PDB-2o9z: Crystal Structure of RebH, a FAD-dependent halogenase from Lechev...

ID or keywords:

Entry

Database: PDB / ID: 2o9z

Title

Crystal Structure of RebH, a FAD-dependent halogenase from Lechevalieria aerocolonigenes, the Apo form

Components

Tryptophan halogenase

Keywords

BIOSYNTHETIC PROTEIN / FLAVOPROTEIN / RebH / Rebeccamycin / halogenase / flavin

Function / homology

Function and homology information

tryptophan 7-halogenase / monooxygenase activity / nucleotide binding
Similarity search - Function

Biological species

Lechevalieria aerocolonigenes (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.494 Å

Authors

Bitto, E. / Bingman, C.A. / Phillips Jr., G.N.

Citation

Journal: Proteins / Year: 2008
Title: The structure of flavin-dependent tryptophan 7-halogenase RebH.
Authors: Bitto, E. / Huang, Y. / Bingman, C.A. / Singh, S. / Thorson, J.S. / Phillips Jr., G.N.

History

Deposition	Dec 14, 2006	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Apr 10, 2007	Provider: repository / Type: Initial release
Revision 1.1	Jan 15, 2008	Group: Version format compliance
Revision 1.2	Jul 13, 2011	Group: Advisory / Version format compliance
Revision 1.3	Oct 18, 2017	Group: Refinement description / Category: software Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.4	Aug 30, 2023	Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim / struct_ref_seq_dif / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/o9/2o9z ftp://data.pdbj.org/pub/pdb/validation_reports/o9/2o9z	HTTPS FTP

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Related structure data

Related structure data	2oa1C 2aqjS C: citing same article (ref.) S: Starting model for refinement
Similar structure data	2e4g-d 6p2v-d 2oa1-d 2oal-d 2oam-d 7cu0-d 6p00-d 4lu6-d 6h44-d 7ju0-d Search similar-shape structures by Omokage search (details) 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: Tryptophan halogenase

B: Tryptophan halogenase

hetero molecules

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

1

Idetical with deposited unit
defined by author

Unit cell

Length a, b, c (Å)	114.786, 114.787, 230.584
Angle α, β, γ (deg.)	90.00, 90.00, 120.00
Int Tables number	171
Space group name H-M	P6₂

Components on special symmetry positions

ID	Model	Components
1	1	A-533- HOH
2	1	B-675- HOH

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLY / Beg label comp-ID: GLY / Refine code: 4

Dom-ID	End auth comp-ID	End label comp-ID	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	GLN	GLN	A	A	3 - 526	23 - 546
2	HIS	HIS	B	B	3 - 528	23 - 548

#1: Protein	Tryptophan halogenase / Putative halogenase of tryptophan / Halogenase Mass: 62540.070 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lechevalieria aerocolonigenes (bacteria) Strain: 39243 / Gene: rbmJ, rebH / Plasmid: pET28a / Species (production host): Escherichia coli / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: Q8KHZ8
#2: Chemical	ChemComp-PO4 / PHOSPHATE ION Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO₄
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 455 / Source method: isolated from a natural source / Formula: H₂O

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 3.7 Å³/Da / Density % sol: 66.3 %
Crystal grow	Temperature: 277 K / Method: vapor diffusion, hanging drop Details: Protein Solution (18 mg/ml protein, 0.050 M sodium chloride, 0.010 M TRIS pH 8.0) mixed in a 1:1 ratio with the Well Solution (0.9 M K2HPO4, 0.5 M NaH2PO4) Cryoprotected with: well solution ...Details: Protein Solution (18 mg/ml protein, 0.050 M sodium chloride, 0.010 M TRIS pH 8.0) mixed in a 1:1 ratio with the Well Solution (0.9 M K2HPO4, 0.5 M NaH2PO4) Cryoprotected with: well solution supplemented with up to 30% glycerol, vapor diffusion, hanging drop, temperature 277K

Crystal

Density Matthews: 3.7 Å³/Da / Density % sol: 66.3 %

Crystal grow

Temperature: 277 K / Method: vapor diffusion, hanging drop
Details: Protein Solution (18 mg/ml protein, 0.050 M sodium chloride, 0.010 M TRIS pH 8.0) mixed in a 1:1 ratio with the Well Solution (0.9 M K2HPO4, 0.5 M NaH2PO4) Cryoprotected with: well solution ...

