PDB-7n2w: The crystal structure of an FMN-dependent NADH-azoreductase, AzoA...

ID or keywords:

Entry

Database: PDB / ID: 7n2w

Title

The crystal structure of an FMN-dependent NADH-azoreductase, AzoA in complex with Red 40

Components

FMN dependent NADH:quinone oxidoreductase

Keywords

OXIDOREDUCTASE / FMN / NADH / Azoreductase / AzoA

Function / homology

Function and homology information

FMN-dependent NADH-azoreductase / Oxidoreductases; Acting on NADH or NADPH; With a quinone or similar compound as acceptor / oxidoreductase activity, acting on NAD(P)H as acceptor / oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor / FMN binding / electron transfer activity
Similarity search - Function

Biological species

Klebsiella pneumoniae subsp. pneumoniae (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.65 Å

Authors

Arcinas, A.J. / Fedorov, E. / Kelly, L. / Almo, S.C. / Ghosh, A.

Citation

Journal: To Be Published
Title: Uncovering a novel mechanism of enzyme activation in multimeric azoreductases
Authors: Hitchings, R. / Ryan, A. / Arcinas, A.J. / Ghosh, A. / Almo, S.C. / Kelly, L.

History

Deposition	May 30, 2021	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jun 1, 2022	Provider: repository / Type: Initial release
Revision 1.1	Oct 18, 2023	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	7n2w.cif.gz	312.6 KB	Display	PDBx/mmCIF format
PDB format	pdb7n2w.ent.gz	255 KB	Display	PDB format
PDBx/mmJSON format	7n2w.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	7n2w_validation.pdf.gz	2.5 MB	Display	wwPDB validaton report
Full document	7n2w_full_validation.pdf.gz	2.5 MB	Display
Data in XML	7n2w_validation.xml.gz	33.8 KB	Display
Data in CIF	7n2w_validation.cif.gz	42.8 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/n2/7n2w ftp://data.pdbj.org/pub/pdb/validation_reports/n2/7n2w	HTTPS FTP

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Related structure data

Related structure data	7n2xC 6dxpS S: Starting model for refinement 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

Deposited unit

A: FMN dependent NADH:quinone oxidoreductase

B: FMN dependent NADH:quinone oxidoreductase

C: FMN dependent NADH:quinone oxidoreductase

D: FMN dependent NADH:quinone oxidoreductase

hetero molecules

101 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: FMN dependent NADH:quinone oxidoreductase

B: FMN dependent NADH:quinone oxidoreductase

hetero molecules

defined by author&software
Evidence: assay for oligomerization, Size Exclusion Chromatography and Dynamic Light Scattering
50.3 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

C: FMN dependent NADH:quinone oxidoreductase

D: FMN dependent NADH:quinone oxidoreductase

hetero molecules

defined by author&software
50.3 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	60.407, 83.336, 87.305
Angle α, β, γ (deg.)	90.00, 100.26, 90.00
Int Tables number	4
Space group name H-M	P12₁1

#1: Protein	FMN dependent NADH:quinone oxidoreductase / Azo-dye reductase / FMN-dependent NADH-azo compound oxidoreductase / FMN-dependent NADH-azoreductase Mass: 24257.512 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578) (bacteria) Strain: ATCC 700721 / MGH 78578 / Gene: acpD, azoR, KPN_02994 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: A6TCS9, Oxidoreductases; Acting on NADH or NADPH; With a quinone or similar compound as acceptor, FMN-dependent NADH-azoreductase
#2: Chemical	ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE Mass: 456.344 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₁₇H₂₁N₄O₉P
#3: Chemical	ChemComp-05G / 6-hydroxy-5-[(E)-(2-methoxy-5-methyl-4-sulfophenyl)diazenyl]naphthalene-2-sulfonic acid Mass: 452.458 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₁₈H₁₆N₂O₈S₂
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 44 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.23 Å³/Da / Density % sol: 44.81 %
Crystal grow	Temperature: 291 K / Method: vapor diffusion, sitting drop / pH: 9.5 Details: 1.26 M ammonium sulfate, 0.2 M NaCl and 0.1 M CHES pH 9.5

