3 and (name N or name...C0
411(chain D and ((resid 3 and (name N or name...D3
421(chain D and ((resid 3 and (name N or name...D1 - 455
431(chain D and ((resid 3 and (name N or name...D1 - 455
441(chain D and ((resid 3 and (name N or name...D1 - 455
451(chain D and ((resid 3 and (name N or name...D1 - 455
511(chain E and ((resid 3 and (name N or name...E0
611(chain F and ((resid 3 and (name N or name...F0
711(chain G and ((resid 3 and (name N or name...G0
811(chain H and (resseq 3 or (resid 4 and (name...H0

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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: 54098.574 Da / Num. of mol.: 8 / Fragment: heme domain, residues 2-456 / Mutation: G265F,T269V,L272W,L322I,F405M,A406S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (strain ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / VKM B-512) (bacteria)
Strain: ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / VKM B-512
Gene: cyp102A1, cyp102, BG04_163 / Plasmid: pCWori / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 AI
References: UniProt: P14779, unspecific monooxygenase, NADPH-hemoprotein reductase
#2: Chemical
ChemComp-FDE / FE(III) DEUTEROPORPHYRIN IX


Mass: 564.413 Da / Num. of mol.: 8 / Fragment: iron(III) deuteroporphyrin IX / Source method: obtained synthetically / Formula: C30H28FeN4O4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1196 / 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: 2.41 Å3/Da / Density % sol: 49.02 %
Crystal growTemperature: 298 K / Method: vapor diffusion / pH: 7.5 / Details: 0.25 M MgCl2, 17% PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1.04 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Oct 31, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.04 Å / Relative weight: 1
ReflectionResolution: 2.16→28.85 Å / Num. obs: 213435 / % possible obs: 98.9 % / Redundancy: 4.9 % / Rmerge(I) obs: 0.107 / Net I/σ(I): 4.9
Reflection shellResolution: 2.16→2.2 Å / Redundancy: 4.4 % / Rmerge(I) obs: 0.47 / Mean I/σ(I) obs: 2.6 / % possible all: 99.3

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIXrefinement
PDB_EXTRACT3.2data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2IJ2

2ij2


Resolution: 2.162→28.848 Å / SU ML: 0.68 / Cross valid method: FREE R-VALUE / σ(F): 1.52 / Phase error: 42.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3335 2000 0.94 %
Rwork0.2584 211435 -
obs0.2591 213435 98.85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 127.21 Å2 / Biso mean: 15.4318 Å2 / Biso min: 2 Å2
Refinement stepCycle: final / Resolution: 2.162→28.848 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms28392 0 312 1196 29900
Biso mean--7.6 10.82 -
Num. residues----3619
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01129475
X-RAY DIFFRACTIONf_angle_d1.24740104
X-RAY DIFFRACTIONf_chiral_restr0.0664356
X-RAY DIFFRACTIONf_plane_restr0.0095206
X-RAY DIFFRACTIONf_dihedral_angle_d15.70717713
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A14576X-RAY DIFFRACTION13.656TORSIONAL
12B14576X-RAY DIFFRACTION13.656TORSIONAL
13C14576X-RAY DIFFRACTION13.656TORSIONAL
14D14576X-RAY DIFFRACTION13.656TORSIONAL
15E14576X-RAY DIFFRACTION13.656TORSIONAL
16F14576X-RAY DIFFRACTION13.656TORSIONAL
17G14576X-RAY DIFFRACTION13.656TORSIONAL
18H14576X-RAY DIFFRACTION13.656TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.1625-2.21650.44271420.3911149851512799
2.2165-2.27640.4641380.4078146021474097
2.2764-2.34340.37221420.3694150531519599
2.3434-2.4190.37651420.3399150101515299
2.419-2.50540.40481420.3388150271516999
2.5054-2.60570.38431440.3327150881523299
2.6057-2.72420.40351400.3155148651500598
2.7242-2.86770.35981430.2878151281527199
2.8677-3.04710.37331430.2762151031524699
3.0471-3.28210.34971430.248151941533799
3.2821-3.61180.31861430.2103150651520898
3.6118-4.13320.27641430.1806151181526198
4.1332-5.20240.22771460.15841544915595100
5.2024-28.85050.25491490.1685157481589799

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