PDB-7ux8: Crystal structure of MfnG, an L- and D-tyrosine O-methyltransfera...

ID or keywords:

Entry

Database: PDB / ID: 7ux8

Title

Crystal structure of MfnG, an L- and D-tyrosine O-methyltransferase from the marformycin biosynthesis pathway of Streptomyces drozdowiczii, with SAH and L-Tyrosine bound at 1.4 A resolution (P212121 - form II)

Components

MfnG

Keywords

TRANSFERASE / O-methyltransferase / O-methyl-tyrosine / Marformycin synthesis / SAM-dependent methyltransferase

Function / homology

Function and homology information

O-methyltransferase activity / methylation / protein dimerization activity
Similarity search - Function

Biological species

Streptomyces drozdowiczii (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

FOURIER SYNTHESIS / Resolution: 1.4 Å

Authors

Miller, M.D. / Wu, K.-L. / Xu, W. / Xiao, H. / Philips Jr., G.N.

Funding support

United States, 9items

Organization	Grant number	Country
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	R01-GM115261	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01-CA217255	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	R35-GM133706	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R21-CA255894	United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	R01-AI165079	United States
Cancer Prevention and Research Institute of Texas (CPRIT)	RR170014	United States
Robert A. Welch Foundation	C-1970	United States
Department of Defense (DOD, United States)	W81XWH-21-1-0789	United States
National Science Foundation (NSF, United States)	STC 1231306	United States

Citation

Journal: Protein Sci. / Year: 2022
Title: Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.
Authors: Wu, K.L. / Moore, J.A. / Miller, M.D. / Chen, Y. / Lee, C. / Xu, W. / Peng, Z. / Duan, Q. / Phillips Jr., G.N. / Uribe, R.A. / Xiao, H.

History

Deposition	May 5, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Sep 28, 2022	Provider: repository / Type: Initial release
Revision 1.1	Oct 12, 2022	Group: Database references / Category: citation / citation_author Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2	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	7ux8.cif.gz	470.1 KB	Display	PDBx/mmCIF format
PDB format	pdb7ux8.ent.gz	394.2 KB	Display	PDB format
PDBx/mmJSON format	7ux8.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	7ux8_validation.pdf.gz	972.4 KB	Display	wwPDB validaton report
Full document	7ux8_full_validation.pdf.gz	979.1 KB	Display
Data in XML	7ux8_validation.xml.gz	36.1 KB	Display
Data in CIF	7ux8_validation.cif.gz	55.9 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ux/7ux8 ftp://data.pdbj.org/pub/pdb/validation_reports/ux/7ux8	HTTPS FTP

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Related structure data

Related structure data	7ux6C 7ux7SC C: citing same article (ref.) S: Starting model for refinement
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	#22 - Oct 2001 Photosystem I similarity (1) #137 - May 2011 Cytochrome bc1 similarity (1) #191 - Nov 2015 Glutamate-gated Chloride Receptors similarity (1) #142 - Oct 2011 PDB Pioneers similarity (1)

Deposited unit

A: MfnG

B: MfnG

hetero molecules

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

1

Idetical with deposited unit
defined by author&software
Evidence: gel filtration, Size Exclusion Chromatography (SEC) supports the assignment of a dimer as the assembly in solution

Unit cell

Length a, b, c (Å)	60.837, 87.167, 134.910
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein	MfnG Mass: 42042.246 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptomyces drozdowiczii (bacteria) / Plasmid: pET22b-T5-MfnG-TEV-His / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0D4WTP2
#2: Chemical	ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₁₄H₂₀N₆O₅S
#3: Chemical	ChemComp-TYR / TYROSINE Type: L-peptide linking / Mass: 181.189 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₉H₁₁NO₃
#4: Chemical	ChemComp-UNL / UNKNOWN LIGAND Num. of mol.: 2 / Source method: obtained synthetically
#5: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 798 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y
Sequence details	The construct was cloned into the EcoRI and HindIII sites of pET22b with an N-terminal Met (0) ...The construct was cloned into the EcoRI and HindIII sites of pET22b with an N-terminal Met (0) added and the native GUG-start codon being expressed as V. A C-terminal expression and 6-His tag ASENLYFQ/GGGHHHHHHG leaving a C-terminal ASENLYFQ after removal of the 6-His tag with TEV protease

