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- PDB-3r1b: Open crystal structure of cytochrome P450 2B4 covalently bound to... -

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Basic information

Entry
Database: PDB / ID: 3r1b
TitleOpen crystal structure of cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene
ComponentsCytochrome P450 2B4
KeywordsOXIDOREDUCTASE/OXIDOREDUCTASE INHIBITOR / cytochrome P450 2B4 / monooxygenase / membrane protein / OXIDOREDUCTASE-OXIDOREDUCTASE INHIBITOR complex
Function / homology
Function and homology information


arachidonate epoxygenase activity / epoxygenase P450 pathway / 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 / xenobiotic metabolic process / iron ion binding / heme binding / endoplasmic reticulum membrane
Similarity search - Function
Cytochrome P450, E-class, group I, CYP2B-like / : / Cytochrome P450, E-class, group I / Cytochrome p450 / Cytochrome P450 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 ...Cytochrome P450, E-class, group I, CYP2B-like / : / Cytochrome P450, E-class, group I / Cytochrome p450 / Cytochrome P450 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
sucrose / PROTOPORPHYRIN IX CONTAINING FE / (4-tert-butylphenyl)acetaldehyde / Cytochrome P450 2B4
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3 Å
AuthorsGay, S.C. / Zhang, H. / Stout, C.D. / Hollenberg, P.F. / Halpert, J.R.
Citation
Journal: Biochemistry / Year: 2011
Title: Structural Analysis of Mammalian Cytochrome P450 2B4 Covalently Bound to the Mechanism-Based Inactivator tert-Butylphenylacetylene: Insight into Partial Enzymatic Activity.
Authors: Gay, S.C. / Zhang, H. / Wilderman, P.R. / Roberts, A.G. / Liu, T. / Li, S. / Lin, H.L. / Zhang, Q. / Woods, V.L. / Stout, C.D. / Hollenberg, P.F. / Halpert, J.R.
#1: Journal: J.Biol.Chem. / Year: 2006
Title: Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction.
Authors: Zhao, Y. / White, M.A. / Muralidhara, B.K. / Sun, L. / Halpert, J.R. / Stout, C.D.
#2: Journal: J.Biol.Chem. / Year: 2004
Title: Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
Authors: Scott, E.E. / White, M.A. / He, Y.A. / Johnson, E.F. / Stout, C.D. / Halpert, J.R.
#3: Journal: Proc.Natl.Acad.Sci.USA / Year: 2003
Title: An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution.
Authors: Scott, E.E. / He, Y.A. / Wester, M.R. / White, M.A. / Chin, C.C. / Halpert, J.R. / Johnson, E.F. / Stout, C.D.
#4: Journal: Biochemistry / Year: 2007
Title: Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4.
Authors: Zhao, Y. / Sun, L. / Muralidhara, B.K. / Kumar, S. / White, M.A. / Stout, C.D. / Halpert, J.R.
#5: Journal: Biochemistry / Year: 2009
Title: Crystal structures of cytochrome P450 2B4 in complex with the inhibitor 1-biphenyl-4-methyl-1H-imidazole: ligand-induced structural response through alpha-helical repositioning.
Authors: Gay, S.C. / Sun, L. / Maekawa, K. / Halpert, J.R. / Stout, C.D.
#6: Journal: Biochemistry / Year: 2010
Title: Structures of cytochrome P450 2B4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .
Authors: Gay, S.C. / Roberts, A.G. / Maekawa, K. / Talakad, J.C. / Hong, W.X. / Zhang, Q. / Stout, C.D. / Halpert, J.R.
#7: Journal: J.Biol.Chem. / Year: 2010
Title: Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography.
Authors: Wilderman, P.R. / Shah, M.B. / Liu, T. / Li, S. / Hsu, S. / Roberts, A.G. / Goodlett, D.R. / Zhang, Q. / Woods, V.L. / Stout, C.D. / Halpert, J.R.
History
DepositionMar 9, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 11, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / struct_asym / struct_conn / struct_conn_type / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.value / _struct_asym.entity_id / _struct_conn_type.id / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Sep 13, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.2Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome P450 2B4
B: Cytochrome P450 2B4
C: Cytochrome P450 2B4
D: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,68921
Polymers216,6764
Non-polymers7,01317
Water00
1
A: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,2936
Polymers54,1691
Non-polymers2,1245
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,2936
Polymers54,1691
Non-polymers2,1245
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,3044
Polymers54,1691
Non-polymers1,1353
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,7995
Polymers54,1691
Non-polymers1,6304
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
A: Cytochrome P450 2B4
D: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,09211
Polymers108,3382
Non-polymers3,7549
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9730 Å2
ΔGint-85 kcal/mol
Surface area42590 Å2
MethodPISA
6
B: Cytochrome P450 2B4
C: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,59710
Polymers108,3382
Non-polymers3,2598
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8440 Å2
ΔGint-82 kcal/mol
Surface area42430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)193.924, 153.691, 129.665
Angle α, β, γ (deg.)90.00, 122.22, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
Cytochrome P450 2B4 / CYPIIB4 / Cytochrome P450 isozyme 2 / Cytochrome P450 LM2 / Cytochrome P450 type B0 / Cytochrome P450 type B1


Mass: 54169.082 Da / Num. of mol.: 4
Mutation: E2A, G22K, H23K, P24T, K25S, A26S, H27K, R29K, H226Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: CYP2B4 / Plasmid: PKK / Production host: Escherichia coli (E. coli) / Strain (production host): TOPP3 / References: UniProt: P00178, unspecific monooxygenase
#2: Polysaccharide
beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose / sucrose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose
DescriptorTypeProgram
DFrufb2-1DGlcpaGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[ha122h-2b_2-5][a2122h-1a_1-5]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(2+1)][a-D-Glcp]{}}LINUCSPDB-CARE
#3: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#4: Chemical
ChemComp-TB2 / (4-tert-butylphenyl)acetaldehyde


