PDB-7ux6: Crystal structure of MfnG, an L- and D-tyrosine O-methyltransfera...

ID or keywords:

Entry

Database: PDB / ID: 7ux6

Title

Crystal structure of MfnG, an L- and D-tyrosine O-methyltransferase from the marformycin biosynthesis pathway of Streptomyces drozdowiczii, with SAH bound at 1.35 A resolution (P212121 - form I)

Components

MfnG

Keywords

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

Function / homology

Function and homology information

methyltransferase activity
Similarity search - Function

Biological species

Streptomyces drozdowiczii (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT /

molecular replacement / Resolution: 1.35 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	Oct 12, 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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PDBx/mmCIF format	7ux6.cif.gz	426.1 KB	Display	PDBx/mmCIF format
PDB format	pdb7ux6.ent.gz	353.6 KB	Display	PDB format
PDBx/mmJSON format	7ux6.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	7ux6_validation.pdf.gz	965.1 KB	Display	wwPDB validaton report
Full document	7ux6_full_validation.pdf.gz	970 KB	Display
Data in XML	7ux6_validation.xml.gz	31.3 KB	Display
Data in CIF	7ux6_validation.cif.gz	46.7 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ux/7ux6 ftp://data.pdbj.org/pub/pdb/validation_reports/ux/7ux6	HTTPS FTP

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Related structure data

Related structure data	7ux7C 7ux8C 2r3sS 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	#137 - May 2011 Cytochrome bc1 similarity (1) #191 - Nov 2015 Glutamate-gated Chloride Receptors similarity (1)

Deposited unit

A: MfnG

B: MfnG

hetero molecules

84.9 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)	64.059, 78.229, 131.680
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID
1	1
2	1

NCS domain segments:

Dom-ID	Component-ID	Ens-ID	Selection details
1	1	1	(CHAIN A AND (RESID 8 THROUGH 61 OR RESID 63...
2	1	1	(CHAIN B AND (RESID 8 THROUGH 61 OR RESID 63...

#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-UNL / UNKNOWN LIGAND Num. of mol.: 2 / Source method: obtained synthetically
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 551 / 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: 1.96 Å³/Da / Density % sol: 37.31 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, sitting drop Details: 0.2 M ammonium formate, 20% (w/v) PEG 3350, Additive: 0.002 M S-Adenosyl methionine (SAM/AdoMet)

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Data collection

Diffraction

Mean temperature: 100 K
Crystal treatment: 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: Feb 10, 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: 1.311→67.256 Å / Num. obs: 101542 / % possible obs: 94.2 % / Redundancy: 12.43 % / Details: Staraniso processed data / CC_1/2: 0.999 / R_merge(I) obs: 0.0506 / R_pim(I) all: 0.0146 / R_rim(I) all: 0.0527 / Net I/σ(I): 19.4 / Num. measured all: 1262670

Reflection shell

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. measured all	Num. measured obs	Num. unique all	Num. unique obs	CC_1/2	R_pim(I) all	R_rim(I) all	% possible all
1.864-1.926	13.85	0.1614	10.52	70294	70294	5074	5074	0.997	0.0447	0.1675	99.2
1.804-1.864	14.11	0.1905	9.31	71645	71645	5078	5078	0.995	0.0524	0.1976	95.4
1.747-1.804	14.29	0.2155	8.43	72569	72569	5079	5079	0.995	0.0588	0.2234	95.3
1.692-1.747	14.46	0.2589	7.29	73393	73393	5076	5076	0.994	0.0703	0.2684	95.3
1.641-1.692	14.63	0.3105	6.33	74314	74314	5078	5078	0.992	0.0838	0.3217	95.8
1.592-1.641	14.3	0.359	5.52	72575	72575	5075	5075	0.988	0.0978	0.3722	97.2
1.542-1.592	10.13	0.4009	4.15	51444	51444	5077	5077	0.974	0.1297	0.4222	94.6
1.49-1.542	7.77	0.4573	3.16	39480	39480	5081	5081	0.951	0.1702	0.4898	90.2
1.429-1.49	6.4	0.5608	2.37	32472	32472	5072	5072	0.905	0.2332	0.6102	78.6
1.311-1.429	5.2	0.7081	1.71	26440	26440	5080	5080	0.806	0.3382	0.7878	62.5