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.13 Å³/Da / Density % sol: 42.17 %
Crystal grow	Temperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8 Details: 0.1 M Tris pH 8, 30% Polyethylene glycol monomethyl ether (PEG MME) 2000, Additive: 0.002 M S-Adenosyl methionine (SAM/AdoMet), Soaking: added crystaline L-Tyrosine to the drop after MfnG crystals were formed

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Data collection

Diffraction

Mean temperature: 100 K
Crystal treatment: crystaline L-Tyrosine was added to the drop after the MfnG crystals were formed. After 46 hours, the MfnG crystals were harvested and flash cooled by immersion in liquid nitrogen
Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

APS

/ Beamline: 21-ID-D / Wavelength: 0.9762 Å

Detector

Type: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Mar 5, 2021

Radiation

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

Radiation wavelength

Wavelength: 0.9762 Å / Relative weight: 1

Reflection

Resolution: 1.4→31.3 Å / Num. obs: 141301 / % possible obs: 99.7 % / Redundancy: 13.109 % / B_iso Wilson estimate: 18 Å² / CC_1/2: 0.999 / R_merge(I) obs: 0.11 / R_rim(I) all: 0.114 / Χ²: 0.828 / Net I/σ(I): 15.37

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. measured obs	Num. possible	Num. unique obs	CC_1/2	R_rim(I) all	% possible all
1.4-1.48	10.496	1.903	0.94	235008	22706	22390	0.456	2	98.6
1.48-1.59	13.942	1.062	2.43	297124	21316	21311	0.845	1.102	100
1.59-1.71	13.866	0.624	4.5	275552	19876	19872	0.943	0.647	100
1.71-1.88	13.509	0.351	8.04	247409	18323	18314	0.982	0.364	100
1.88-2.1	12.933	0.181	14.79	215813	16694	16687	0.994	0.189	100
2.1-2.42	14.189	0.112	25.41	209341	14756	14754	0.997	0.116	100
2.42-2.96	13.818	0.078	35.21	172768	12503	12503	0.998	0.081	100
2.96-4.19	12.386	0.055	47.47	121496	9817	9809	0.999	0.057	99.9
4.19-31.3	13.755	0.048	59.2	77867	5668	5661	0.999	0.049	99.9

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Processing

Software

Name	Version	Classification
PHENIX	1.20.1	refinement
XDS	Feb 5, 2021	data reduction
XDS	Feb 5, 2021	data scaling
PDB_EXTRACT	3.27	data extraction
PHENIX	1.19	phasing

Refinement

Method to determine structure:

FOURIER SYNTHESIS
Starting model: 7UX7

7ux7

Resolution: 1.4→31.3 Å / SU ML: 0.19 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 17.1 / Stereochemistry target values: ML
Details: 1. HYDROGENS HAVE BEEN INCLUDED AT THEIR RIDING POSITIONS USING THE ELECTRON CLOUD DISTANCES. 2. THE STRUCTURE WAS REFINED USING REFERENCE MODEL RESTRAINTS FROM THE HIGH RESOLUTION STRUCTURE ...

