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- PDB-2q9g: Crystal structure of human cytochrome P450 46A1 -

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Basic information

Entry
Database: PDB / ID: 2q9g
TitleCrystal structure of human cytochrome P450 46A1
ComponentsCytochrome P450 46A1
KeywordsOXIDOREDUCTASE / CYP46A1 / P450 46A1 / P450 / MONOOXYGENASE / CHOLESTEROL METABOLIC ENZYME / HEME
Function / homology
Function and homology information


cholesterol 24-hydroxylase / cholesterol 24-hydroxylase activity / protein localization to membrane raft / testosterone 16-beta-hydroxylase activity / Synthesis of bile acids and bile salts via 24-hydroxycholesterol / progesterone metabolic process / sterol metabolic process / bile acid biosynthetic process / regulation of long-term synaptic potentiation / steroid hydroxylase activity ...cholesterol 24-hydroxylase / cholesterol 24-hydroxylase activity / protein localization to membrane raft / testosterone 16-beta-hydroxylase activity / Synthesis of bile acids and bile salts via 24-hydroxycholesterol / progesterone metabolic process / sterol metabolic process / bile acid biosynthetic process / regulation of long-term synaptic potentiation / steroid hydroxylase activity / Endogenous sterols / cholesterol catabolic process / xenobiotic metabolic process / presynapse / nervous system development / postsynapse / iron ion binding / dendrite / heme binding / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Cholesterol 24-hydroxylase / 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 / Cholesterol 24-hydroxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsWhite, M.A. / Mast, N.V. / Johnson, E.F. / Stout, C.D. / Pikuleva, I.A.
Citation
Journal: Proc.Natl.Acad.Sci.Usa / Year: 2008
Title: Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain.
Authors: Mast, N. / White, M.A. / Bjorkhem, I. / Johnson, E.F. / Stout, C.D. / Pikuleva, I.A.
#1: Journal: Acta Crystallogr.,Sect.D / Year: 2008
Title: Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1.
Authors: White, M.A. / Mast, N. / Bjorkhem, I. / Johnson, E.F. / Stout, C.D. / Pikuleva, I.A.
History
DepositionJun 12, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.2Aug 30, 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 / 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 / _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

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Assembly

Deposited unit
A: Cytochrome P450 46A1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,9264
Polymers52,1251
Non-polymers8013
Water45025
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)121.350, 121.350, 142.570
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number98
Space group name H-MI4122

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Components

#1: Protein Cytochrome P450 46A1 / Cholesterol 24-hydroxylase / CH24H


Mass: 52125.086 Da / Num. of mol.: 1 / Fragment: RESIDUES 51-500
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYP46A1, CYP46 / Plasmid: PUC18 / Production host: Escherichia coli (E. coli) / Strain (production host): DH5ALPHA / References: UniProt: Q9Y6A2, EC: 1.14.13.98
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 25 / 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: 51.65 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: PEG8000, Potassium Phosphate, Glycerol, NaCl, PH 4.6, VAPOR DIFFUSION, HANGING DROP, TEMPERATURE 291K, VAPOR DIFFUSION, SITTING DROP

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL11-1 / Wavelength: 0.97945 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jan 30, 2007
Details: FLAT MIRROR (VERTICAL FOCUSING), SINGLE CRYSTAL SI(111) BENT MONOCHROMATOR (HO RIZONTAL FOCUSING)
RadiationMonochromator: SIDE SCATTERING BENT CUBE- ROOT I-BEAM SINGLE CRYSTAL, ASYMMETRIC CUT 4.965 DEGS
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97945 Å / Relative weight: 1
ReflectionResolution: 2.4→92.45 Å / Num. all: 21106 / Num. obs: 21106 / % possible obs: 99.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7 % / Biso Wilson estimate: 48 Å2 / Rmerge(I) obs: 0.098 / Rsym value: 0.098 / Net I/σ(I): 34.6
Reflection shellResolution: 2.4→2.46 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.9 / Mean I/σ(I) obs: 1.8 / Rsym value: 0.9 / % possible all: 100

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Processing

Software
NameVersionClassification
CNS1.1refinement
Blu-Icedata collection
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: CYP46A1-C3S, PDB ENTRY 2q9f

2q9f


Resolution: 2.4→46.21 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 4160886 / Data cutoff low absF: 0 / Isotropic thermal model: variable / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
Details: Refmac5 and TLS used in intermediate refinement steps. TLS not used in CNS refinement. TLS groups determined using the TLSMD server. TLSMD REF: J Painter & E A Merritt (2006) J. Appl. Cryst. ...Details: Refmac5 and TLS used in intermediate refinement steps. TLS not used in CNS refinement. TLS groups determined using the TLSMD server. TLSMD REF: J Painter & E A Merritt (2006) J. Appl. Cryst. 39, 109-111. PMB used in all CNS refinements. PMB BOND target set to 0.011. PMB Variable Sigma-B used default parameters. PMB Hydrogen bond NOE restraints used: D=2.90, 0.4, 0.7, K=75, PMB Hydrogen bond NOE restraints used: Number of NOEs=272. PMB Local scale reject set to 35 sigma, PMB Local Scale rejected 103 reflections of 21096. PMB Local Scale (h,k,l) box sizes = +/- 4 3 5, PMB Local Scale maximum scale used (localscale method): 32.710, PMB Local Scale minimum scale used (localscale method): 1.053. Note: WEAK DENSITY OVER HEME WAS NOT MODELLED. IT COULD BE A HISTIDINE FROM the BUFFER OR IT COULD BE DUE TO ANOTHER UNKNOWN MOLECULE FROM EXPRESSION. Note: The F'-G' LOOP, residues 229-239, was not built due to weak and ambiguous electron density
RfactorNum. reflection% reflectionSelection details
Rfree0.242 1018 5 %THICK SHELLS
Rwork0.189 ---
all0.21799 20181 --
obs0.189 20181 95.3 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 59.9685 Å2 / ksol: 0.356954 e/Å3
Displacement parametersBiso mean: 73.6 Å2
Baniso -1Baniso -2Baniso -3
1--0.56 Å20 Å20 Å2
2---0.56 Å20 Å2
3---1.12 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.38 Å0.29 Å
Luzzati d res low-5 Å
Luzzati sigma a0.48 Å0.38 Å
Refinement stepCycle: LAST / Resolution: 2.4→46.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3392 0 55 25 3472
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.6
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d16.8
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d2.2
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
LS refinement shellResolution: 2.4→2.44 Å / Rfactor Rfree error: 0.048 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.367 58 6.6 %
Rwork0.326 822 -
obs-877 84.7 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep_pmb.paramprotein_pmb.top
X-RAY DIFFRACTION2water_rep.paramwater.top
X-RAY DIFFRACTION3ion.paramion.top
X-RAY DIFFRACTION4ligand.paramligand.top
X-RAY DIFFRACTION5carbohydrate.param

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