Mass: 176.255 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C12H16O
#5: Chemical
ChemComp-CM5 / 5-CYCLOHEXYL-1-PENTYL-BETA-D-MALTOSIDE / 5-CYCLOHEXYLPENTYL 4-O-ALPHA-D-GLUCOPYRANOSYL-BETA-D-GLUCOPYRANOSIDE / CYMAL-5


Mass: 494.573 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C23H42O11 / Comment: detergent*YM
Has protein modificationY
Nonpolymer detailsTHE PROTEIN WAS INCUBATED WITH TERT-BUTYLPHENYLACETYLENE, CATALASE, CYTOCHROME P450 REDUCTASE, AND ...THE PROTEIN WAS INCUBATED WITH TERT-BUTYLPHENYLACETYLENE, CATALASE, CYTOCHROME P450 REDUCTASE, AND CYTOCHROME B5. A SINGLE CATALYTIC P450 2B4 TURNOVER RESULTS IN A KETENE INTERMEDIATE, WHICH CONJUGATES TO THR 302. THIS CONJUGATED PROTEIN IS PUT DOWN FOR CRYSTALLIZATION. lIGAND CM5 A 605 IS MISSING 10 ATOMS SINCE THEY WERE NOT OBSERVED IN THE ELECTRON DENSITY MAPS.

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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: 3.77 Å3/Da / Density % sol: 67.39 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M HEPES, 12% (w/v) PEG 3350, pH 7.5, vapor diffusion, sitting drop, temperature 291K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL11-1 / Wavelength: 0.98 Å
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: May 22, 2010
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 3→112.161 Å / Num. all: 63932 / Num. obs: 63932 / % possible obs: 99.4 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.2 % / Biso Wilson estimate: 84.22 Å2 / Rmerge(I) obs: 0.092 / Rsym value: 0.092 / Net I/σ(I): 7.5
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsRsym value% possible all
3-3.163.10.5861.30.58699.9
3.16-3.353.20.3212.40.32199.8
3.35-3.593.20.2273.40.22799.7
3.59-3.873.20.145.20.1499.6
3.87-4.243.20.1046.40.10499.5
4.24-4.743.20.0887.30.08899.3
4.74-5.483.20.0798.10.07999.2
5.48-6.713.20.0758.40.07598.7
6.71-9.493.20.0648.60.06498.4
9.49-53.4323.10.0638.70.06397.1

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 51.63 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3 Å53.43 Å
Translation3 Å53.43 Å

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.3.9data scaling
PHASER2.1.4phasing
PHENIX1.6.4_486refinement
PDB_EXTRACT3.1data extraction
SSRLBlue-Icedata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1SUO

1suo


Resolution: 3→53.43 Å / Occupancy max: 1 / Occupancy min: 0 / SU ML: 0.44 / σ(F): 0 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.288 3092 5.07 %
Rwork0.242 --
obs0.244 61019 94.8 %
all-64352 -
Solvent computationShrinkage radii: 0.72 Å / VDW probe radii: 1 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 81.04 Å2 / ksol: 0.33 e/Å3
Displacement parametersBiso mean: 100.79 Å2
Baniso -1Baniso -2Baniso -3
1--4.4092 Å2-0 Å23.4714 Å2
2---9.6467 Å2-0 Å2
3---14.0559 Å2
Refinement stepCycle: LAST / Resolution: 3→53.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14023 0 476 0 14499
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00814884
X-RAY DIFFRACTIONf_angle_d1.35120347
X-RAY DIFFRACTIONf_dihedral_angle_d16.4135345
X-RAY DIFFRACTIONf_chiral_restr0.0782303
X-RAY DIFFRACTIONf_plane_restr0.012623
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.04690.42791460.35592344X-RAY DIFFRACTION85
3.0469-3.09680.3461290.3222385X-RAY DIFFRACTION86
3.0968-3.15020.3591190.3162426X-RAY DIFFRACTION88
3.1502-3.20750.40391310.29912504X-RAY DIFFRACTION91
3.2075-3.26920.34741260.29422588X-RAY DIFFRACTION93
3.2692-3.33590.33481560.28932595X-RAY DIFFRACTION93
3.3359-3.40840.29831330.27742556X-RAY DIFFRACTION93
3.4084-3.48770.32721490.2682528X-RAY DIFFRACTION93
3.4877-3.57490.30381120.26292518X-RAY DIFFRACTION91
3.5749-3.67150.29931490.27952708X-RAY DIFFRACTION97
3.6715-3.77960.32391440.25392731X-RAY DIFFRACTION99
3.7796-3.90150.25491500.23652705X-RAY DIFFRACTION97
3.9015-4.04090.27931420.23352659X-RAY DIFFRACTION96
4.0409-4.20260.28311380.23062682X-RAY DIFFRACTION98
4.2026-4.39380.27911450.22092751X-RAY DIFFRACTION98
4.3938-4.62540.30921560.21192719X-RAY DIFFRACTION98
4.6254-4.9150.2771510.21382744X-RAY DIFFRACTION99
4.915-5.29410.24461490.22552733X-RAY DIFFRACTION99
5.2941-5.82630.34611380.26192751X-RAY DIFFRACTION99
5.8263-6.6680.26991340.27042758X-RAY DIFFRACTION98
6.668-8.39580.26541510.22412760X-RAY DIFFRACTION98
8.3958-53.44060.23661440.20992782X-RAY DIFFRACTION97

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