	R_factor	Num. reflection	% reflection
R_free	0.172	7030	4.98 %
R_work	0.153	-	-
obs	0.154	141291	99.9 %

Solvent computation

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

Displacement parameters

B_iso mean: 26.08 Å²

Refinement step

Cycle: LAST / Resolution: 1.4→31.3 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5690	0	96	798	6584

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	6538
X-RAY DIFFRACTION	f_angle_d	1.004	8968
X-RAY DIFFRACTION	f_dihedral_angle_d	13.826	2400
X-RAY DIFFRACTION	f_chiral_restr	0.079	965
X-RAY DIFFRACTION	f_plane_restr	0.012	1238

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 (%)
1.4-1.42	0.4445	236	0.406	4357	X-RAY DIFFRACTION	98
1.42-1.43	0.3457	231	0.3549	4335	X-RAY DIFFRACTION	99
1.43-1.45	0.3394	241	0.3138	4439	X-RAY DIFFRACTION	100
1.45-1.47	0.2812	227	0.2666	4408	X-RAY DIFFRACTION	100
1.47-1.49	0.2652	240	0.2464	4448	X-RAY DIFFRACTION	100
1.49-1.51	0.2595	227	0.2302	4427	X-RAY DIFFRACTION	100
1.51-1.53	0.2571	247	0.2264	4427	X-RAY DIFFRACTION	100
1.53-1.55	0.2333	223	0.2289	4453	X-RAY DIFFRACTION	100
1.55-1.58	0.2135	239	0.2133	4433	X-RAY DIFFRACTION	100
1.58-1.6	0.2291	245	0.1927	4413	X-RAY DIFFRACTION	100
1.6-1.63	0.1784	238	0.1742	4446	X-RAY DIFFRACTION	100
1.63-1.66	0.1884	235	0.1621	4448	X-RAY DIFFRACTION	100
1.66-1.69	0.1689	252	0.1562	4448	X-RAY DIFFRACTION	100
1.69-1.73	0.1755	229	0.1486	4440	X-RAY DIFFRACTION	100
1.73-1.76	0.1832	212	0.1469	4481	X-RAY DIFFRACTION	100
1.76-1.8	0.1636	240	0.1397	4467	X-RAY DIFFRACTION	100
1.8-1.85	0.1766	241	0.1427	4452	X-RAY DIFFRACTION	100
1.85-1.9	0.1787	240	0.1454	4460	X-RAY DIFFRACTION	100
1.9-1.96	0.1833	241	0.1554	4450	X-RAY DIFFRACTION	100
1.96-2.02	0.1847	228	0.1472	4477	X-RAY DIFFRACTION	100
2.02-2.09	0.1634	207	0.1385	4503	X-RAY DIFFRACTION	100
2.09-2.17	0.1413	241	0.1331	4513	X-RAY DIFFRACTION	100
2.17-2.27	0.1636	246	0.1287	4448	X-RAY DIFFRACTION	100
2.27-2.39	0.1525	230	0.1307	4487	X-RAY DIFFRACTION	100
2.39-2.54	0.1539	249	0.1291	4524	X-RAY DIFFRACTION	100
2.54-2.74	0.1495	236	0.1377	4502	X-RAY DIFFRACTION	100
2.74-3.02	0.1538	226	0.1386	4573	X-RAY DIFFRACTION	100
3.02-3.45	0.1619	241	0.1426	4539	X-RAY DIFFRACTION	100
3.45-4.35	0.1458	215	0.1327	4661	X-RAY DIFFRACTION	100
4.35-31.3	0.166	227	0.1613	4802	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	2.0899	0.6805	-2.4069	1.6083	-1.6828	4.9168	-0.0299	0.0925	-0.075	0.0705	0.067	0.0469	0.143	-0.2724	0.0087	0.1273	-0.0288	-0.0101	0.1409	-0.0123	0.1369	30.656	30.7753	47.1295
2	3.6375	0.5345	-1.4311	1.7971	-0.0345	3.2446	0.033	-0.0958	-0.0419	-0.0597	0.037	-0.2412	0.1028	0.2348	-0.1038	0.1118	0.016	0.0106	0.1586	-0.01	0.1731	51.3162	31.4602	32.7137
3	0.7678	0.7169	0.1057	1.3247	0.307	1.8292	0.0504	0.1227	-0.1284	-0.0081	0.0263	-0.162	0.0831	0.1776	-0.0024	0.146	0.0022	0.018	0.1748	0.0064	0.1938	42.646	30.9253	25.1211
