PDB-8agm: Limonene epoxide low pH soak of epoxide hydrolase from metagenomi...

ID or keywords:

Entry

Database: PDB / ID: 8agm

Title

Limonene epoxide low pH soak of epoxide hydrolase from metagenomic source ch65

Components

Alpha/beta epoxide hydrolase

Keywords

HYDROLASE / Alpha/beta hydrolase fold / complex with substrate / limonene epoxide

Function / homology

Epoxide hydrolase-like / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / Alpha/Beta hydrolase fold / hydrolase activity / D-LIMONENE 1,2-EPOXIDE / Alpha/beta epoxide hydrolase

Function and homology information

Biological species

metagenome (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.966 Å

Authors

Isupov, M.N. / De Rose, S.A. / Mitchell, D. / Littlechild, J.A.

Funding support

European Union, 1items

Citation

Journal: To Be Published
Title: Complexes of epoxide hydrolase from metagenomic source ch65
Authors: Isupov, M.N. / De Rose, S.A. / Mitchell, D. / Littlechild, J.A. / Parker, E. / Ferrandi, E. / Guazzelli, E. / Monti, D.

History

Deposition	Jul 20, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Aug 16, 2023	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	8agm.cif.gz	261.2 KB	Display	PDBx/mmCIF format
PDB format	pdb8agm.ent.gz		Display	PDB format
PDBx/mmJSON format	8agm.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8agm_validation.pdf.gz	2.5 MB	Display	wwPDB validaton report
Full document	8agm_full_validation.pdf.gz	2.5 MB	Display
Data in XML	8agm_validation.xml.gz	44.1 KB	Display
Data in CIF	8agm_validation.cif.gz	61.3 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ag/8agm ftp://data.pdbj.org/pub/pdb/validation_reports/ag/8agm	HTTPS FTP

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Related structure data

Related structure data	8agnC 8agpC 8agsC 5ng7S 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
Related items in Molecule of the Month	#165 - Sep 2013 Designed Protein Cages similarity (3) #220 - Apr 2018 Dehalogenases similarity (3) #216 - Dec 2017 Biodegradable Plastic similarity (1)

Deposited unit

AAA: Alpha/beta epoxide hydrolase

BBB: Alpha/beta epoxide hydrolase

CCC: Alpha/beta epoxide hydrolase

DDD: Alpha/beta epoxide hydrolase

hetero molecules

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

1

AAA: Alpha/beta epoxide hydrolase

BBB: Alpha/beta epoxide hydrolase

hetero molecules

defined by author&software
Evidence: gel filtration
70.7 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

CCC: Alpha/beta epoxide hydrolase

DDD: Alpha/beta epoxide hydrolase

hetero molecules

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

Unit cell

Length a, b, c (Å)	95.985, 47.416, 142.814
Angle α, β, γ (deg.)	90.000, 91.660, 90.000
Int Tables number	4
Space group name H-M	P12₁1

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	AAA
2	1	BBB
3	2	AAA
4	2	CCC
5	3	AAA
6	3	DDD
7	4	BBB
8	4	CCC
9	5	BBB
10	5	DDD
11	6	CCC
12	6	DDD

NCS domain segments:

Dom-ID	Component-ID	Ens-ID	Beg auth comp-ID	Beg label comp-ID	End auth comp-ID	End label comp-ID	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	1	MET	MET	LEU	LEU	AAA	A	4 - 289	4 - 289
2	2	1	MET	MET	LEU	LEU	BBB	B	4 - 289	4 - 289
3	3	2	MET	MET	LEU	LEU	AAA	A	4 - 289	4 - 289
4	4	2	MET	MET	LEU	LEU	CCC	C	4 - 289	4 - 289
5	5	3	MET	MET	LEU	LEU	AAA	A	4 - 289	4 - 289
6	6	3	MET	MET	LEU	LEU	DDD	D	4 - 289	4 - 289
7	7	4	GLU	GLU	LYS	LYS	BBB	B	3 - 290	3 - 290
8	8	4	GLU	GLU	LYS	LYS	CCC	C	3 - 290	3 - 290
9	9	5	GLU	GLU	LYS	LYS	BBB	B	3 - 290	3 - 290
10	10	5	GLU	GLU	LYS	LYS	DDD	D	3 - 290	3 - 290
11	11	6	GLU	GLU	LYS	LYS	CCC	C	3 - 290	3 - 290
12	12	6	GLU	GLU	LYS	LYS	DDD	D	3 - 290	3 - 290

