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- PDB-8og3: E. coli NfsB triple mutant T41L/N71S/F124T bound to citrate -

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Basic information

Entry
Database: PDB / ID: 8og3
TitleE. coli NfsB triple mutant T41L/N71S/F124T bound to citrate
ComponentsOxygen-insensitive NAD(P)H nitroreductase
KeywordsOXIDOREDUCTASE / Nitroreductase / citrate complex
Function / homology
Function and homology information


6,7-dihydropteridine reductase / 2,4,6-trinitrotoluene catabolic process / 6,7-dihydropteridine reductase activity / NAD(P)H dehydrogenase (quinone) activity / Oxidoreductases / FMN binding / oxidoreductase activity / protein homodimerization activity / identical protein binding / membrane / cytosol
Similarity search - Function
Oxygen-insensitive NAD(P)H nitroreductase NfsB-like / : / Nitroreductase / Nitroreductase family / Nitroreductase-like
Similarity search - Domain/homology
CITRIC ACID / FLAVIN MONONUCLEOTIDE / Oxygen-insensitive NAD(P)H nitroreductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.09 Å
AuthorsDay, M.A. / White, S.A. / Hyde, E.I. / Searle, P.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: Int J Mol Sci / Year: 2023
Title: Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.
Authors: Day, M.A. / Christofferson, A.J. / Anderson, J.L.R. / Vass, S.O. / Evans, A. / Searle, P.F. / White, S.A. / Hyde, E.I.
History
DepositionMar 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Oxygen-insensitive NAD(P)H nitroreductase
B: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,0039
Polymers47,5202
Non-polymers1,4837
Water5,981332
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9840 Å2
ΔGint-26 kcal/mol
Surface area17120 Å2
Unit cell
Length a, b, c (Å)57.263, 57.263, 263.827
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein Oxygen-insensitive NAD(P)H nitroreductase / Dihydropteridine reductase / FMN-dependent nitroreductase


Mass: 23759.916 Da / Num. of mol.: 2 / Mutation: T41L, N71S. F124T
Source method: isolated from a genetically manipulated source
Details: Triple mutant of NfsB / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: DH5a / Gene: nfsB, dprA, nfnB, nfsI, ntr, b0578, JW0567 / Plasmid: pET11c / Details (production host): T7 expression vector / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P38489, Oxidoreductases, 6,7-dihydropteridine reductase
#2: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P
#3: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 332 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.28 Å3/Da / Density % sol: 45.95 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 13% PEG 4,000; 100 mM Na Acetate, 15% Ethylene glycol, 100 mM Citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 0.933 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Jan 29, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.933 Å / Relative weight: 1
ReflectionResolution: 2.09→47.99 Å / Num. obs: 27043 / % possible obs: 99.8 % / Redundancy: 7.16 % / Rsym value: 0.083 / Net I/σ(I): 21.17
Reflection shellResolution: 2.094→2.148 Å / Mean I/σ(I) obs: 5.54 / Num. unique obs: 1910 / Rsym value: 0.369 / % possible all: 98.45

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Processing

Software
NameVersionClassification
REFMAC5.8.0405refinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.09→47.99 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.93 / SU B: 4.615 / SU ML: 0.121 / Cross valid method: THROUGHOUT / ESU R: 0.211 / ESU R Free: 0.179 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.21796 1347 5 %RANDOM
Rwork0.16451 ---
obs0.16717 25696 99.88 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 21.551 Å2
Baniso -1Baniso -2Baniso -3
1--0.89 Å20 Å20 Å2
2---0.89 Å20 Å2
3---1.79 Å2
Refinement stepCycle: LAST / Resolution: 2.09→47.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3346 0 100 332 3778
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0123530
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163323
X-RAY DIFFRACTIONr_angle_refined_deg1.5891.6444791
X-RAY DIFFRACTIONr_angle_other_deg0.5411.5827673
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1635434
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.449514
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.15510592
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.0750.2536
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.024055
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02749
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.4312.111733
X-RAY DIFFRACTIONr_mcbond_other2.4312.111733
X-RAY DIFFRACTIONr_mcangle_it3.4463.7842165
X-RAY DIFFRACTIONr_mcangle_other3.4453.7842166
X-RAY DIFFRACTIONr_scbond_it3.622.4871797
X-RAY DIFFRACTIONr_scbond_other3.5782.4821794
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.4524.3812626
X-RAY DIFFRACTIONr_long_range_B_refined7.00522.414227
X-RAY DIFFRACTIONr_long_range_B_other7.00422.424228
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.094→2.148 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.278 86 -
Rwork0.211 1824 -
obs--98.45 %

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