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- PDB-8c5p: E. coli NfsB mutant N71S T41L with acetate -

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Basic information

Entry
Database: PDB / ID: 8c5p
TitleE. coli NfsB mutant N71S T41L with acetate
ComponentsOxygen-insensitive NAD(P)H nitroreductase
KeywordsOXIDOREDUCTASE / Oxygen insensitive NAD(P)H Nitroreductase / Double mutant
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
ACETATE ION / FLAVIN MONONUCLEOTIDE / Oxygen-insensitive NAD(P)H nitroreductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.69 Å
AuthorsDay, M.A. / White, S.A. / Hyde, E.I.
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
DepositionJan 10, 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

Downloads & links

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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)49,40114
Polymers47,8442
Non-polymers1,55612
Water9,404522
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10930 Å2
ΔGint-26 kcal/mol
Surface area16940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.223, 57.223, 262.944
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-974-

HOH

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Components

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Protein , 1 types, 2 molecules AB

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


Mass: 23922.209 Da / Num. of mol.: 2 / Mutation: T41L N71S
Source method: isolated from a genetically manipulated source
Details: Double 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 system / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P38489, Oxidoreductases, 6,7-dihydropteridine reductase

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Non-polymers , 5 types, 534 molecules

#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-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#5: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: C2H3O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 522 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.25 Å3/Da / Density % sol: 45.32 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 10-18% PEG 4000, 50-200 mM sodium acetate buffer pH 4.6, 15% ethylene glycol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 0.9334 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Jul 17, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9334 Å / Relative weight: 1
ReflectionResolution: 1.69→47.92 Å / Num. obs: 49521 / % possible obs: 99 % / Redundancy: 13 % / Rsym value: 0.05 / Net I/σ(I): 36.9
Reflection shellResolution: 1.69→1.8 Å / Redundancy: 3.54 % / Num. unique obs: 3178 / Rsym value: 0.348

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Processing

Software
NameVersionClassification
REFMAC5refinement
iMOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.69→43.16 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.951 / SU B: 1.889 / SU ML: 0.061 / Cross valid method: THROUGHOUT / ESU R: 0.096 / ESU R Free: 0.096 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.18793 2499 5 %RANDOM
Rwork0.15 ---
obs0.15189 47036 98.99 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 16.507 Å2
Baniso -1Baniso -2Baniso -3
1--0.54 Å2-0 Å20 Å2
2---0.54 Å20 Å2
3---1.07 Å2
Refinement stepCycle: 1 / Resolution: 1.69→43.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3352 0 102 527 3981
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0123676
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163493
X-RAY DIFFRACTIONr_angle_refined_deg1.8181.6444986
X-RAY DIFFRACTIONr_angle_other_deg0.6211.5838079
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8185462
X-RAY DIFFRACTIONr_dihedral_angle_2_deg6.004514
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.56610629
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.1020.2550
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.024268
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02784
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_it2.1611.5681810
X-RAY DIFFRACTIONr_mcbond_other2.1391.5651806
X-RAY DIFFRACTIONr_mcangle_it2.8052.8122282
X-RAY DIFFRACTIONr_mcangle_other2.8052.8122283
X-RAY DIFFRACTIONr_scbond_it3.4391.9051866
X-RAY DIFFRACTIONr_scbond_other3.4391.9061867
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.0753.3022705
X-RAY DIFFRACTIONr_long_range_B_refined6.43418.534572
X-RAY DIFFRACTIONr_long_range_B_other6.2117.274401
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.694→1.738 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.242 154 -
Rwork0.219 3011 -
obs--87.46 %

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