NCS ensembles :

ID	Details
1	Local NCS retraints between domains: 1 2
2	Local NCS retraints between domains: 3 4
3	Local NCS retraints between domains: 5 6
4	Local NCS retraints between domains: 7 8
5	Local NCS retraints between domains: 9 10
6	Local NCS retraints between domains: 11 12

#1: Protein	Alpha/beta epoxide hydrolase Mass: 34940.449 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) metagenome (others) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A1U9WZ52
#2: Chemical	ChemComp-LEO / D-LIMONENE 1,2-EPOXIDE Mass: 152.233 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C₁₀H₁₆O / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Cl
#4: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#5: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 231 / 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: 2.32 Å³/Da / Density % sol: 47.08 %
Crystal grow	Temperature: 291 K / Method: microbatch / pH: 6.5 Details: Morpheus crystal screen condition C3, 40% v/v Glycerol; 20% w/v PEG 4000 . The crystal was soaked for two hours in cryoprotectant containing 1mM mixture of cis (1R,2S,4R) and trans (1S,2R,4R) ...Details: Morpheus crystal screen condition C3, 40% v/v Glycerol; 20% w/v PEG 4000 . The crystal was soaked for two hours in cryoprotectant containing 1mM mixture of cis (1R,2S,4R) and trans (1S,2R,4R) isomers of (+)-limonene oxide at pH 4.5.

Crystal

Density Matthews: 2.32 Å³/Da / Density % sol: 47.08 %

Crystal grow

Temperature: 291 K / Method: microbatch / pH: 6.5
Details: Morpheus crystal screen condition C3, 40% v/v Glycerol; 20% w/v PEG 4000 . The crystal was soaked for two hours in cryoprotectant containing 1mM mixture of cis (1R,2S,4R) and trans (1S,2R,4R) ...

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
Detector	Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 20, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.9795 Å / Relative weight: 1
Reflection	Resolution: 1.966→96.129 Å / Num. obs: 91956 / % possible obs: 99.6 % / Redundancy: 3.4 % / CC_1/2: 0.998 / Net I/σ(I): 7.3
Reflection shell	Resolution: 1.966→2 Å / Num. unique obs: 4525 / CC_1/2: 0.322

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
XDS		data reduction
XSCALE		data scaling
MOLREP		phasing
BUSTER		refinement
Coot		model building
PARROT		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5ng7

5ng7

Resolution: 1.966→96.129 Å / Cor.coef. F_o:F_c: 0.963 / Cor.coef. F_o:F_c free: 0.951 / Cross valid method: FREE R-VALUE / ESU R: 0.217 / ESU R Free: 0.185 / Details: Hydrogens have not been used

	R_factor	Num. reflection	% reflection
R_free	0.2704	4467	4.858 %
R_work	0.2316	87488	-
all	0.233	-	-
obs	-	91955	99.594 %

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL PLUS MASK

Displacement parameters

B_iso mean: 63.288 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-5.799 Å²	-0 Å²	-0.553 Å²
2-	-	8.397 Å²	0 Å²
3-	-	-	-2.561 Å²

Refinement step

Cycle: LAST / Resolution: 1.966→96.129 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	9549	0	100	231	9880

