PDB-8sp2: Crystal structure of metformin hydrolase (MfmAB) from Pseudomonas...

ID or keywords:

Entry

Database: PDB / ID: 8sp2

Title

Crystal structure of metformin hydrolase (MfmAB) from Pseudomonas mendocina sp. MET-2 apo form

Components

metformin hydrolase subunit A
metformin hydrolase subunit B

Keywords

HYDROLASE / ureohydrolase / metformin / metalloenzyme / guanylurea

Function / homology

NICKEL (II) ION

Function and homology information

Biological species

Pseudomonas mendocina (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.2 Å

Authors

Tassoulas, L.J. / Rankin, J.A. / Elias, M.H. / Wackett, L.P.

Funding support

United States, 1items

Citation

Journal: Proc.Natl.Acad.Sci.USA / Year: 2024
Title: Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes.
Authors: Tassoulas, L.J. / Rankin, J.A. / Elias, M.H. / Wackett, L.P.

History

Deposition	May 1, 2023	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Mar 6, 2024	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8sp2.cif.gz	1.9 MB	Display	PDBx/mmCIF format
PDB format	pdb8sp2.ent.gz	1.2 MB	Display	PDB format
PDBx/mmJSON format	8sp2.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8sp2_validation.pdf.gz	4 MB	Display	wwPDB validaton report
Full document	8sp2_full_validation.pdf.gz	4 MB	Display
Data in XML	8sp2_validation.xml.gz	143.2 KB	Display
Data in CIF	8sp2_validation.cif.gz	199.2 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/sp/8sp2 ftp://data.pdbj.org/pub/pdb/validation_reports/sp/8sp2	HTTPS FTP

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Related structure data

Related structure data	8snfC 8snkC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: metformin hydrolase subunit B

B: metformin hydrolase subunit B

C: metformin hydrolase subunit B

D: metformin hydrolase subunit B

E: metformin hydrolase subunit B

F: metformin hydrolase subunit B

G: metformin hydrolase subunit A

H: metformin hydrolase subunit A

I: metformin hydrolase subunit A

J: metformin hydrolase subunit A

K: metformin hydrolase subunit B

L: metformin hydrolase subunit B

hetero molecules

468 kDa, 12 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: metformin hydrolase subunit B

E: metformin hydrolase subunit B

I: metformin hydrolase subunit A

hetero molecules

defined by author&software
Evidence: gel filtration
117 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

J: metformin hydrolase subunit A

hetero molecules

B: metformin hydrolase subunit B

F: metformin hydrolase subunit B

defined by author&software
Evidence: gel filtration
117 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

3

C: metformin hydrolase subunit B

G: metformin hydrolase subunit A

K: metformin hydrolase subunit B

hetero molecules

defined by author&software
Evidence: gel filtration
117 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

4

H: metformin hydrolase subunit A

L: metformin hydrolase subunit B

hetero molecules

D: metformin hydrolase subunit B

defined by author&software
Evidence: gel filtration
117 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	103.530, 107.880, 114.510
Angle α, β, γ (deg.)	93.429, 97.625, 98.105
Int Tables number	1
Space group name H-M	P1

#1: Protein	metformin hydrolase subunit B Mass: 38024.281 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas mendocina (bacteria) / Strain: MET-2 / Gene: mfmB / Plasmid: pET28 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein	metformin hydrolase subunit A Mass: 40723.984 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas mendocina (bacteria) / Strain: MET-2 / Gene: mfmA / Plasmid: pET28 / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical	ChemComp-NI / NICKEL (II) ION Mass: 58.693 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 910 / 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: 2.68 Å³/Da / Density % sol: 54.07 %
Crystal grow	Temperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5 Details: 1 uL 10 mg/mL protein + 1 uL 13.5% w/v PEG3350, 0.1 M Bis-Tris propane, 0.2 M NaNO3, pH 7.5

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

APS

/ Beamline: 24-ID-E / Wavelength: 0.97918 Å

Detector

Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 28, 2022

Radiation

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

Radiation wavelength

Wavelength: 0.97918 Å / Relative weight: 1

Reflection

Resolution: 2.2→80 Å / Num. obs: 224389 / % possible obs: 91.2 % / Redundancy: 2.0558 % / CC_1/2: 0.999 / Net I/σ(I): 12.75

Reflection shell

Resolution (Å)	Mean I/σ(I) obs	Num. unique obs	CC_1/2	Diffraction-ID	% possible all
2.2-2.3	1.59	27288	0.745	1	89
2.3-2.4	2.2	24279	0.845	1	93.9