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Phasing

Phasing	Method: molecular replacement

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Processing

Software

Name	Version	Classification
PHENIX	1.19.2	refinement
autoPROC	1.0.5 20201211	data processing
XDS	Jan 31, 2020	data reduction
Aimless	0.7.4	data scaling
STARANISO	2.3.54	data scaling
PHASER	2.8.3	phasing
PDB_EXTRACT	3.27	data extraction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 2r3s

2r3s

Resolution: 1.35→33.63 Å / SU ML: 0.12 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 29.91 / Stereochemistry target values: ML
Details: 1. HYDROGENS HAVE BEEN INCLUDED AT THEIR RIDING POSITIONS USING THE ELECTRON CLOUD DISTANCES. 2. THE STRUCTURE CONTAINS AND UNKNOWN LIGAND UNL BOUND IN THE ACTIVE SITE. THE COMPOUND IS A ...

	R_factor	Num. reflection	% reflection
R_free	0.19	4933	4.91 %
R_work	0.166	-	-
obs	0.167	100565	69.1 %

Solvent computation

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

Displacement parameters

B_iso mean: 26.47 Å²

Refinement step

Cycle: LAST / Resolution: 1.35→33.63 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5458	0	70	551	6079

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	5899
X-RAY DIFFRACTION	f_angle_d	0.801	8062
X-RAY DIFFRACTION	f_dihedral_angle_d	12.17	2136
X-RAY DIFFRACTION	f_chiral_restr	0.066	886
X-RAY DIFFRACTION	f_plane_restr	0.008	1097

Refine LS restraints NCS

Ens-ID	Dom-ID	Auth asym-ID	Number	Refine-ID	Type
1	1	A	3130	X-RAY DIFFRACTION	POSITIONAL
1	2	B	3130	X-RAY DIFFRACTION	POSITIONAL

