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

ID or keywords:

Entry

Database: PDB / ID: 8c5f

Title

E. coli NfsB-T41Q/N71S/F124T mutant bound to acetate

Components

Oxygen-insensitive NAD(P)H nitroreductase

Keywords

OXIDOREDUCTASE / 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

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.6 Å

Authors

White, S.A. / Hyde, E.I. / Day, M.A.

Funding support

United Kingdom, 1items

Citation

Journal: 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

Deposition	Jan 7, 2023	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Apr 19, 2023	Provider: repository / Type: Initial release
Revision 1.1	Jun 19, 2024	Group: Data collection / Category: chem_comp_atom / chem_comp_bond

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	8c5f.cif.gz	196.2 KB	Display	PDBx/mmCIF format
PDB format	pdb8c5f.ent.gz	156.4 KB	Display	PDB format
PDBx/mmJSON format	8c5f.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8c5f_validation.pdf.gz	1 MB	Display	wwPDB validaton report
Full document	8c5f_full_validation.pdf.gz	1 MB	Display
Data in XML	8c5f_validation.xml.gz	22.9 KB	Display
Data in CIF	8c5f_validation.cif.gz	34.6 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/c5/8c5f ftp://data.pdbj.org/pub/pdb/validation_reports/c5/8c5f	HTTPS FTP

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Related structure data

Related structure data	8c5eC 8c5pC 8ccvC 8cj0C 8og3C C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI
Related items in Molecule of the Month	#188 - Aug 2015 Tetrahydrobiopterin Biosynthesis similarity (1)

Deposited unit

A: Oxygen-insensitive NAD(P)H nitroreductase

B: Oxygen-insensitive NAD(P)H nitroreductase

hetero molecules

49.2 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

Idetical with deposited unit
defined by author
Evidence: gel filtration

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 number	92
Space group name H-M	P4₁2₁2

Components on special symmetry positions

ID	Model	Components
1	1	A-647- HOH

#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 interest	N

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 45.14 Å³/Da / Density % sol: 45.44 %
Crystal grow	Temperature: 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

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 0.934 Å
Detector	Type: MAR CCD 165 mm / Detector: CCD / Date: Jul 17, 2010
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.934 Å / Relative weight: 1
Reflection	Resolution: 1.6→47.89 Å / Num. obs: 57801 / % possible obs: 98.6 % / Redundancy: 5.9 % / R_sym value: 0.046 / Net I/σ(I): 27.21
Reflection shell	Resolution: 1.6→1.69 Å / Redundancy: 4.41 % / Num. unique obs: 37708 / R_sym value: 0.293 / % possible all: 92.3

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
PDB_EXTRACT	3.27	data extraction
MOSFLM		data reduction
SCALA		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.6→47.89 Å / Cor.coef. F_o:F_c: 0.97 / Cor.coef. F_o:F_c 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

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.1705	2896	5 %	RANDOM
R_work	0.1438	-	-	-
obs	0.1451	54982	99.05 %	-

Solvent computation

Ion probe radii: 0.7 Å / Shrinkage radii: 0.7 Å / VDW probe radii: 1 Å / Solvent model: MASK

Displacement parameters

B_iso max: 60.08 Å² / B_iso mean: 8.846 Å² / B_iso min: 7.03 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.39 Å²	0 Å²	0 Å²
2-	-	-0.39 Å²	0 Å²
3-	-	-	0.78 Å²

Refinement step

Cycle: final / Resolution: 1.6→47.89 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3346	0	114	500	3960
B_iso mean	-	-	22.02	29.63	-
Num. residues	-	-	-	-	432

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.014	0.018	3650
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.019	3467
X-RAY DIFFRACTION	r_angle_refined_deg	1.518	1.87	4945
X-RAY DIFFRACTION	r_angle_other_deg	1.226	2.743	8014
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.492	5	457
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	31.319	23.793	174
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	12.325	15	617
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	12.389	15	15
X-RAY DIFFRACTION	r_chiral_restr	0.101	0.2	545
X-RAY DIFFRACTION	r_gen_planes_refined	0.01	0.02	4142
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	772
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 1.605→1.646 Å / R_factor R_free error: 0 / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.224	181	-
R_work	0.202	3544	-
all	-	3725	-
obs	-	-	87.94 %

Refinement TLS params.