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: APS / Beamline: 31-ID / Wavelength: 0.98 Å
Detector	Type: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Mar 16, 2018 / Details: KB MIRRORS
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.98 Å / Relative weight: 1
Reflection	Resolution: 2.65→19.96 Å / Num. obs: 23952 / % possible obs: 96.2 % / Redundancy: 3.5 % / B_iso Wilson estimate: 34.63 Å² / R_merge(I) obs: 0.174 / Net I/σ(I): 5.4
Reflection shell	Resolution: 2.65→2.78 Å / Redundancy: 2.5 % / R_merge(I) obs: 0.703 / % possible all: 81.4

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Processing

Software

Name	Version	Classification
PHENIX	1.19.1_4122	refinement
MOSFLM		data reduction
Aimless	0.7.4	data scaling
PDB_EXTRACT	3.27	data extraction
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 6DXP

6dxp

Resolution: 2.65→19.96 Å / SU ML: 0.4 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 33.61 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.302	1205	5.04 %
R_work	0.249	-	-
obs	0.252	23893	96.2 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL

Displacement parameters

B_iso mean: 38.71 Å²

Refinement step

Cycle: LAST / Resolution: 2.65→19.96 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6003	0	244	44	6291

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.65-2.76	0.336	96	0.3376	2059	X-RAY DIFFRACTION	79
2.76-2.88	0.3798	132	0.3361	2439	X-RAY DIFFRACTION	94
2.88-3.03	0.3824	144	0.3193	2600	X-RAY DIFFRACTION	100
3.03-3.22	0.3383	149	0.2859	2594	X-RAY DIFFRACTION	100
3.22-3.47	0.3609	129	0.2689	2562	X-RAY DIFFRACTION	98
3.47-3.82	0.3636	132	0.2792	2566	X-RAY DIFFRACTION	98
3.82-4.36	0.2624	139	0.2204	2559	X-RAY DIFFRACTION	98
4.36-5.48	0.2311	148	0.1925	2641	X-RAY DIFFRACTION	100
5.48-19.96	0.2408	136	0.1904	2668	X-RAY DIFFRACTION	100

