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- PDB-2bf4: A second FMN-binding site in yeast NADPH-cytochrome P450 reductas... -

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Basic information

Entry
Database: PDB / ID: 2bf4
TitleA second FMN-binding site in yeast NADPH-cytochrome P450 reductase suggests a novel mechanism of electron transfer by diflavin reductases.
ComponentsNADPH-CYTOCHROME P450 REDUCTASE
KeywordsREDUCTASE / NADPH-CYTOCHROME P450 REDUCTASE / CPR / DIFLAVIN REDUCTASE / FAD / FMN / NADP / ELECTRON TRANSFER
Function / homology
Function and homology information


Nitric oxide stimulates guanylate cyclase / Ion homeostasis / Peroxisomal protein import / ergosterol biosynthetic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / eNOS activation / Extra-nuclear estrogen signaling / VEGFR2 mediated vascular permeability / ROS and RNS production in phagocytes / NADPH dehydrogenase activity ...Nitric oxide stimulates guanylate cyclase / Ion homeostasis / Peroxisomal protein import / ergosterol biosynthetic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / eNOS activation / Extra-nuclear estrogen signaling / VEGFR2 mediated vascular permeability / ROS and RNS production in phagocytes / NADPH dehydrogenase activity / NADPH-hemoprotein reductase / NADPH-hemoprotein reductase activity / FMN binding / flavin adenine dinucleotide binding / NADP binding / mitochondrial outer membrane / electron transfer activity / endoplasmic reticulum membrane / mitochondrion / plasma membrane / cytosol
Similarity search - Function
NADPH-cytochrome p450 Reductase; Chain A, domain 3 / NADPH-cytochrome p450 Reductase; Chain A, domain 3 / NADPH-cytochrome P450 reductase / Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module / Flavodoxin domain / Translation factors / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like ...NADPH-cytochrome p450 Reductase; Chain A, domain 3 / NADPH-cytochrome p450 Reductase; Chain A, domain 3 / NADPH-cytochrome P450 reductase / Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module / Flavodoxin domain / Translation factors / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Elongation Factor Tu (Ef-tu); domain 3 / 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 / Flavoprotein-like superfamily / Up-down Bundle / Beta Barrel / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / FLAVIN MONONUCLEOTIDE / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / NADPH--cytochrome P450 reductase
Similarity search - Component
Biological speciesSACCHAROMYCES CEREVISIAE (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3 Å
AuthorsPodust, L.M. / Lepesheva, G.I. / Kim, Y. / Yermalitskaya, L.V. / Yermalitsky, V.N. / Lamb, D.C. / Kelly, S.L. / Waterman, M.R.
CitationJournal: Structure / Year: 2006
Title: A Second Fmn-Binding Site in Yeast Nadph-Cytochrome P450 Reductase Suggests a Mechanism of Electron Transfer by Diflavin Reductases.
Authors: Lamb, D.C. / Kim, Y. / Yermalitskaya, L.V. / Yermalitsky, V.N. / Lepesheva, G.I. / Kelly, S.L. / Waterman, M.R. / Podust, L.M.
History
DepositionDec 3, 2004Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2006Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3May 8, 2019Group: Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Remark 700 SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN ... SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NADPH-CYTOCHROME P450 REDUCTASE
B: NADPH-CYTOCHROME P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,85813
Polymers151,6862
Non-polymers5,17111
Water2,720151
1
A: NADPH-CYTOCHROME P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,5738
Polymers75,8431
Non-polymers2,7307
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: NADPH-CYTOCHROME P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,2855
Polymers75,8431
Non-polymers2,4424
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)77.751, 87.094, 259.590
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (-0.17445, -0.98425, 0.02856), (0.9844, -0.175, -0.01823), (0.02294, 0.02494, 0.99943)
Vector: 62.15195, 38.60347, 63.59497)

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein NADPH-CYTOCHROME P450 REDUCTASE / CPR / P450R


Mass: 75843.047 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PET-15B / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): HMS174(DE3) / References: UniProt: P16603, NADPH-hemoprotein reductase

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Non-polymers , 5 types, 162 molecules

#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H21N4O9P
#4: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#5: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 151 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsFUNCTION: TRANSFER OF TWO REDUCING EQUIVALENTS IN A FORM OF THE HYDRID ION FROM NADPH VIA FAD AND ...FUNCTION: TRANSFER OF TWO REDUCING EQUIVALENTS IN A FORM OF THE HYDRID ION FROM NADPH VIA FAD AND FMN TO CYTOCHROME P450 IN ONE-ELECTRON TRANSFER STEPS
Sequence details33 N-TERMINAL RESIDUES ARE TRUNCATED. 6XHIS RESIDUES AT THE N-TERMINUS ARE PRECEDED BY 4 AND ...33 N-TERMINAL RESIDUES ARE TRUNCATED. 6XHIS RESIDUES AT THE N-TERMINUS ARE PRECEDED BY 4 AND FOLLOWED BY 15 RESIDUES FROM THE CLONING SITE. RESIDUES NUMBERING IN THE STRUCTURE BEGINS FROM THE START METHIONINE

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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: 2.85 Å3/Da / Density % sol: 56 %
Crystal growTemperature: 293 K / pH: 5
Details: 1.6 M AMMONIUM SULFATE,100 MM SODIUM CITRATE (PH 5.0),T=20 C

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 0.9794
DetectorType: MARRESEARCH / Detector: CCD / Date: Aug 13, 2003
Details: SAGITTAL FOCUSING CRYSTAL AND VERTICALLY FOCUSING MIRROR
RadiationMonochromator: SI (220) DOUBLE CRYSTAL / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 3→50 Å / Num. obs: 67872 / % possible obs: 99.9 % / Observed criterion σ(I): 0 / Redundancy: 7.6 % / Biso Wilson estimate: 74.5 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 28
Reflection shellResolution: 3→3.11 Å / Redundancy: 4.7 % / Rmerge(I) obs: 0.3 / Mean I/σ(I) obs: 4.3 / % possible all: 99.8

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: SAD / Resolution: 3→43.55 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 190408.62 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
Stereochemistry target values: MAXIMUM LIKELIHOOD USING AMPLITUDES AND PHASE PROBABILITY DISTRIBUTION
RfactorNum. reflection% reflectionSelection details
Rfree0.261 6560 10 %RANDOM
Rwork0.197 ---
obs0.197 65903 96.8 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 42.4716 Å2 / ksol: 0.365566 e/Å3
Displacement parametersBiso mean: 53.1 Å2
Baniso -1Baniso -2Baniso -3
1-6.05 Å20 Å20 Å2
2--14.42 Å20 Å2
3----20.47 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.45 Å0.32 Å
Luzzati d res low-5 Å
Luzzati sigma a0.65 Å0.48 Å
Refinement stepCycle: LAST / Resolution: 3→43.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10148 0 325 151 10624
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.008
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_deg1.4
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.8
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.88
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.221.5
X-RAY DIFFRACTIONc_mcangle_it2.112
X-RAY DIFFRACTIONc_scbond_it1.782
X-RAY DIFFRACTIONc_scangle_it2.862.5
LS refinement shellResolution: 3→3.19 Å / Rfactor Rfree error: 0.012 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.371 975 9.3 %
Rwork0.292 9525 -
obs--92.8 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER_REP.TOP
X-RAY DIFFRACTION3ION.PARAMION.TOP
X-RAY DIFFRACTION4PROSTETIC_2.PARPROSTETIC_1.TOP

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