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Data collection

Diffraction

Mean temperature: 100 K

Diffraction source

Source:

SYNCHROTRON / Site:

APS

/ Beamline: 23-ID-D / Wavelength: 0.97919 Å

Detector

Type: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Mar 26, 2006 / Details: Adjustable focusing mirrors in K-B geometry

Radiation

Monochromator: Si(111) Double Crystal Monochromater / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.97919 Å / Relative weight: 1

Reflection

Resolution: 2.5→29.129 Å / Num. obs: 59677 / % possible obs: 100 % / Redundancy: 12.8 % / R_merge(I) obs: 0.131 / Χ²: 1.522 / Net I/σ(I): 24.484

Reflection shell

Diffraction-ID: 1 / % possible all: 100

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. unique all	Χ²
2.5-2.59	10.3	0.432	8.095	5912	1.004
2.59-2.69	11.1	0.381		5998	1.037
2.69-2.82	12	0.335		5924	1.109
2.82-2.96	12.9	0.298		5978	1.233
2.96-3.15	13.5	0.254		5927	1.561
3.15-3.39	13.6	0.176		5990	1.906
3.39-3.73	13.6	0.118		5935	1.914
3.73-4.27	13.6	0.096		5978	1.876
4.27-5.38	13.6	0.091		5993	2.002
5.38-30	13.5	0.066		6042	1.28

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Phasing

Phasing MR

R_factor: 0.462 / Cor.coef. F_o:F_c: 0.49

	Highest resolution	Lowest resolution
Rotation	3 Å	29.13 Å
Translation	3 Å	29.13 Å

Phasing dm

Method: Solvent flattening and Histogram matching / Reflection: 59619

Phasing dm shell

Resolution (Å)	Delta phi final	FOM	Reflection
12.02-100	32	0.604	516
8.98-12.02	32.4	0.884	750
7.48-8.98	37.2	0.873	950
6.55-7.48	42.3	0.844	1105
5.89-6.55	40.8	0.841	1229
5.4-5.89	36.3	0.866	1356
5.02-5.4	34.3	0.88	1462
4.7-5.02	30.9	0.91	1574
4.44-4.7	29.9	0.911	1660
4.22-4.44	31.1	0.898	1748
4.03-4.22	29.9	0.89	1866
3.86-4.03	32.3	0.878	1915
3.71-3.86	30.8	0.889	2005
3.58-3.71	31.6	0.871	2088
3.46-3.58	31.4	0.87	2168
3.35-3.46	32.7	0.865	2243
3.25-3.35	33.3	0.859	2281
3.16-3.25	32.4	0.859	2376
3.08-3.16	33.6	0.847	2439
3-3.08	33.2	0.855	2488
2.93-3	36.2	0.838	2538
2.87-2.93	33.9	0.84	2630
2.8-2.87	34.1	0.845	2657
2.74-2.8	33.6	0.849	2755
2.69-2.74	34.4	0.855	2779
2.64-2.69	34.7	0.844	2894
2.59-2.64	34	0.845	2873
2.54-2.59	35.5	0.831	2936
2.49-2.54	40.4	0.798	3338

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Processing

Software

Name	Version	Classification
DENZO		data reduction
SCALEPACK		data scaling
DM	5	phasing
MOLREP		phasing
REFMAC	5.2.0005	refinement
PDB_EXTRACT	2	data extraction
HKL-2000		data reduction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 2AQJ monomer with no ligands

2aqj

Resolution: 2.494→29.129 Å / Cor.coef. F_o:F_c: 0.958 / Cor.coef. F_o:F_c free: 0.932 / WR_factor R_free: 0.204 / WR_factor R_work: 0.159 / SU B: 12.078 / SU ML: 0.142 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.267 / ESU R Free: 0.209 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.208	3008	5.045 %	RANDOM
R_work	0.161	-	-	-
obs	0.164	59619	99.921 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT

Displacement parameters

B_iso mean: 2.559 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.995 Å²	0.498 Å²	0 Å²
2-	-	0.995 Å²	0 Å²
3-	-	-	-1.493 Å²

Refinement step

Cycle: LAST / Resolution: 2.494→29.129 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	8437	0	10	455	8902

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.014	0.022	8678
X-RAY DIFFRACTION	r_angle_refined_deg	1.407	1.936	11779
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.012	5	1048
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.102	23.224	456
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.825	15	1365
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	18.633	15	74
X-RAY DIFFRACTION	r_chiral_restr	0.1	0.2	1224
X-RAY DIFFRACTION	r_gen_planes_refined	0.006	0.02	6856
X-RAY DIFFRACTION	r_nbd_refined	0.203	0.2	3879
X-RAY DIFFRACTION	r_nbtor_refined	0.312	0.2	6015
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.141	0.2	577
X-RAY DIFFRACTION	r_symmetry_vdw_refined	0.153	0.2	32
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.246	0.2	2
X-RAY DIFFRACTION	r_mcbond_it	0.195	1.5	5325
X-RAY DIFFRACTION	r_mcangle_it	0.251	2	8398
X-RAY DIFFRACTION	r_scbond_it	1.1	3	3802
X-RAY DIFFRACTION	r_scangle_it	1.853	4.5	3381

Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Number: 4209 / Refine-ID: X-RAY DIFFRACTION

Type	Rms dev position (Å)	Weight position
MEDIUM POSITIONAL	0.25	0.5
MEDIUM THERMAL	0.3	2

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Num. reflection all	% reflection obs (%)
2.494-2.559	0.267	219	0.221	4180	4407	99.818
2.559-2.629	0.235	186	0.199	4072	4258	100
2.629-2.705	0.278	231	0.206	3953	4184	100
2.705-2.788	0.261	229	0.195	3831	4060	100
2.788-2.88	0.253	188	0.193	3736	3924	100
2.88-2.981	0.234	206	0.176	3589	3795	100
2.981-3.093	0.239	170	0.176	3523	3693	100
3.093-3.219	0.239	187	0.17	3327	3514	100
3.219-3.362	0.208	155	0.162	3235	3390	100
3.362-3.526	0.198	152	0.151	3071	3223	100
3.526-3.717	0.203	170	0.156	2932	3102	100
3.717-3.942	0.198	144	0.146	2773	2917	100
3.942-4.214	0.157	136	0.128	2600	2736	100
4.214-4.55	0.16	111	0.128	2453	2564	100
4.55-4.984	0.187	129	0.132	2234	2363	100
4.984-5.57	0.193	132	0.15	1984	2116	100
5.57-6.429	0.24	75	0.182	1816	1891	100
6.429-7.865	0.193	92	0.169	1492	1584	100
7.865-11.089	0.139	65	0.126	1178	1243	100
11.089-29.129	0.141	31	0.172	632	702	94.444