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	0.4359	0.0994	-0.4856	-0.0082	0.241	0.5471	0.1005	0.0363	-0.2362	-0.0863	-0.0181	-0.0204	-0.1692	0.0869	0	0.2507	-0.0009	-0.0189	0.2733	-0.0044	0.2879	7.9861	2.47	38.0152
2	-0.009	-0.0204	-0.0262	-0.0388	-0.0581	-0.0367	-0.0478	0.3411	0.2368	-0.2611	-0.1037	-0.1008	-0.1612	0.5007	0	0.4075	0.0262	0.0574	0.3748	-0.0782	0.3002	20.557	5.4306	39.6187
3	0.2206	-0.1258	0.0252	-0.0364	0.1316	0.2121	-0.0615	-0.0338	0.0089	-0.124	-0.0047	0.002	-0.2154	0.16	-0	0.4367	-0.0332	-0.0025	0.2551	-0.0013	0.2122	9.7687	6.6781	46.2402
4	0.1071	-0.0035	-0.0206	0.018	-0.0792	-0.0227	0.6193	-0.1168	-0.2784	0.526	-0.3006	0.4344	-0.6267	0.2468	0	0.7513	-0.0535	-0.0429	0.353	-0.0321	0.2081	6.5033	16.0008	45.5576
5	0.0194	-0.0573	-0.0332	0.0719	0.0038	0.0831	-0.019	-0.3295	0.361	-0.2608	0.0436	0.3738	-0.429	-0.2025	-0	0.3742	0.0072	-0.0019	0.2764	-0.0355	0.2872	-2.7982	6.3135	52.1249
6	0.4519	-0.4322	0.2267	0.0652	0.0685	0.2888	0.3804	0.0608	-0.2475	0.5624	-0.0248	0.1613	-0.3886	-0.1978	-0	0.5239	0.0485	-0.0047	0.3703	0.013	0.3648	-9.6544	4.719	41.5482
7	0.122	0.3725	-0.3137	0.5448	-0.1914	0.4714	-0.1281	-0.0326	0.0498	0.1021	0.0919	0.1936	-0.0146	-0.0542	0	0.2429	-0.0199	-0.0221	0.2821	0.0286	0.3261	4.0605	-10.2758	60.8307
8	0.0479	0.0894	0.1899	0.0298	-0.1454	0.1029	0.0914	-0.0224	-0.1956	0.2243	0.0378	0.3948	-0.6228	-0.0219	-0	0.3873	0.0227	0.032	0.2263	-0.0397	0.2438	3.5486	4.8776	64.0363
9	0.1713	-0.2402	0.1806	0.1515	0.0628	0.4154	-0.106	0.2467	0.3501	-0.2374	0.2618	0.29	-0.2857	-0.1117	-0	0.4539	0.0319	0.0009	0.2842	-0.0335	0.3226	8.6486	8.4997	68.4037
10	0.0942	-0.0888	-0.0104	0.061	0.0717	0.1038	-0.3726	-0.0725	0.0431	-0.309	0.2104	0.2317	-0.4196	-0.4233	0	0.4458	0.0163	-0.0872	0.3857	-0.1015	0.3968	12.0875	9.7058	76.929
11	0.8011	-0.4516	-0.0202	0.2299	0.2153	0.1692	0.0595	0.0277	0.0604	-0.0602	0.018	0.0088	0.0229	-0.084	-0	0.3078	0.007	-0.0149	0.2212	-0.0072	0.2164	-19.1229	-9.795	-1.9772
12	0.2507	0.1186	0.2285	-0.0597	-0.0227	0.0887	1.2845	0.3254	-0.4039	-2.0663	-0.9154	0.4577	-0.3028	-0.0818	0	-3.4201	-0.8283	0.4355	0.0004	0.2054	0.2416	-17.0386	-11.4272	3.473
13	0.0204	0.0714	-0.0276	0.1107	-0.0623	0.1001	-0.2094	-0.3087	-0.4899	0.3402	-0.1115	-0.2011	0.6443	0.8628	0	0.5117	0.1261	-0.0669	0.4277	0.1394	0.4206	-14.3462	-24.7685	13.0166
14	-0.131	0.2264	-0.0652	-0.0427	0.0861	-0.0271	0.7978	-0.7404	-2.0864	-0.0801	0.5909	0.5813	0.3816	-0.506	-0	0.6297	-0.2109	-0.4417	-0.7618	-1.7276	-2.5394	-14.05	-21.832	-2.1487
15	-0.0212	0.0033	0.0032	-0.0077	0.0158	-0.0082	-0.2146	0.2487	-0.2128	-0.1682	0.0303	-0.4348	0.0705	0.5182	-0	0.7044	0.1744	0.0112	0.3317	0.0163	0.3084	3.5726	-10.8807	-6.3899
16	0.3131	-0.2266	0.1107	0.4165	0.2386	0.3997	-0.012	0.194	-0.132	-0.2086	0.1159	-0.1113	0.2787	0.1146	-0	0.354	-0.0045	-0.0232	0.3039	-0.0732	0.2423	-6.2051	-14.2048	-11.2324
17	0.0299	-0.104	0.0693	0.1868	-0.1017	0.0121	-0.1614	-0.7371	-0.1628	0.1064	0.5107	-0.3708	-0.4882	1.2261	-0	0.4978	0.124	-0.0955	-0.1826	0.2722	0.0663	1.0555	-4.0867	22.1255
18	-0.1355	-0.301	0.0732	0.3328	0.3326	0.2653	-0.1678	-0.0465	-0.0189	0.2153	0.1291	0.0921	-0.5049	-0.0808	-0	0.2964	0.009	0.0193	0.2903	0.0345	0.2686	0.1717	1.3464	15.8682
19	0.1766	-0.4186	0.183	0.0691	-0.1572	0.1554	-0.0384	0.1589	-0.0156	-0.2704	-0.0769	-0.1445	-0.0238	-0.0212	0	0.3079	-0.0094	0.0198	0.1968	-0.0244	0.2312	0.1793	2.1021	9.017
20	-0.0083	-0.0112	-0.0236	-0.0122	0.0058	0.0004	-0.1161	0.5794	0.1117	-0.4448	-0.0803	0.1458	0.0144	0.1341	-0	-1.2416	-0.4751	0.2112	0.5787	-0.0471	0.5022	12.3881	3.1919	0.9443
21	0.3994	-0.0055	0.5809	0.2035	-0.285	0.412	0.0431	-0.0856	-0.1199	0.0247	0.007	-0.1288	0.1099	0.0987	-0	0.2389	0.023	0.0172	0.2434	-0.0237	0.2804	6.4853	-15.1391	14.9786
22	0.6057	-0.1561	-0.432	0.2762	-0.0576	0.2844	0.1453	0.0205	-0.2404	0.0543	0.1051	-0.0928	0.1766	0.3049	0	0.2997	0.0288	-0.0142	0.3246	-0.0392	0.2545	13.3132	-8.223	65.4056