4	1.6587	1.4245	-1.1396	2.5061	0.8338	3.3439	0.0522	0.1784	0.1893	-0.1457	-0.1139	0.2032	-0.4703	-0.2474	0.1146	0.2666	0.0446	0.004	0.1848	0.0212	0.2392	20.1707	49.3528	31.2524
5	1.9121	0.6132	0.7354	2.4807	-0.0438	1.9284	0.0637	0.1478	-0.1166	-0.1902	-0.0157	0.0149	0.0449	0.0534	-0.0681	0.1595	0.0323	0.0221	0.1619	-0.0175	0.1372	22.0496	26.9384	11.2371
6	4.3749	0.491	0.1501	3.7735	0.3028	2.5528	0.0362	0.2475	-0.0676	-0.202	-0.021	-0.1544	0.0304	0.2074	-0.0201	0.1866	0.0226	0.0357	0.1879	0.01	0.1069	26.954	30.3059	5.7908
7	1.8097	-0.3148	-0.0256	1.062	0.193	1.2864	0.0502	0.1481	0.1347	-0.1535	-0.0241	-0.0092	-0.0986	-0.0419	-0.0334	0.1765	0.015	0.0058	0.1439	0.0194	0.1375	14.7388	36.6552	8.4956
8	1.0095	-0.2017	0.4974	0.3234	0.0072	1.2813	0.018	-0.0425	-0.0734	-0.0354	0.0116	0.0594	0.0393	-0.1267	-0.0321	0.1409	-0.0037	0.0006	0.1541	0.0143	0.1499	11.1962	29.5791	23.1985
9	6.1428	-2.1075	0.2161	4.2747	-1.9027	2.0144	0.1933	0.0482	-0.4191	0.1107	-0.2942	-0.0401	0.0593	0.1313	0.0732	0.2595	0.0001	0.0344	0.2743	0.0505	0.227	-11.1714	36.8804	13.3251
10	1.6942	-0.4324	1.9117	0.5493	-0.6371	2.8827	-0.1277	-0.3228	-0.0304	0.3946	0.1314	-0.0459	-0.256	-0.1179	-0.0267	0.2263	0.0137	0.0033	0.1976	0.0185	0.1952	23.378	38.7866	19.0305
11	1.2319	-1.1128	-1.0738	4.3477	3.1464	3.1788	-0.0331	-0.0395	-0.0965	0.0112	0.1205	-0.1166	0.1589	0.1838	-0.0653	0.1325	0.0207	-0.0147	0.1449	0.0207	0.1415	29.1843	28.5636	31.5008
12	2.1978	-0.3449	0.8608	1.8445	-0.3093	1.9563	0.0478	-0.2327	0.0608	0.0409	-0.0356	0.1499	-0.0999	-0.294	-0.0031	0.1241	0.0085	0.0138	0.2206	-0.0137	0.1475	11.0618	40.1618	43.1373
13	1.3479	-0.417	0.9771	0.709	0.1915	1.1244	0.0093	-0.0683	-0.0908	0.0593	0.0159	0.0186	0.0169	-0.2743	-0.0195	0.1197	0.0006	0.0144	0.1744	-0.0103	0.1196	19.7985	39.5482	51.0673
14	0.5021	-0.4699	-0.165	1.5535	0.1809	3.6461	0.0172	-0.0861	0.0314	-0.1366	0.1062	-0.2994	-0.356	0.3441	-0.0282	0.2298	-0.0578	0.0543	0.1948	-0.0246	0.2807	46.3622	46.4398	41.0846
15	1.6351	0.1256	0.3159	1.2813	0.5499	2.0041	-0.0074	-0.1447	-0.0907	0.2329	0.0925	-0.051	0.2842	0.0747	-0.0354	0.1712	0.0321	-0.0117	0.128	-0.0096	0.1114	37.6992	33.0854	66.1423
16	3.3958	0.3694	-0.1425	3.0008	-0.4769	3.2436	-0.0371	-0.1356	0.1308	0.1839	0.0321	0.0358	-0.0388	-0.0742	-0.0137	0.1555	0.0196	-0.013	0.1245	-0.0382	0.0924	34.3177	40.2816	70.3232
17	1.2631	-0.3023	-0.7069	1.0266	0.0772	1.7897	-0.012	-0.1145	0.2263	0.1352	0.0892	-0.2172	-0.086	0.1634	-0.0842	0.1651	0.0063	-0.0441	0.1683	-0.0423	0.2132	48.641	41.5569	66.9427
18	0.9971	-0.1892	0.28	0.4795	-0.172	1.7979	-0.0534	-0.0289	-0.0494	0.0767	0.0576	-0.1601	0.1716	0.2052	-0.0161	0.1661	0.0343	-0.0123	0.1374	-0.0156	0.1672	48.5763	28.5234	55.6579
19	1.6204	0.1681	1.9248	0.4627	0.3728	4.0645	-0.0248	0.1973	0.0897	-0.2855	-0.0118	-0.0236	-0.015	0.1711	0.0247	0.2128	-0.0066	-0.0158	0.1917	-0.0054	0.2203	40.2419	41.9043	55.7268

