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- PDB-8cj0: E. coli NfsB-T41Q/N71S/F124T/M127V mutant bound to nicotinate -

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Basic information

Entry
Database: PDB / ID: 8cj0
TitleE. coli NfsB-T41Q/N71S/F124T/M127V mutant bound to nicotinate
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: 1.99 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
DepositionFeb 11, 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)48,9397
Polymers47,7182
Non-polymers1,2215
Water2,774154
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9400 Å2
ΔGint-26 kcal/mol
Surface area17490 Å2
Unit cell
Length a, b, c (Å)57.310, 57.310, 265.670
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: 23859.045 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: 4 mutations T41Q, N71S, F124T, M127V. / 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 (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.: 154 / 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: 2.29 Å3/Da / Density % sol: 46.19 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 12% PEG 4000, 100 mM Na Acetate, 20 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: ID14-1 / Wavelength: 0.934 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Jul 28, 2009
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.934 Å / Relative weight: 1
ReflectionResolution: 1.99→35.06 Å / Num. obs: 31129 / % possible obs: 98.9 % / Redundancy: 9 % / Rsym value: 0.075 / Net I/σ(I): 25.3
Reflection shellResolution: 1.994→2.046 Å / Redundancy: 8.93 % / Num. unique obs: 2176 / Rsym value: 0.55 / % possible all: 95.4

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Processing

Software
NameClassification
PDB-REDOrefinement
iMOSFLMdata reduction
SCALAdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.99→35.06 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.945 / SU B: 6.685 / SU ML: 0.092 / Cross valid method: THROUGHOUT / ESU R: 0.16 / ESU R Free: 0.142 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.19869 1575 5.1 %RANDOM
Rwork0.16095 ---
obs0.16283 29553 98.88 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 38.944 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å20 Å20 Å2
2---0.04 Å20 Å2
3---0.07 Å2
Refinement stepCycle: LAST / Resolution: 1.99→35.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3344 0 84 154 3582
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0163533
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163247
X-RAY DIFFRACTIONr_angle_refined_deg1.2751.8144802
X-RAY DIFFRACTIONr_angle_other_deg0.491.5757566
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1235.166453
X-RAY DIFFRACTIONr_dihedral_angle_2_deg
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.67510591
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.0690.2539
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023992
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02674
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_it4.0571.4611740
X-RAY DIFFRACTIONr_mcbond_other4.0561.461740
X-RAY DIFFRACTIONr_mcangle_it5.4252.1722176
X-RAY DIFFRACTIONr_mcangle_other5.4422.1742177
X-RAY DIFFRACTIONr_scbond_it7.4382.2471793
X-RAY DIFFRACTIONr_scbond_other7.442.2491794
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other10.1763.0142625
X-RAY DIFFRACTIONr_long_range_B_refined11.79221.6694045
X-RAY DIFFRACTIONr_long_range_B_other11.80821.4964025
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.994→2.046 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.228 112 -
Rwork0.202 2056 -
obs--95.3 %
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)
12.46860.4311-0.35822.54390.06491.916-0.06690.27410.2749-0.09920.17720.0324-0.0892-0.0822-0.11020.0254-0.0192-0.03040.07430.03370.087315.8117.6918.128
22.13610.554-0.08642.93790.05561.9944-0.11560.198-0.0969-0.10320.1943-0.49430.19190.2764-0.07870.03140.0027-0.00690.1067-0.01950.150128.465-2.03418.256
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A-10 - 9999
2X-RAY DIFFRACTION2B-10 - 9999

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