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	CHAIN 'A' AND (RESID 1 THROUGH 66 )
2	X-RAY DIFFRACTION	2	CHAIN 'A' AND (RESID 67 THROUGH 84 )
3	X-RAY DIFFRACTION	3	CHAIN 'A' AND (RESID 85 THROUGH 121 )
4	X-RAY DIFFRACTION	4	CHAIN 'A' AND (RESID 122 THROUGH 139 )
5	X-RAY DIFFRACTION	5	CHAIN 'A' AND (RESID 140 THROUGH 162 )
6	X-RAY DIFFRACTION	6	CHAIN 'A' AND (RESID 163 THROUGH 201 )
7	X-RAY DIFFRACTION	7	CHAIN 'B' AND (RESID 67 THROUGH 132 )
8	X-RAY DIFFRACTION	8	CHAIN 'B' AND (RESID 133 THROUGH 163 )
9	X-RAY DIFFRACTION	9	CHAIN 'B' AND (RESID 164 THROUGH 180 )
10	X-RAY DIFFRACTION	10	CHAIN 'B' AND (RESID 181 THROUGH 201 )
11	X-RAY DIFFRACTION	11	CHAIN 'C' AND (RESID 2 THROUGH 66 )
12	X-RAY DIFFRACTION	12	CHAIN 'C' AND (RESID 67 THROUGH 111 )
13	X-RAY DIFFRACTION	13	CHAIN 'C' AND (RESID 112 THROUGH 124 )
14	X-RAY DIFFRACTION	14	CHAIN 'C' AND (RESID 125 THROUGH 139 )
15	X-RAY DIFFRACTION	15	CHAIN 'C' AND (RESID 140 THROUGH 151 )
16	X-RAY DIFFRACTION	16	CHAIN 'C' AND (RESID 152 THROUGH 201 )
17	X-RAY DIFFRACTION	17	CHAIN 'D' AND (RESID 1 THROUGH 16 )
18	X-RAY DIFFRACTION	18	CHAIN 'D' AND (RESID 17 THROUGH 66 )
19	X-RAY DIFFRACTION	19	CHAIN 'D' AND (RESID 67 THROUGH 121 )
20	X-RAY DIFFRACTION	20	CHAIN 'D' AND (RESID 122 THROUGH 132 )
21	X-RAY DIFFRACTION	21	CHAIN 'D' AND (RESID 133 THROUGH 201 )
22	X-RAY DIFFRACTION	22	CHAIN 'B' AND (RESID 0 THROUGH 66)

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