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 (%)
1.35-1.37	0.303	30	0.2759	550	X-RAY DIFFRACTION	12
1.37-1.38	0.2575	35	0.2652	675	X-RAY DIFFRACTION	15
1.38-1.4	0.3109	43	0.269	822	X-RAY DIFFRACTION	18
1.4-1.42	0.3673	53	0.2769	1013	X-RAY DIFFRACTION	22
1.42-1.43	0.3015	63	0.2863	1231	X-RAY DIFFRACTION	27
1.43-1.45	0.2868	77	0.2654	1449	X-RAY DIFFRACTION	32
1.45-1.48	0.2798	69	0.2591	1665	X-RAY DIFFRACTION	36
1.48-1.5	0.2456	99	0.2481	1960	X-RAY DIFFRACTION	43
1.5-1.52	0.3255	109	0.2589	2186	X-RAY DIFFRACTION	48
1.52-1.55	0.2422	112	0.2438	2411	X-RAY DIFFRACTION	52
1.55-1.57	0.2565	148	0.2242	2558	X-RAY DIFFRACTION	56
1.57-1.6	0.2234	167	0.2161	2813	X-RAY DIFFRACTION	61
1.6-1.63	0.2423	164	0.1995	3012	X-RAY DIFFRACTION	66
1.63-1.66	0.2351	173	0.2047	3162	X-RAY DIFFRACTION	69
1.66-1.7	0.2271	148	0.1982	3323	X-RAY DIFFRACTION	72
1.7-1.74	0.2333	177	0.1937	3536	X-RAY DIFFRACTION	77
1.74-1.78	0.2035	187	0.1984	3750	X-RAY DIFFRACTION	81
1.78-1.83	0.2163	215	0.1854	3961	X-RAY DIFFRACTION	86
1.83-1.89	0.2335	224	0.1946	4223	X-RAY DIFFRACTION	92
1.89-1.95	0.2398	241	0.1866	4598	X-RAY DIFFRACTION	100
1.95-2.02	0.2148	248	0.1659	4585	X-RAY DIFFRACTION	100
2.02-2.1	0.1835	247	0.1548	4589	X-RAY DIFFRACTION	100
2.1-2.19	0.164	232	0.1439	4619	X-RAY DIFFRACTION	100
2.19-2.31	0.1918	239	0.14	4620	X-RAY DIFFRACTION	100
2.31-2.45	0.182	265	0.1488	4611	X-RAY DIFFRACTION	100
2.45-2.64	0.1761	251	0.1469	4636	X-RAY DIFFRACTION	100
2.64-2.91	0.1678	223	0.1545	4687	X-RAY DIFFRACTION	100
2.91-3.33	0.171	240	0.1505	4699	X-RAY DIFFRACTION	100
3.33-4.19	0.1576	219	0.1417	4750	X-RAY DIFFRACTION	100
4.19-33.63	0.1798	235	0.1753	4938	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	2.6081	0.2984	-0.2108	0.5409	-0.1288	1.1053	0.0118	-0.0854	0.1728	0.0352	-0.0166	0.0351	-0.0607	-0.0981	-0.0366	0.113	0.0322	0.0017	0.0873	0.0124	0.0773	21.2083	26.7144	43.895
2	1.3462	0.0512	0.1684	0.4176	-0.1479	1.3995	0.0003	0.0534	-0.1469	-0.1074	0.0352	0.028	0.0772	-0.0084	-0.015	0.1375	-0.0006	-0.0009	0.073	-0.0051	0.1091	16.8556	19.8074	22.9653
3	2.4261	0.1449	-0.024	0.8831	-0.1598	2.4828	-0.0384	0.2991	-0.1307	-0.0445	-0.0299	0.0522	0.0691	-0.3522	-0.0441	0.0887	0.009	-0.0031	0.2099	-0.0065	0.0901	2.3212	22.9633	9.7186
4	1.459	-0.0696	0.1462	0.5674	0.2499	1.9706	-0.0273	0.4019	0.0417	-0.0628	-0.0059	-0.0567	-0.1691	0.2295	0.0493	0.1423	-0.0152	0.0051	0.2237	0.0503	0.1325	20.5718	26.9869	5.9192
5	1.5686	-0.5186	-0.3433	4.2928	1.6269	1.9051	0.1228	0.1057	-0.2125	0.0245	-0.1836	-0.1174	0.1501	-0.2125	0.0506	0.1919	0.0374	0.0084	0.2014	-0.0296	0.1698	13.0967	16.4989	12.3512
6	1.7756	0.1532	0.1713	0.3411	0.0313	1.1929	-0.0155	0.2487	0.1716	-0.0512	0.0193	-0.0685	-0.097	0.2759	-0.0652	0.1168	-0.0092	0.0069	0.1626	0.0244	0.082	34.1206	26.7258	21.954
7	1.8784	-0.0097	0.3987	0.5176	0.3243	1.0018	0.0382	0.1557	-0.1587	0.0746	0.062	-0.0263	0.0693	0.0665	-0.0072	0.1448	0.0369	-0.0012	0.07	0.0159	0.1063	38.5211	19.459	43.3288
8	2.5145	-0.1831	0.051	1.3924	-0.0741	2.6156	-0.041	-0.0784	0.0599	-0.0081	-0.0052	-0.0405	-0.0625	0.3263	-0.0303	0.0752	0.0287	-0.003	0.0705	0.0025	0.094	52.9785	22.4285	56.1978
9	1.9467	-0.1099	0.1981	0.6372	-0.2257	1.5364	-0.0302	-0.2262	0.0634	0.0503	0.0291	0.0266	-0.0961	-0.1507	0.0011	0.1244	0.0273	0.0123	0.0844	-0.0155	0.1041	34.8204	26.9823	60.0818
10	1.3954	-0.0504	0.3044	2.0002	-5.363	3.7661	0.0781	0.138	-0.133	-0.164	0.1497	0.2058	0.079	-0.0524	-0.2322	0.1665	0.0135	0.0066	0.0748	0.0015	0.1554	42.2906	16.4866	53.4931

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	CHAIN 'A' AND (RESID 8 THROUGH 104 )
2	X-RAY DIFFRACTION	2	CHAIN 'A' AND (RESID 105 THROUGH 186 )
3	X-RAY DIFFRACTION	3	CHAIN 'A' AND (RESID 187 THROUGH 254 )
4	X-RAY DIFFRACTION	4	CHAIN 'A' AND (RESID 255 THROUGH 366 )
5	X-RAY DIFFRACTION	5	CHAIN 'A' AND (RESID 401 THROUGH 401 )
6	X-RAY DIFFRACTION	6	CHAIN 'B' AND (RESID 8 THROUGH 104 )
7	X-RAY DIFFRACTION	7	CHAIN 'B' AND (RESID 105 THROUGH 186 )
8	X-RAY DIFFRACTION	8	CHAIN 'B' AND (RESID 187 THROUGH 254 )
9	X-RAY DIFFRACTION	9	CHAIN 'B' AND (RESID 255 THROUGH 366 )
10	X-RAY DIFFRACTION	10	CHAIN 'B' AND (RESID 401 THROUGH 401 )

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