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0411	refinement
XDS	VERSION Jan 10, 2022 BUILT=20220820	data reduction
XSCALE	VERSION Jan 10, 2022 BUILT=20220820	data scaling
MOLREP	Version 11.0	phasing
Coot	0.9.8.1	model building

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 2.2→74.608 Å / Cor.coef. F_o:F_c: 0.962 / Cor.coef. F_o:F_c free: 0.94 / WR_factor R_free: 0.257 / WR_factor R_work: 0.212 / SU B: 15.769 / SU ML: 0.184 / Average fsc free: 0.9456 / Average fsc work: 0.9605 / Cross valid method: THROUGHOUT / ESU R: 0.301 / ESU R Free: 0.23
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.257	11220	5 %
R_work	0.2125	213168	-
all	0.215	-	-
obs	-	224388	91.205 %

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: 50.927 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.408 Å²	-0.565 Å²	-0.543 Å²
2-	-	0.2 Å²	0.612 Å²
3-	-	-	-0.402 Å²

Refinement step

Cycle: LAST / Resolution: 2.2→74.608 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	31104	0	8	910	32022

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.005	0.012	31808
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.016	29660
X-RAY DIFFRACTION	r_angle_refined_deg	1.314	1.645	43052
X-RAY DIFFRACTION	r_angle_other_deg	0.457	1.573	68308
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.219	5	4012
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	7.954	5	252
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	17.624	10	5200
X-RAY DIFFRACTION	r_dihedral_angle_6_deg	15.9	10	1460
X-RAY DIFFRACTION	r_chiral_restr	0.068	0.2	4620
X-RAY DIFFRACTION	r_gen_planes_refined	0.005	0.02	37936
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	7424
X-RAY DIFFRACTION	r_nbd_refined	0.217	0.2	7534
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.206	0.2	28776
X-RAY DIFFRACTION	r_nbtor_refined	0.181	0.2	15672
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.08	0.2	16771
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.175	0.2	1323
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.085	0.2	6
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.177	0.2	88
X-RAY DIFFRACTION	r_nbd_other	0.22	0.2	416
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.142	0.2	70
X-RAY DIFFRACTION	r_mcbond_it	2.77	4.02	16108
X-RAY DIFFRACTION	r_mcbond_other	2.77	4.02	16108
X-RAY DIFFRACTION	r_mcangle_it	4.253	7.211	20100
X-RAY DIFFRACTION	r_mcangle_other	4.253	7.211	20101
X-RAY DIFFRACTION	r_scbond_it	2.878	4.322	15700
X-RAY DIFFRACTION	r_scbond_other	2.878	4.322	15699
X-RAY DIFFRACTION	r_scangle_it	4.558	7.825	22952
X-RAY DIFFRACTION	r_scangle_other	4.558	7.825	22953
X-RAY DIFFRACTION	r_lrange_it	7.761	50.007	136889
X-RAY DIFFRACTION	r_lrange_other	7.752	49.889	136475

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	R_factor all	Num. reflection all	Fsc free	Fsc work	% reflection obs (%)	WR_factor R_work
2.2-2.257	0.394	777	0.375	14769	0.376	18157	0.878	0.89	85.6199	0.375
2.257-2.319	0.365	833	0.33	15822	0.332	17738	0.911	0.924	93.8945	0.33
2.319-2.386	0.333	813	0.304	15451	0.306	17294	0.922	0.937	94.0442	0.304
2.386-2.459	0.321	784	0.283	14891	0.284	16748	0.928	0.948	93.5933	0.283
2.459-2.54	0.288	756	0.255	14369	0.257	16201	0.944	0.96	93.3584	0.255
2.54-2.629	0.3	731	0.249	13888	0.251	15714	0.947	0.964	93.0317	0.249
2.629-2.728	0.285	701	0.23	13307	0.232	15137	0.953	0.97	92.5415	0.23
2.728-2.839	0.261	669	0.211	12714	0.213	14586	0.958	0.975	91.7524	0.211
2.839-2.965	0.285	633	0.215	12027	0.218	13951	0.951	0.974	90.7462	0.215
2.965-3.11	0.275	594	0.223	11287	0.226	13365	0.955	0.972	88.8964	0.223
3.11-3.278	0.273	552	0.223	10482	0.225	12754	0.957	0.973	86.514	0.223
3.278-3.476	0.264	546	0.219	10389	0.221	12030	0.961	0.975	90.8978	0.219
3.476-3.716	0.252	509	0.206	9665	0.208	11290	0.964	0.978	90.1152	0.206
3.716-4.013	0.23	476	0.189	9038	0.191	10524	0.971	0.982	90.4029	0.189
4.013-4.394	0.211	424	0.167	8063	0.169	9648	0.975	0.985	87.9664	0.167
4.394-4.911	0.198	393	0.158	7460	0.16	8785	0.979	0.987	89.391	0.158
4.911-5.666	0.202	350	0.173	6645	0.175	7707	0.98	0.987	90.7616	0.173
5.666-6.93	0.215	298	0.183	5664	0.185	6535	0.976	0.984	91.2318	0.183
6.93-9.758	0.24	245	0.177	4653	0.18	5042	0.971	0.985	97.144	0.177
9.758-74.608	0.269	136	0.214	2585	0.217	2815	0.962	0.974	96.6607	0.214

