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- PDB-8c5f: E. coli NfsB-T41Q/N71S/F124T mutant bound to acetate -

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Basic information

Entry
Database: PDB / ID: 8c5f
TitleE. coli NfsB-T41Q/N71S/F124T mutant bound to acetate
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
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: 1.6 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 7, 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)49,16516
Polymers47,5202
Non-polymers1,64514
Water8,629479
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10970 Å2
ΔGint-13 kcal/mol
Surface area17220 Å2
Unit cell
Length a, b, c (Å)57.193, 57.193, 262.010
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-647-

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 E. coli 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
#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.: 8 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H3O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 479 / Source method: isolated from a natural source / Formula: H2O
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: 45.14 Å3/Da / Density % sol: 45.44 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 10-18% PEG 4,000, 50-200 mM sodium acetate buffer, 15% ethyelene 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: MAR CCD 165 mm / Detector: CCD / Date: Jul 17, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.934 Å / Relative weight: 1
ReflectionResolution: 1.6→47.89 Å / Num. obs: 57801 / % possible obs: 98.6 % / Redundancy: 5.9 % / Rsym value: 0.046 / Net I/σ(I): 27.21
Reflection shellResolution: 1.6→1.69 Å / Redundancy: 4.41 % / Num. unique obs: 37708 / Rsym value: 0.293 / % possible all: 92.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→47.89 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.961 / SU B: 2.655 / SU ML: 0.049 / Cross valid method: THROUGHOUT / ESU R: 0.077 / ESU R Free: 0.076 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : RESIDUAL ONLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1705 2896 5 %RANDOM
Rwork0.1438 ---
obs0.1451 54982 99.05 %-
Solvent computationIon probe radii: 0.7 Å / Shrinkage radii: 0.7 Å / VDW probe radii: 1 Å / Solvent model: MASK
Displacement parametersBiso max: 60.08 Å2 / Biso mean: 8.846 Å2 / Biso min: 7.03 Å2
Baniso -1Baniso -2Baniso -3
1--0.39 Å20 Å20 Å2
2---0.39 Å20 Å2
3---0.78 Å2
Refinement stepCycle: final / Resolution: 1.6→47.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3346 0 114 500 3960
Biso mean--22.02 29.63 -
Num. residues----432
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0183650
X-RAY DIFFRACTIONr_bond_other_d0.0010.0193467
X-RAY DIFFRACTIONr_angle_refined_deg1.5181.874945
X-RAY DIFFRACTIONr_angle_other_deg1.2262.7438014
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.4925457
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.31923.793174
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.32515617
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.3891515
X-RAY DIFFRACTIONr_chiral_restr0.1010.2545
X-RAY DIFFRACTIONr_gen_planes_refined0.010.024142
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02772
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_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.605→1.646 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.224 181 -
Rwork0.202 3544 -
all-3725 -
obs--87.94 %
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)
11.3902-0.0197-0.35712.07350.13980.4040.00040.2417-0.0405-0.23470.01780.0732-0.0012-0.0709-0.01820.0988-0.0094-0.01320.10710.01170.0434-2.3259.367-29.198
21.14730.49850.22881.96460.0251.48380.00730.11790.013-0.07970.0229-0.1530.04970.1004-0.03020.01770.00010.00850.02470.01790.05912.79116.001-22.855
34.7163-1.3954-2.4173.12232.49697.3651-0.0412-0.0337-0.36210.18580.0269-0.10750.52930.01620.01430.1235-0.0042-0.03510.01540.01940.09229.338-1.549-14.337
43.3889-1.3193-3.93434.8783.306214.0818-0.078-0.1385-0.26180.2915-0.09410.36420.5108-0.52880.17220.118-0.04470.00310.08960.0440.1305-1.101-0.491-13.564
50.84040.3759-0.1821.0419-0.32310.92570.0928-0.05920.08840.1248-0.1054-0.2331-0.13740.18720.01260.0557-0.0201-0.01480.06510.02340.118619.26322.774-16.818
67.09733.1916-7.78362.506-4.32369.97580.4106-0.38230.44770.4671-0.2159-0.0208-0.68110.4181-0.19470.2277-0.0837-0.0190.1066-0.03730.183818.83334.462-8.975
70.9853-0.00030.33940.7859-0.19561.23760.025-0.04130.02380.0567-0.0062-0.05680.04250.0434-0.01880.0328-0.0099-0.00260.020.01060.02626.8913.232-14.567
81.7063-0.9899-0.61343.88611.39081.04570.0560.10040.0217-0.0314-0.10950.34050.0197-0.16860.05350.04510.0006-0.01560.09010.04130.0777-12.79316.412-22.382
92.68981.025-0.4812.7733-0.9930.5866-0.00280.41750.4666-0.2660.16720.0366-0.0663-0.1405-0.16440.17360.03610.06920.11990.13560.21145.16532.94-31.332
101.92440.7904-0.26662.5608-0.15241.0430.10970.03990.50370.0872-0.02320.1573-0.2256-0.0788-0.08650.06740.02870.03260.03210.0380.1754-5.13634.824-18.685
115.03151.47510.88275.81213.1043.82340.22-0.32050.09270.3457-0.26590.33580.1624-0.42950.04590.0489-0.04750.03620.14290.02520.1023-19.35215.104-13.083
129.46524.09548.34722.28343.99067.67750.2594-0.6642-0.27240.3066-0.18230.05830.4371-0.5352-0.07710.2207-0.03590.08310.22210.07580.1626-11.74411.146-4.001
131.56650.2109-0.66461.11950.04961.57090.0946-0.05260.2750.0561-0.00460.0383-0.1833-0.0334-0.090.04440.00290.01220.0170.01410.0956-0.11629.66-16.454
143.70630.6965-2.52991.826-0.66814.02030.20870.02130.54240.1091-0.01350.1565-0.2669-0.1103-0.19520.09260.0060.020.0080.01410.2049-0.40336.98-17.858
150.78880.2813-0.23337.595-2.15861.30340.09810.0681-0.0342-0.1179-0.2307-0.33670.05950.19730.13260.0452-0.00120.00890.07110.00720.097918.86719.788-23.311
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 24
2X-RAY DIFFRACTION2A25 - 53
3X-RAY DIFFRACTION3A54 - 65
4X-RAY DIFFRACTION4A66 - 77
5X-RAY DIFFRACTION5A78 - 110
6X-RAY DIFFRACTION6A111 - 134
7X-RAY DIFFRACTION7A135 - 192
8X-RAY DIFFRACTION8A193 - 217
9X-RAY DIFFRACTION9B2 - 23
10X-RAY DIFFRACTION10B24 - 91
11X-RAY DIFFRACTION11B92 - 110
12X-RAY DIFFRACTION12B111 - 134
13X-RAY DIFFRACTION13B135 - 175
14X-RAY DIFFRACTION14B176 - 201
15X-RAY DIFFRACTION15B202 - 217

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