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- PDB-2oz0: Mechanistic and Structural Studies of H373Q Flavocytochrome b2: E... -

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Basic information

Entry
Database: PDB / ID: 2oz0
TitleMechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base
ComponentsCytochrome b2
KeywordsOXIDOREDUCTASE / Flavocytochrome b2 / FMN / Pyruvate
Function / homology
Function and homology information


L-lactate dehydrogenase (cytochrome) / L-lactate dehydrogenase (cytochrome) activity / lactate metabolic process / : / mitochondrial intermembrane space / mitochondrial inner membrane / heme binding / mitochondrion / nucleus / metal ion binding / cytosol
Similarity search - Function
L-mandelate/L-lactate dehydrogenase, FMN-binding domain / Flavocytochrome B2; Chain A, domain 1 / Cytochrome b5-like heme/steroid binding domain / Cytochrome b5, heme-binding site / Cytochrome b5 family, heme-binding domain signature. / FMN-dependent alpha-hydroxy acid dehydrogenase, active site / FMN hydroxy acid dehydrogenase domain / FMN-dependent alpha-hydroxy acid dehydrogenases active site. / FMN-dependent alpha-hydroxy acid dehydrogenase domain profile. / Cytochrome b5 family, heme-binding domain profile. ...L-mandelate/L-lactate dehydrogenase, FMN-binding domain / Flavocytochrome B2; Chain A, domain 1 / Cytochrome b5-like heme/steroid binding domain / Cytochrome b5, heme-binding site / Cytochrome b5 family, heme-binding domain signature. / FMN-dependent alpha-hydroxy acid dehydrogenase, active site / FMN hydroxy acid dehydrogenase domain / FMN-dependent alpha-hydroxy acid dehydrogenases active site. / FMN-dependent alpha-hydroxy acid dehydrogenase domain profile. / Cytochrome b5 family, heme-binding domain profile. / Cytochrome b5-like heme/steroid binding domain / Cytochrome b5-like heme/steroid binding domain superfamily / Cytochrome b5-like Heme/Steroid binding domain / Cytochrome b5-like Heme/Steroid binding domain / FMN-dependent dehydrogenase / FMN-dependent dehydrogenase / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Roll / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
FLAVIN MONONUCLEOTIDE / PROTOPORPHYRIN IX CONTAINING FE / PYRUVIC ACID / L-lactate dehydrogenase (cytochrome)
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsTsai, C.-L. / Gokulan, K. / Sobrado, P. / Sacchettini, J.C. / Fitzpatrick, P.F.
CitationJournal: Biochemistry / Year: 2007
Title: Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.
Authors: Tsai, C.L. / Gokulan, K. / Sobrado, P. / Sacchettini, J.C. / Fitzpatrick, P.F.
History
DepositionFeb 23, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2007Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / 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
Revision 1.3Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Category: atom_site / chem_comp_atom / chem_comp_bond
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cytochrome b2
B: Cytochrome b2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,9256
Polymers113,3082
Non-polymers1,6174
Water30617
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Cytochrome b2
B: Cytochrome b2
hetero molecules

A: Cytochrome b2
B: Cytochrome b2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,85012
Polymers226,6164
Non-polymers3,2348
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation6_555-x,-x+y,-z+2/31
Buried area33040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)163.689, 163.689, 112.021
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein Cytochrome b2 / L-lactate dehydrogenase [Cytochrome] / L-lactate ferricytochrome C oxidoreductase / L-LCR


Mass: 56653.883 Da / Num. of mol.: 2 / Fragment: Flavocytochrome b2 / Mutation: H373Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CYB2 / Plasmid: pDSb2 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21DE3
References: UniProt: P00175, L-lactate dehydrogenase (cytochrome)
#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-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P
#4: Chemical ChemComp-PYR / PYRUVIC ACID


Mass: 88.062 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H4O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 17 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 3.82 Å3/Da / Density % sol: 67.81 %
Crystal growTemperature: 291 K / Method: evaporation / pH: 4.6
Details: 100 mM Sodium citrate, 17% PEG 3K, 0.2mM Sucrose monolaurate, pH 4.6, EVAPORATION, temperature 291K

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Data collection

DiffractionMean temperature: 120 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Jul 27, 2006
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.8→50 Å / Num. all: 69254 / Num. obs: 36191 / % possible obs: 88 % / Rmerge(I) obs: 0.26 / Net I/σ(I): 14.5

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
ADSCQuantumdata collection
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 1FCB

1fcb


Resolution: 2.8→50 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.892 / SU B: 15.461 / SU ML: 0.291 / Cross valid method: THROUGHOUT / ESU R: 0.604 / ESU R Free: 0.369 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.278 1941 5.2 %RANDOM
Rwork0.206 ---
obs0.21 35636 87.6 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 69.71 Å2
Baniso -1Baniso -2Baniso -3
1-1.08 Å20.54 Å20 Å2
2--1.08 Å20 Å2
3----1.61 Å2
Refinement stepCycle: LAST / Resolution: 2.8→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6914 0 111 17 7042
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.050.0227213
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg4.2842.029786
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg13.795891
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.97725300
X-RAY DIFFRACTIONr_dihedral_angle_3_deg27.967151294
X-RAY DIFFRACTIONr_dihedral_angle_4_deg26.6221539
X-RAY DIFFRACTIONr_chiral_restr0.3020.21102
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.025340
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.3720.24531
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.40.24995
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.2860.2423
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined0.1250.21
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.3170.2136
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2820.26
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.8941.54639
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it3.09727213
X-RAY DIFFRACTIONr_scbond_it4.58633065
X-RAY DIFFRACTIONr_scangle_it7.2924.52571
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.8→2.87 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.484 46 -
Rwork0.474 1116 -
obs--36.91 %

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