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	0.2458	0.0795	0.2817	0.1584	-0.0026	0.607	-0.0284	-0.1062	0.038	-0.0093	-0.0933	0.0446	-0.0342	-0.0869	0.1217	0.0724	0.0386	0.0365	0.1015	-0.0467	0.1273	-0.0556	-0.8803	0.4537
2	0.257	0.0913	0.3086	0.1332	0.0426	0.5169	-0.0096	-0.0977	0.0683	-0.0085	-0.0945	0.0066	0.01	-0.1164	0.1041	0.0713	0.0257	0.035	0.1136	-0.0446	0.1209	-36.4919	-49.2707	-56.4615
3	0.2309	0.3302	0.1389	0.5486	0.1287	0.1947	0.0339	0.0581	-0.1218	0.038	0.0462	-0.1241	0.0293	0.0547	-0.0801	0.073	0.0638	0.0255	0.0943	-0.0304	0.1642	5.5627	-40.0991	-40.6393
4	0.2226	0.2043	0.0572	0.3867	-0.135	0.3442	0.0273	0.0329	-0.0816	-0.0629	0.008	-0.0656	0.1218	0.1193	-0.0353	0.1247	0.09	0.0302	0.1258	-0.0163	0.1008	-31.5987	-89.4691	-96.7463
5	0.1779	-0.1974	0.0561	0.449	-0.036	0.4064	0.0931	0.0293	-0.1028	0.0455	0.011	0.0499	0.037	0.0882	-0.104	0.1417	0.053	-0.0715	0.0665	0.0118	0.1586	25.739	-29.7007	-2.587
6	0.2599	-0.2481	0.0712	0.4164	-0.1216	0.5368	0.1304	0.062	-0.1218	0.0443	-0.0256	0.0896	0.0707	0.0969	-0.1048	0.2209	0.0666	-0.104	0.0416	-0.0242	0.1538	-11.5627	-78.9835	-58.6784
7	0.1413	0.1124	0.1337	0.0968	0.1434	0.3463	-0.1034	-0.039	0.0669	-0.0704	-0.0358	0.0751	-0.0315	-0.0196	0.1391	0.0983	0.048	-0.0133	0.0689	-0.0231	0.1625	-16.6596	-9.8025	-37.7188
8	0.1599	0.1114	0.1344	0.0863	0.1119	0.3443	-0.1074	-0.0183	0.066	-0.0692	-0.0181	0.0769	-0.0379	-0.0098	0.1255	0.0965	0.0468	-0.0165	0.0905	-0.0023	0.1413	20.3386	39.3391	18.445
9	0.2109	0.1259	0.1541	0.1691	0.166	0.2037	-0.1244	0.0488	-0.0127	-0.0537	0.0967	0.0386	-0.0921	0.146	0.0277	0.0926	-0.0441	0.0357	0.1589	0.0327	0.082	21.4142	-1.9793	-30.4437
10	0.1359	0.1253	0.1624	0.2328	0.187	0.2445	-0.0932	0.0879	-0.0089	-0.0329	0.0899	0.0222	-0.0818	0.183	0.0032	0.0917	-0.0263	0.0417	0.2085	0.0321	0.0686	-45.0071	46.0561	26.0437
11	0.334	0.007	-0.2827	0.4073	0.0184	0.433	0.1147	-0.1457	-0.0102	0.0613	-0.1673	-0.0037	0.0858	0.0278	0.0526	0.2089	-0.1488	0.0354	0.1806	-0.0064	0.0126	-19.6849	-38.8362	-11.087
12	0.1764	0.063	-0.1474	0.2581	-0.107	0.3951	0.0865	-0.1141	-0.0011	0.0082	-0.1361	0.0129	0.0678	0.0149	0.0496	0.1261	-0.0786	0.0447	0.1853	0.0063	0.0414	16.1673	10.8634	45.5797

Refinement TLS group

Selection: ALL

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