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	3.0648	1.1436	1.6653	1.351	0.603	2.7388	0.146	-0.1192	-0.3145	0.2335	-0.1518	-0.0973	0.6014	0.0083	0.0058	0.0541	-0.0516	0.074	-0.0767	0.0287	-0.0175	-21.7857	24.1705	5.9291
2	16.7579	-5.0082	-3.2383	8.7561	0.3454	10.8738	-0.3207	-0.0453	-0.553	0.2339	0.3569	-0.5481	1.4214	1.4387	-0.0362	0.2547	0.0515	0.0529	0.213	-0.1349	-0.0659	-23.4713	18.595	-24.3333
3	2.2285	0.5965	1.1313	1.1223	0.3549	1.6021	0.0407	0.1805	-0.3177	0.0745	-0.1061	-0.0514	0.8968	0.1874	0.0653	0.2134	-0.0312	0.1086	-0.0469	-0.0145	0.0717	-23.2373	15.1988	-1.5985
4	2.0274	0.128	2.4869	0.9935	-0.1862	7.216	0.0468	-0.3328	0.1555	0.396	-0.1628	0.021	-0.2559	0.0633	0.116	-0.0464	-0.1086	0.0313	-0.0066	0.0083	-0.0286	-15.5382	45.6112	18.0693
5	1.1592	-0.4199	0.1236	3.3053	-0.4019	3.2503	-0.0558	-0.0969	-0.1337	0.4349	0.0247	0.4125	0.3952	-0.4571	0.031	-0.0248	-0.1773	0.1008	0.0278	-0.0165	-0.0209	-39.5678	25.0127	6.9906
6	1.5392	0.9713	0.3757	1.974	-0.0825	2.422	-0.0506	0.0259	0.0457	0.0979	-0.0839	0.0078	0.2012	0.1819	0.1345	-0.1471	-0.0285	0.0085	-0.0352	0.0326	-0.0969	-15.8364	39.1049	3.5485
7	2.1044	0.5175	0.1835	0.5	0.4647	4.2916	-0.0949	0.4901	0.3976	-0.1235	0.065	0.0993	-0.1185	-0.0713	0.0299	-0.1013	-0.0784	0.0166	-0.0004	0.0018	-0.0197	-32.449	36.5597	-10.3402
8	8.5541	0.3908	11.4827	0.3842	-0.7337	19.7365	0.5056	0.1024	-0.9749	-0.3721	-0.0445	-0.4796	1.6971	1.028	-0.4611	0.3159	0.011	0.0586	0.4235	-0.0574	0.3143	-15.2985	25.8406	-13.1942
9	1.6833	0.8132	-0.3199	0.3939	-0.2497	8.3996	-0.2793	0.3592	-0.3	-0.0888	0.1784	-0.1154	1.1016	0.6044	0.1009	0.1524	-0.0443	0.0931	0.1417	-0.09	0.0167	-26.2058	20.4125	-23.5666
10	3.5974	0.1427	0.3367	10.8568	-4.5337	28.1928	-0.0914	0.1677	-1.2502	-0.0764	-0.3638	0.0501	2.6029	0.8116	0.4552	0.1932	0.0992	0.1405	0.1614	-0.0718	0.2663	-26.2915	2.1567	-3.1619
11	6.2864	-0.1756	1.409	1.1595	0.1794	1.8472	0.0206	0.5403	-0.1809	-0.1275	0.017	0.0232	0.2663	-0.1165	-0.0376	-0.1044	-0.0169	0.022	0.0818	-0.1001	-0.0265	-6.7274	30.0661	-42.1759
12	4.9754	3.2335	2.2191	6.4237	2.8291	4.4729	0.1946	0.0375	-0.4206	0.42	0.006	-0.1148	0.5017	0.3088	-0.2006	-0.2616	0.0964	0.0263	0.0205	0.038	-0.0738	15.8438	36.4153	-26.9981
13	11.7559	6.893	0.8727	17.0031	0.8458	7.0707	0.1115	-0.3488	-0.622	-0.03	-0.2022	-0.1398	1.0652	0.0052	0.0907	0.0458	0.0048	0.0446	0.1628	0.1009	-0.1121	8.747	33.3114	-3.8491
14	2.5665	-0.2985	0.8993	0.5384	0.0329	1.7775	0.0624	0.3274	-0.1049	-0.0311	-0.0699	0.0296	0.2334	-0.0174	0.0074	-0.1448	-0.0426	0.0623	0.0468	0.0126	-0.0176	-1.9736	35.0334	-38.077
15	2.5431	-0.282	-0.3161	2.3497	0.1696	3.1153	0.0492	0.399	0.1414	-0.163	-0.072	-0.0356	-0.0419	0.2533	0.0227	-0.2533	-0.0172	-0.0028	0.1162	0.0167	-0.1034	15.7176	45.9428	-37.2621
16	4.9748	0.2771	0.5998	1.1733	-0.1154	1.1917	0.0148	0.029	0.0864	0.0659	-0.0369	0.1164	0.1229	-0.2019	0.0221	-0.1814	-0.0352	0.0334	0.0706	-0.0279	-0.074	-11.5397	39.6865	-33.2361
17	2.9808	1.0545	0.1109	0.3735	0.0006	3.1829	0.0973	-0.3203	0.5336	0.1479	-0.0555	0.398	-0.1169	-0.2471	-0.0418	-0.1819	0.0317	0.0357	0.0466	-0.0043	0.0139	3.2697	50.741	-20.0174
18	8.1116	5.5771	9.934	4.4776	8.9431	19.1087	0.178	-0.1686	-0.9891	0.4503	0.2738	-0.0546	1.5712	-0.137	-0.4517	0.2668	-0.1259	0.0605	0.3327	0.0943	0.3084	-2.5975	30.7055	-16.3655
19	2.0116	-0.2269	-1.0223	1.0402	0.0645	7.113	0.0495	-0.4296	-0.241	0.0681	-0.133	0.0715	0.7794	0.0762	0.0835	-0.0759	-0.0251	0.0332	0.1364	0.0648	0.0051	8.4387	34.91	-6.3259
20	5.7435	0.7503	-3.6117	12.873	4.0402	24.5029	-0.3321	0.1866	-0.5505	-0.042	-0.2254	-0.3429	1.9652	1.0969	0.5575	0.0827	0.2137	0.0912	0.0533	0.0572	0.1666	21.2738	20.4998	-27.0163

Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Selection: ALL

ID	Refine TLS-ID	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	A	A	12 - 120	32 - 140
2	2	A	A	121 - 138	141 - 158
3	3	A	A	139 - 199	159 - 219
4	4	A	A	200 - 254	220 - 274
5	5	A	A	255 - 336	275 - 356
6	6	A	A	337 - 398	357 - 418
7	7	A	A	399 - 446	419 - 466
8	8	A	A	447 - 473	467 - 493
9	9	A	A	474 - 517	494 - 537
10	10	A	A	518 - 526	538 - 546
11	11	B	B	12 - 75	32 - 95
12	12	B	B	76 - 122	96 - 142
13	13	B	B	123 - 140	143 - 160
14	14	B	B	141 - 254	161 - 274
15	15	B	B	255 - 337	275 - 357
16	16	B	B	338 - 400	358 - 420
17	17	B	B	401 - 446	421 - 466
18	18	B	B	447 - 472	467 - 492
19	19	B	B	473 - 517	493 - 537
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