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- PDB-8ccv: E. coli NfsB mutant T41LN71S with nicotinate -

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Basic information

Entry
Database: PDB / ID: 8ccv
TitleE. coli NfsB mutant T41LN71S with nicotinate
ComponentsOxygen-insensitive NAD(P)H nitroreductase
KeywordsOXIDOREDUCTASE / Oxygen insensitve NAD(P)H nitroreductase / double mutant
Function / homology
Function and homology information


oxidoreductase activity, acting on other nitrogenous compounds as donors, with NAD or NADP as acceptor / 6,7-dihydropteridine reductase / 2,4,6-trinitrotoluene catabolic process / 6,7-dihydropteridine reductase activity / NAD(P)H dehydrogenase (quinone) activity / Oxidoreductases / FMN binding / 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: 2.2 Å
AuthorsDay, M.A. / White, S.A. / Hyde, E.I. / Searle, P.F.
Funding support1items
OrganizationGrant numberCountry
Not funded
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 27, 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)49,0036
Polymers47,8442
Non-polymers1,1594
Water3,009167
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9390 Å2
ΔGint-37 kcal/mol
Surface area17390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.862, 56.862, 263.866
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: 23922.209 Da / Num. of mol.: 2 / Mutation: T41L, N71S
Source method: isolated from a genetically manipulated source
Details: Double mutant of E. coli NfsB bound to nicotinate / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: nfsB, dprA, nfnB, nfsI, ntr, b0578, JW0567 / Variant: DH5a / Plasmid: pET11c / Details (production host): T7 expression system / 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-NIO / NICOTINIC ACID


Mass: 123.109 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H5NO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 167 / 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: 2.23 Å3/Da / Density % sol: 44.82 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 18% PEG 4,000; 200 mM Na Acetate, 15 mM 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: 0.934 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jul 28, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.934 Å / Relative weight: 1
ReflectionResolution: 2.2→47.8 Å / Num. obs: 22338 / % possible obs: 96.52 % / Redundancy: 7.7 % / Rsym value: 0.07 / Net I/σ(I): 24.9
Reflection shellResolution: 2.2→2.257 Å / Num. unique obs: 1206 / Rsym value: 0.53 / % possible all: 77.11

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Processing

Software
NameVersionClassification
REFMAC5.8.0403refinement
iMOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.2→47.8 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.94 / SU B: 15.12 / SU ML: 0.191 / Cross valid method: THROUGHOUT / ESU R: 0.293 / ESU R Free: 0.221 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23657 1138 5.1 %RANDOM
Rwork0.17784 ---
obs0.18077 21200 96.52 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 52.256 Å2
Baniso -1Baniso -2Baniso -3
1-0.96 Å2-0 Å2-0 Å2
2--0.96 Å2-0 Å2
3----1.93 Å2
Refinement stepCycle: 1 / Resolution: 2.2→47.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3352 0 80 167 3599
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0123506
X-RAY DIFFRACTIONr_bond_other_d0.0030.0163302
X-RAY DIFFRACTIONr_angle_refined_deg1.6961.6394758
X-RAY DIFFRACTIONr_angle_other_deg0.6381.5817616
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7095430
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.821516
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.31810586
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.0870.2532
X-RAY DIFFRACTIONr_gen_planes_refined0.010.024030
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02760
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_it2.9142.81726
X-RAY DIFFRACTIONr_mcbond_other2.912.81726
X-RAY DIFFRACTIONr_mcangle_it4.2785.0392154
X-RAY DIFFRACTIONr_mcangle_other4.2775.0382155
X-RAY DIFFRACTIONr_scbond_it3.5933.2611780
X-RAY DIFFRACTIONr_scbond_other3.5813.261779
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.3715.8182604
X-RAY DIFFRACTIONr_long_range_B_refined8.33931.634077
X-RAY DIFFRACTIONr_long_range_B_other8.3431.34054
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.367 74 -
Rwork0.339 1206 -
obs--77.11 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.9434-0.0039-0.20693.26160.42161.7946-0.09260.42860.2932-0.18430.1818-0.0686-0.1091-0.0126-0.08920.1081-0.0145-0.04930.17730.04110.057815.66017.426117.9637
22.27750.3148-0.1973.29310.07251.7295-0.04860.287-0.204-0.18150.1571-0.52830.19740.2287-0.10850.10510.0117-0.02110.2009-0.02580.132227.9551-2.591217.7408
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 217
2X-RAY DIFFRACTION2B2 - 217

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