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- PDB-2bpo: Crystal structure of the yeast CPR triple mutant: D74G, Y75F, K78A. -

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Basic information

Entry
Database: PDB / ID: 2bpo
TitleCrystal structure of the yeast CPR triple mutant: D74G, Y75F, K78A.
ComponentsNADPH-CYTOCHROM P450 REDUCTASE
KeywordsREDUCTASE / NADPH-CYTOCHROME P450 REDUCTASE / CPR / DIFLAVIN REDUCTASE / FAD / FMN-BINDING / 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 / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsYermalitskaya, L.V. / Kim, Y. / Waterman, M.R. / Podust, L.M.
CitationJournal: To be Published
Title: Crystal Structure of the Yeast Cpr Triple Mutant: D74G, Y75F, K78A.
Authors: Yermalitskaya, L.V. / Kim, Y. / Waterman, M.R. / Podust, L.M.
History
DepositionApr 21, 2005Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 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: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.4Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
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-CYTOCHROM P450 REDUCTASE
B: NADPH-CYTOCHROM P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,68111
Polymers151,4222
Non-polymers4,2599
Water3,333185
1
A: NADPH-CYTOCHROM P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,8886
Polymers75,7111
Non-polymers2,1775
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: NADPH-CYTOCHROM P450 REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,7925
Polymers75,7111
Non-polymers2,0814
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)78.357, 86.596, 259.717
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: (-0.16692, -0.98565, 0.02499), (0.98573, -0.16738, -0.01771), (0.02164, 0.02168, 0.99953)
Vector: 62.6435, 37.7158, 63.81243)

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Components

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Protein , 1 types, 2 molecules AB

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


Mass: 75710.906 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PET15B / 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, 194 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.: 2 / 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.: 185 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsRESIDUE NUMBERS REFER TO THE UNIPROT SEQUENCE, WITH THE PDB NUMBERING GIVEN IN PARENTHESES. ...RESIDUE NUMBERS REFER TO THE UNIPROT SEQUENCE, WITH THE PDB NUMBERING GIVEN IN PARENTHESES. ENGINEERED RESIDUE IN CHAIN A, GLY 73 (74) TO ASP ENGINEERED RESIDUE IN CHAIN A, TYR 74 (75) TO PHE ENGINEERED RESIDUE IN CHAIN A, LYS 77 (78) TO ALA ENGINEERED RESIDUE IN CHAIN B, GLY 73 (74) TO ASP ENGINEERED RESIDUE IN CHAIN B, TYR 74 (75) TO PHE ENGINEERED RESIDUE IN CHAIN B, LYS 77 (78) TO ALA
Sequence detailsRESIDUES NUMBERING IN THE ENTRY BEGINS FROM THE START METHIONINE 33 N-TERMINUS RESIDUES ARE ...RESIDUES NUMBERING IN THE ENTRY BEGINS FROM THE START METHIONINE 33 N-TERMINUS RESIDUES ARE TRUNCATED, 6XHIS RESIDUES AT THE N-TERMINUS ARE PRECEDED BY 4 AND FOLLOWED BY 15 RESIDUES FROM THE CLONING SITE. MUTATIONS D74G, Y75F, AND K78A ARE ENGINEERED. 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: 295 K / pH: 5
Details: 1.6 M AMMONIUM SULFATE 100 MM SODIUM CITRATE, PH 5.0 5 MM NICKEL CHLORIDE 1 MM FMN 1 MM FAD 1 MM NADP, T=22 C.

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1
DetectorType: MARRESEARCH / Detector: CCD / Date: Feb 17, 2005
Details: SAGITTAL FOCUSING CRYSTAL AND VERTICALLY FOCUSING MIRROR
RadiationMonochromator: SI (220) DOUBLE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. obs: 37243 / % possible obs: 92.6 % / Observed criterion σ(I): 0 / Redundancy: 5.4 % / Biso Wilson estimate: 136.3 Å2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 16.5
Reflection shellResolution: 2.9→3 Å / Redundancy: 4.8 % / Rmerge(I) obs: 0.36 / Mean I/σ(I) obs: 3.8 / % possible all: 78

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2BF4

2bf4


Resolution: 2.9→39.18 Å / Rfactor Rfree error: 0.005 / Data cutoff high absF: 149934.34 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: DISORDERED REGIONS WERE MODELED STEREOCHEMICALLY OMITTED RESIDUES LYS 564, GLY 565, GLY 566, ASN 567 ARE DUE TO LACK OF ELECTRON DENSITY
RfactorNum. reflection% reflectionSelection details
Rfree0.268 3504 10 %RANDOM
Rwork0.205 ---
obs0.205 35007 87.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 36.2759 Å2 / ksol: 0.345189 e/Å3
Displacement parametersBiso mean: 56.1 Å2
Baniso -1Baniso -2Baniso -3
1--2.02 Å20 Å20 Å2
2--25.63 Å20 Å2
3----23.61 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.48 Å0.36 Å
Luzzati d res low-5 Å
Luzzati sigma a0.6 Å0.55 Å
Refinement stepCycle: LAST / Resolution: 2.9→39.18 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10080 0 263 185 10528
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_deg1.3
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d23
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.9
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.241.5
X-RAY DIFFRACTIONc_mcangle_it2.142
X-RAY DIFFRACTIONc_scbond_it1.752
X-RAY DIFFRACTIONc_scangle_it2.812.5
LS refinement shellResolution: 2.9→3.08 Å / Rfactor Rfree error: 0.018 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.387 467 10.3 %
Rwork0.324 4079 -
obs--69.3 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER_REP.TOP
X-RAY DIFFRACTION3PROSTETIC_2.PARPROSTETIC_1.TOP
X-RAY DIFFRACTION4ION.PARAMION.TOP

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