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- PDB-8c5e: E. coli NfsB-T41Q/N71S/F124T mutant bound to nicotinic acid -

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Basic information

Entry
Database: PDB / ID: 8c5e
TitleE. coli NfsB-T41Q/N71S/F124T mutant bound to nicotinic acid
ComponentsOxygen-insensitive NAD(P)H nitroreductase
KeywordsOXIDOREDUCTASE / Nitroreductase mutant / 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
FLAVIN MONONUCLEOTIDE / NICOTINIC ACID / Oxygen-insensitive NAD(P)H nitroreductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.65 Å
AuthorsWhite, S.A. / Hyde, E.I. / Day, M.A.
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 6, 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

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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)48,7417
Polymers47,5202
Non-polymers1,2215
Water9,206511
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9570 Å2
ΔGint-23 kcal/mol
Surface area17120 Å2
Unit cell
Length a, b, c (Å)57.610, 57.610, 264.815
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-965-

HOH

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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: T41Q, N71S, F124T
Source method: isolated from a genetically manipulated source
Details: Triple mutant of NfsB, bound to nicotinate / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: DH5a / Gene: nfsB, dprA, nfnB, nfsI, ntr, b0578, JW0567 / Plasmid: pET11c / Production host: Escherichia coli BL21 (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-NIO / NICOTINIC ACID


Mass: 123.109 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H5NO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 511 / 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: 2.31 Å3/Da / Density % sol: 46.51 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: PEG 4000, 50-200 mM sodium acetate buffer, 15 mM nicotinic acid, 15% ethylene glycol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 1 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Nov 21, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.65→48.24 Å / Num. obs: 53617 / % possible obs: 97.7 % / Redundancy: 5.42 % / Rsym value: 0.078 / Net I/σ(I): 13.2
Reflection shellResolution: 1.65→1.74 Å / Redundancy: 2.79 % / Mean I/σ(I) obs: 1.9 / Num. unique obs: 6747 / Rsym value: 0.557 / % possible all: 86.8

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Processing

Software
NameVersionClassification
REFMAC5refinement
iMOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.65→43.5 Å / SU B: 4.056 / SU ML: 0.072 / Cross valid method: THROUGHOUT / ESU R: 0.102 / ESU R Free: 0.103 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.21666 2751 5.2 %RANDOM
Rwork0.17593 ---
obs0.17798 50633 97.32 %-
Displacement parametersBiso mean: 13.041 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å2-0 Å2-0 Å2
2--0.09 Å2-0 Å2
3----0.18 Å2
Refinement stepCycle: LAST / Resolution: 1.65→43.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3346 0 84 514 3944
LS refinement shellResolution: 1.653→1.696 Å
RfactorNum. reflection% reflection
Rfree0.393 193 -
Rwork0.346 3051 -
obs--81.38 %

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