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.012	0.012	10333
X-RAY DIFFRACTION	r_angle_refined_deg	1.635	1.629	14107
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	2.834	5	1254
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	35.337	22.303	547
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	16.069	15	1859
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	22	15	58
X-RAY DIFFRACTION	r_chiral_restr	0.115	0.2	1308
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.02	7800
X-RAY DIFFRACTION	r_nbd_refined	0.177	0.2	4714
X-RAY DIFFRACTION	r_nbtor_refined	0.285	0.2	6712
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.059	0.2	379
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.166	0.2	53
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.065	0.2	19
X-RAY DIFFRACTION	r_mcbond_it	3.455	15.883	4752
X-RAY DIFFRACTION	r_mcangle_it	4.467	26.595	5962
X-RAY DIFFRACTION	r_scbond_it	6.749	17.432	5581
X-RAY DIFFRACTION	r_scangle_it	8.892	28.406	8070
X-RAY DIFFRACTION	r_lrange_it	10.003	88.229	15644
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.096	0.05	9696
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.09	0.05	9791
X-RAY DIFFRACTION	r_ncsr_local_group_3	0.085	0.05	9812
X-RAY DIFFRACTION	r_ncsr_local_group_4	0.093	0.05	9813
X-RAY DIFFRACTION	r_ncsr_local_group_5	0.089	0.05	9814
X-RAY DIFFRACTION	r_ncsr_local_group_6	0.09	0.05	9957

Refine LS restraints NCS

Ens-ID	Dom-ID	Auth asym-ID	Refine-ID	Type	Rms dev position (Å)	Weight position
1	1	AAA	X-RAY DIFFRACTION	Local ncs	0.09604	0.05008
1	2	BBB	X-RAY DIFFRACTION	Local ncs	0.09604	0.05008
2	3	AAA	X-RAY DIFFRACTION	Local ncs	0.09045	0.05008
2	4	CCC	X-RAY DIFFRACTION	Local ncs	0.09045	0.05008
3	5	AAA	X-RAY DIFFRACTION	Local ncs	0.08475	0.05008
3	6	DDD	X-RAY DIFFRACTION	Local ncs	0.08475	0.05008
4	7	BBB	X-RAY DIFFRACTION	Local ncs	0.09331	0.05008
4	8	CCC	X-RAY DIFFRACTION	Local ncs	0.09331	0.05008
5	9	BBB	X-RAY DIFFRACTION	Local ncs	0.08947	0.05008
5	10	DDD	X-RAY DIFFRACTION	Local ncs	0.08947	0.05008
6	11	CCC	X-RAY DIFFRACTION	Local ncs	0.08969	0.05008
6	12	DDD	X-RAY DIFFRACTION	Local ncs	0.08969	0.05008

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 (%)
1.966-2.017	0.47	361	0.454	6345	X-RAY DIFFRACTION	99.304
2.017-2.072	0.462	332	0.423	6295	X-RAY DIFFRACTION	99.7892
2.072-2.132	0.414	299	0.395	6079	X-RAY DIFFRACTION	99.7498
2.132-2.198	0.372	315	0.373	5891	X-RAY DIFFRACTION	99.6948
2.198-2.27	0.379	271	0.345	5783	X-RAY DIFFRACTION	99.8186
2.27-2.35	0.366	289	0.33	5568	X-RAY DIFFRACTION	99.8977
2.35-2.438	0.312	269	0.306	5326	X-RAY DIFFRACTION	99.5375
2.438-2.538	0.353	267	0.285	5175	X-RAY DIFFRACTION	99.7069
2.538-2.651	0.332	272	0.265	4977	X-RAY DIFFRACTION	99.6015
2.651-2.78	0.306	244	0.257	4738	X-RAY DIFFRACTION	99.6998
2.78-2.93	0.327	216	0.252	4514	X-RAY DIFFRACTION	99.7259
2.93-3.108	0.308	208	0.249	4286	X-RAY DIFFRACTION	99.6673
3.108-3.323	0.281	186	0.235	4055	X-RAY DIFFRACTION	99.6008
3.323-3.589	0.271	218	0.216	3726	X-RAY DIFFRACTION	99.3701
3.589-3.931	0.23	170	0.201	3474	X-RAY DIFFRACTION	99.3999
3.931-4.394	0.204	138	0.174	3158	X-RAY DIFFRACTION	99.2771
4.394-5.074	0.214	157	0.169	2760	X-RAY DIFFRACTION	99.2177
5.074-6.212	0.203	111	0.176	2382	X-RAY DIFFRACTION	99.4812
6.212-8.777	0.23	104	0.173	1856	X-RAY DIFFRACTION	99.2405
8.777-96.129	0.146	40	0.191	1100	X-RAY DIFFRACTION	99.1304

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