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- PDB-5og9: The BM3 mutant WIFI-WC heme domain in complex with testosterone -

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Basic information

Entry
Database: PDB / ID: 5og9
TitleThe BM3 mutant WIFI-WC heme domain in complex with testosterone
ComponentsBifunctional cytochrome P450/NADPH--P450 reductase
KeywordsOXIDOREDUCTASE / cytochrome P450 / testosterone / laboratory evolution / hydroxylation / steroids
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 / response to hormone / FMN binding / flavin adenine dinucleotide binding / iron ion binding ...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 / response to hormone / 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 / 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 / TESTOSTERONE / Bifunctional cytochrome P450/NADPH--P450 reductase / Bifunctional cytochrome P450/NADPH--P450 reductase
Similarity search - Component
Biological speciesBacillus megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.09 Å
AuthorsGamble, C. / Munro, A.W. / Leys, D.
CitationJournal: To Be Published
Title: Regio- and diastereoselective hydroxylation of steroids using P450-BM3: Readdressing the numbers problem in directed evolution
Authors: Acevedo-Rocha, C.G. / Gamble, C. / Lonsdale, R. / Li, A. / Nett, N. / Hoebenreich, S. / Lingnau, J. / Wirtz, C. / Fares, C. / Hinrichs, H. / Deege, A. / Mulholland, A.J. / Nov, Y. / Leys, D. ...Authors: Acevedo-Rocha, C.G. / Gamble, C. / Lonsdale, R. / Li, A. / Nett, N. / Hoebenreich, S. / Lingnau, J. / Wirtz, C. / Fares, C. / Hinrichs, H. / Deege, A. / Mulholland, A.J. / Nov, Y. / Leys, D. / Munro, A.W. / Reetz, M.T.
History
DepositionJul 12, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: 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 / pdbx_initial_refinement_model / struct_conn / struct_ncs_dom_lim
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 / _struct_conn.ptnr2_label_seq_id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_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)110,81710
Polymers108,3302
Non-polymers2,4888
Water8,431468
1
A: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,4596
Polymers54,1651
Non-polymers1,2945
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Bifunctional cytochrome P450/NADPH--P450 reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,3584
Polymers54,1651
Non-polymers1,1933
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.390, 89.502, 172.550
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:

Component-ID: 0 / Ens-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS / End auth comp-ID: GLY / End label comp-ID: GLY / Refine code: 0 / Auth seq-ID: 4 - 457 / Label seq-ID: 3 - 456

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Bifunctional cytochrome P450/NADPH--P450 reductase


Mass: 54164.848 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus megaterium (bacteria) / Production host: Escherichia coli (E. coli)
References: UniProt: F2Q7T0, UniProt: P14779*PLUS, unspecific monooxygenase, NADPH-hemoprotein reductase

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Non-polymers , 5 types, 476 molecules

#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-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-TES / TESTOSTERONE / Testosterone


Mass: 288.424 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C19H28O2 / Comment: hormone*YM
#5: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 468 / 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: 2.4 Å3/Da / Density % sol: 48.79 %
Crystal growTemperature: 297 K / Method: vapor diffusion, sitting drop
Details: 25% w/v PEG 550 MME, 0.1 M MES pH 6.5, 0.01 M zinc sulfate heptahydrate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS3 R CdTe 300K-W / Detector: PIXEL / Date: Nov 22, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.09→29.64 Å / Num. obs: 62142 / % possible obs: 99 % / Redundancy: 6.7 % / CC1/2: 0.999 / Rmerge(I) obs: 0.075 / Net I/σ(I): 14.7
Reflection shellResolution: 2.09→2.14 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.731 / Mean I/σ(I) obs: 2.4 / Num. unique obs: 4452 / CC1/2: 0.786 / % possible all: 97.3

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Processing

Software
NameVersionClassification
REFMAC5.7.0029refinement
xia2data reduction
xia2data scaling
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1JPZ

1jpz


Resolution: 2.09→29.64 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.941 / SU B: 5.331 / SU ML: 0.143 / Cross valid method: THROUGHOUT / ESU R: 0.224 / ESU R Free: 0.187 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23865 3155 5.1 %RANDOM
Rwork0.19263 ---
obs0.19504 58933 98.83 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 44.692 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å20 Å20 Å2
2---0.06 Å2-0 Å2
3---0.11 Å2
Refinement stepCycle: 1 / Resolution: 2.09→29.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7309 0 172 468 7949
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0197728
X-RAY DIFFRACTIONr_bond_other_d0.0050.027384
X-RAY DIFFRACTIONr_angle_refined_deg1.7681.99610514
X-RAY DIFFRACTIONr_angle_other_deg1.164317061
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2255915
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.54124.891366
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.793151351
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.0641537
X-RAY DIFFRACTIONr_chiral_restr0.1220.21126
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0218667
X-RAY DIFFRACTIONr_gen_planes_other0.0060.021754
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 27725 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.11 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.088→2.142 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.317 234 -
Rwork0.264 4214 -
obs--97.12 %

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