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- PDB-3r1a: Closed crystal structure of cytochrome P450 2B4 covalently bound ... -

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Basic information

Entry
Database: PDB / ID: 3r1a
TitleClosed 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 / oxidoreductase / membrane protein / T302 covalently linked to tert-butylphenylacetylene / OXIDOREDUCTASE-OXIDOREDUCTASE INHIBITOR complex
Function / homology
Function and homology information


arachidonic acid epoxygenase activity / epoxygenase P450 pathway / unspecific monooxygenase / aromatase activity / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
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: 3.5 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 1.2Sep 13, 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_conn_type / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_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_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _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 / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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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
E: Cytochrome P450 2B4
F: Cytochrome P450 2B4
G: Cytochrome P450 2B4
H: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)439,69524
Polymers433,3538
Non-polymers6,34216
Water1448
1
A: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
4
D: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
5
E: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
6
F: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
7
G: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
8
H: Cytochrome P450 2B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9623
Polymers54,1691
Non-polymers7932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)134.381, 144.549, 229.275
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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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.: 8
Mutation: E2A, G22K, H23K, P24T, K25S, A26S, H27K, R29K, P221S, 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: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical
ChemComp-TB2 / (4-tert-butylphenyl)acetaldehyde


Mass: 176.255 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C12H16O
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O
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

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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: 2.57 Å3/Da / Density % sol: 52.13 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.4
Details: 0.1 M magnesium formate, 15% (w/v) PEG 3350, pH 7.4, 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: Jan 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: 3.5→229.275 Å / Num. all: 51302 / Num. obs: 51302 / % possible obs: 90.2 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.2 % / Biso Wilson estimate: 72.64 Å2 / Rmerge(I) obs: 0.156 / Rsym value: 0.156 / Net I/σ(I): 6.5
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
3.5-3.693.20.5771.32012063230.57777.3
3.69-3.913.10.4331.71949062920.43381.2
3.91-4.1830.3162.41918963340.31686.6
4.18-4.5230.2253.31865762100.22591.3
4.52-4.9530.1694.51804459440.16994.7
4.95-5.533.10.1614.71724855020.16196.3
5.53-6.393.30.1634.61639049970.16398.5
6.39-7.833.60.0947.91541843340.094100
7.83-11.073.60.04414.31217134070.04499.8
11.07-115.9353.30.04811642019590.04899.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 44.43 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.5 Å115.93 Å
Translation3.5 Å115.93 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: 3.5→115.93 Å / Occupancy max: 1 / Occupancy min: 1 / SU ML: 0.46 / σ(F): 0 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2849 2484 5.09 %Random
Rwork0.2058 ---
all0.2097 57024 --
obs0.2097 48767 85.52 %-
Solvent computationShrinkage radii: 0.27 Å / VDW probe radii: 0.6 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 53.456 Å2 / ksol: 0.349 e/Å3
Displacement parametersBiso max: 159.12 Å2 / Biso mean: 73.8183 Å2 / Biso min: 29.65 Å2
Baniso -1Baniso -2Baniso -3
1--3.6726 Å20 Å20 Å2
2--2.2938 Å2-0 Å2
3---1.3787 Å2
Refinement stepCycle: LAST / Resolution: 3.5→115.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms28357 0 448 8 28813
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00429594
X-RAY DIFFRACTIONf_angle_d0.88940482
X-RAY DIFFRACTIONf_chiral_restr0.0564540
X-RAY DIFFRACTIONf_plane_restr0.0055285
X-RAY DIFFRACTIONf_dihedral_angle_d14.09810316
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 18

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.5001-3.56740.4086950.27482020211567
3.5674-3.64020.29011170.26062049216669
3.6402-3.71940.31721160.24532133224972
3.7194-3.80590.31221220.22672154227673
3.8059-3.90110.36311330.24472258239177
3.9011-4.00650.31891280.23022311243978
4.0065-4.12440.31131300.20522429255981
4.1244-4.25760.27381370.18542493263084
4.2576-4.40970.23881530.17472598275188
4.4097-4.58630.30211370.18292693283090
4.5863-4.7950.23761400.17482708284890
4.795-5.04780.28481530.1862748290192
5.0478-5.3640.26441500.20292789293993
5.364-5.77820.32411690.21542818298794
5.7782-6.35950.33331430.22062940308397
6.3595-7.27950.25051430.19073011315499
7.2795-9.17040.23171550.167330763231100
9.1704-98.4590.2671630.22783055321895

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