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	CHAIN 'A' AND (RESID 6 THROUGH 50 )
2	X-RAY DIFFRACTION	2	CHAIN 'A' AND (RESID 51 THROUGH 95 )
3	X-RAY DIFFRACTION	3	CHAIN 'A' AND (RESID 96 THROUGH 129 )
4	X-RAY DIFFRACTION	4	CHAIN 'A' AND (RESID 130 THROUGH 158 )
5	X-RAY DIFFRACTION	5	CHAIN 'A' AND (RESID 159 THROUGH 205 )
6	X-RAY DIFFRACTION	6	CHAIN 'A' AND (RESID 206 THROUGH 239 )
7	X-RAY DIFFRACTION	7	CHAIN 'A' AND (RESID 240 THROUGH 288 )
8	X-RAY DIFFRACTION	8	CHAIN 'A' AND (RESID 289 THROUGH 365 )
9	X-RAY DIFFRACTION	9	CHAIN 'A' AND (RESID 366 THROUGH 383 )
10	X-RAY DIFFRACTION	10	CHAIN 'A' AND (RESID 401 THROUGH 403 )
11	X-RAY DIFFRACTION	11	CHAIN 'B' AND (RESID 6 THROUGH 50 )
12	X-RAY DIFFRACTION	12	CHAIN 'B' AND (RESID 51 THROUGH 95 )
13	X-RAY DIFFRACTION	13	CHAIN 'B' AND (RESID 96 THROUGH 129 )
14	X-RAY DIFFRACTION	14	CHAIN 'B' AND (RESID 130 THROUGH 158 )
15	X-RAY DIFFRACTION	15	CHAIN 'B' AND (RESID 159 THROUGH 205 )
16	X-RAY DIFFRACTION	16	CHAIN 'B' AND (RESID 206 THROUGH 239 )
17	X-RAY DIFFRACTION	17	CHAIN 'B' AND (RESID 240 THROUGH 288 )
18	X-RAY DIFFRACTION	18	CHAIN 'B' AND (RESID 289 THROUGH 366 )
19	X-RAY DIFFRACTION	19	CHAIN 'B' AND (RESID 401 THROUGH 403 )

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Structure viewers

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Open data

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Validation report

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Links

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Assembly

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Experimental details

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Experiment

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Sample preparation

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Data collection

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Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

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Yorodumi