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

ID	L11 (°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)
1	1.3902	-0.0197	-0.3571	2.0735	0.1398	0.404	0.0004	0.2417	-0.0405	-0.2347	0.0178	0.0732	-0.0012	-0.0709	-0.0182	0.0988	-0.0094	-0.0132	0.1071	0.0117	0.0434	-2.325	9.367	-29.198
2	1.1473	0.4985	0.2288	1.9646	0.025	1.4838	0.0073	0.1179	0.013	-0.0797	0.0229	-0.153	0.0497	0.1004	-0.0302	0.0177	0.0001	0.0085	0.0247	0.0179	0.059	12.791	16.001	-22.855
3	4.7163	-1.3954	-2.417	3.1223	2.4969	7.3651	-0.0412	-0.0337	-0.3621	0.1858	0.0269	-0.1075	0.5293	0.0162	0.0143	0.1235	-0.0042	-0.0351	0.0154	0.0194	0.0922	9.338	-1.549	-14.337
4	3.3889	-1.3193	-3.9343	4.878	3.3062	14.0818	-0.078	-0.1385	-0.2618	0.2915	-0.0941	0.3642	0.5108	-0.5288	0.1722	0.118	-0.0447	0.0031	0.0896	0.044	0.1305	-1.101	-0.491	-13.564
5	0.8404	0.3759	-0.182	1.0419	-0.3231	0.9257	0.0928	-0.0592	0.0884	0.1248	-0.1054	-0.2331	-0.1374	0.1872	0.0126	0.0557	-0.0201	-0.0148	0.0651	0.0234	0.1186	19.263	22.774	-16.818
6	7.0973	3.1916	-7.7836	2.506	-4.3236	9.9758	0.4106	-0.3823	0.4477	0.4671	-0.2159	-0.0208	-0.6811	0.4181	-0.1947	0.2277	-0.0837	-0.019	0.1066	-0.0373	0.1838	18.833	34.462	-8.975
7	0.9853	-0.0003	0.3394	0.7859	-0.1956	1.2376	0.025	-0.0413	0.0238	0.0567	-0.0062	-0.0568	0.0425	0.0434	-0.0188	0.0328	-0.0099	-0.0026	0.02	0.0106	0.0262	6.89	13.232	-14.567
8	1.7063	-0.9899	-0.6134	3.8861	1.3908	1.0457	0.056	0.1004	0.0217	-0.0314	-0.1095	0.3405	0.0197	-0.1686	0.0535	0.0451	0.0006	-0.0156	0.0901	0.0413	0.0777	-12.793	16.412	-22.382
9	2.6898	1.025	-0.481	2.7733	-0.993	0.5866	-0.0028	0.4175	0.4666	-0.266	0.1672	0.0366	-0.0663	-0.1405	-0.1644	0.1736	0.0361	0.0692	0.1199	0.1356	0.2114	5.165	32.94	-31.332
10	1.9244	0.7904	-0.2666	2.5608	-0.1524	1.043	0.1097	0.0399	0.5037	0.0872	-0.0232	0.1573	-0.2256	-0.0788	-0.0865	0.0674	0.0287	0.0326	0.0321	0.038	0.1754	-5.136	34.824	-18.685
11	5.0315	1.4751	0.8827	5.8121	3.104	3.8234	0.22	-0.3205	0.0927	0.3457	-0.2659	0.3358	0.1624	-0.4295	0.0459	0.0489	-0.0475	0.0362	0.1429	0.0252	0.1023	-19.352	15.104	-13.083
12	9.4652	4.0954	8.3472	2.2834	3.9906	7.6775	0.2594	-0.6642	-0.2724	0.3066	-0.1823	0.0583	0.4371	-0.5352	-0.0771	0.2207	-0.0359	0.0831	0.2221	0.0758	0.1626	-11.744	11.146	-4.001
13	1.5665	0.2109	-0.6646	1.1195	0.0496	1.5709	0.0946	-0.0526	0.275	0.0561	-0.0046	0.0383	-0.1833	-0.0334	-0.09	0.0444	0.0029	0.0122	0.017	0.0141	0.0956	-0.116	29.66	-16.454
14	3.7063	0.6965	-2.5299	1.826	-0.6681	4.0203	0.2087	0.0213	0.5424	0.1091	-0.0135	0.1565	-0.2669	-0.1103	-0.1952	0.0926	0.006	0.02	0.008	0.0141	0.2049	-0.403	36.98	-17.858
15	0.7888	0.2813	-0.2333	7.595	-2.1586	1.3034	0.0981	0.0681	-0.0342	-0.1179	-0.2307	-0.3367	0.0595	0.1973	0.1326	0.0452	-0.0012	0.0089	0.0711	0.0072	0.0979	18.867	19.788	-23.311

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	2 - 24
2	X-RAY DIFFRACTION	2	A	25 - 53
3	X-RAY DIFFRACTION	3	A	54 - 65
4	X-RAY DIFFRACTION	4	A	66 - 77
5	X-RAY DIFFRACTION	5	A	78 - 110
6	X-RAY DIFFRACTION	6	A	111 - 134
7	X-RAY DIFFRACTION	7	A	135 - 192
8	X-RAY DIFFRACTION	8	A	193 - 217
9	X-RAY DIFFRACTION	9	B	2 - 23
10	X-RAY DIFFRACTION	10	B	24 - 91
11	X-RAY DIFFRACTION	11	B	92 - 110
12	X-RAY DIFFRACTION	12	B	111 - 134
13	X-RAY DIFFRACTION	13	B	135 - 175
14	X-RAY DIFFRACTION	14	B	176 - 201
15	X-RAY DIFFRACTION	15	B	202 - 217

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