PDB-8c0i: Structure of E. coli Class 2 L-asparaginase EcAIII, mutant M200L ...

ID or keywords:

Entry

Database: PDB / ID: 8c0i

Title

Structure of E. coli Class 2 L-asparaginase EcAIII, mutant M200L (acyl-enzyme intermediate)

Components

(Isoaspartyl peptidase subunit ...) x 3

Keywords

HYDROLASE / L-asparaginase / Ntn-hydrolase / mutation / acyl-enzyme intermediate

Function / homology

beta-aspartyl-peptidase / Peptidase T2, asparaginase 2 / Asparaginase / asparaginase activity / beta-aspartyl-peptidase activity / protein autoprocessing / Nucleophile aminohydrolases, N-terminal / hydrolase activity / Isoaspartyl peptidase

Function and homology information

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

MOLECULAR REPLACEMENT / Resolution: 1.9 Å

Authors

Sciuk, A. / Jaskolski, M. / Loch, J.I.

Funding support

Poland, 1items

Citation

Journal: Protein Sci. / Year: 2023
Title: The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.
Authors: Janicki, M. / Sciuk, A. / Zielezinski, A. / Ruszkowski, M. / Ludwikow, A. / Karlowski, W.M. / Jaskolski, M. / Loch, J.I.

History

Deposition	Dec 16, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 3, 2023	Provider: repository / Type: Initial release
Revision 1.1	Jun 7, 2023	Group: Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation_author.identifier_ORCID

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Summary document	8c0i_validation.pdf.gz	460.7 KB	Display	wwPDB validaton report
Full document	8c0i_full_validation.pdf.gz	466.5 KB	Display
Data in XML	8c0i_validation.xml.gz	24.1 KB	Display
Data in CIF	8c0i_validation.cif.gz	34 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/c0/8c0i ftp://data.pdbj.org/pub/pdb/validation_reports/c0/8c0i	HTTPS FTP

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Related structure data

Related structure data	8bi3C 8bkfC 8bp9C 8bqoC 8c23C 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	#166 - Oct 2013 Proteasome similarity (1) #80 - Aug 2006 AAA+ Proteases similarity (1) #176 - Aug 2014 Dynein similarity (1) #60 - Dec 2004 Ubiquitin similarity (1)

Deposited unit

AAA: Isoaspartyl peptidase subunit alpha

BBB: Isoaspartyl peptidase subunit beta

CCC: Isoaspartyl peptidase subunit alpha

DDD: Isoaspartyl peptidase subunit beta

hetero molecules

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

1

Idetical with deposited unit
defined by author
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	49.710, 74.851, 146.595
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	AAA
2	1	CCC
3	2	BBB
4	2	DDD

NCS domain segments:

Dom-ID	Component-ID	Ens-ID	Auth asym-ID	Auth seq-ID
1	1	1	AAA	4 - 157
2	2	1	CCC	4 - 157
3	3	2	BBB	179 - 313
4	4	2	DDD	179 - 313

NCS ensembles :

ID	Details
1	Local NCS retraints between domains: 1 2
2	Local NCS retraints between domains: 3 4

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Isoaspartyl peptidase subunit ... , 3 types, 4 molecules AAACCCBBBDDD

#1: Protein	Isoaspartyl peptidase subunit alpha Mass: 19013.773 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold / References: UniProt: P37595
#2: Protein	Isoaspartyl peptidase subunit beta Mass: 14410.107 Da / Num. of mol.: 1 / Mutation: M200L Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pEt11d / Production host: Escherichia coli (E. coli) / Strain (production host): BLG21 GOLD / References: UniProt: P37595
#3: Protein	Isoaspartyl peptidase subunit beta Mass: 14525.197 Da / Num. of mol.: 1 / Mutation: M200L Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pEt11d / Production host: Escherichia coli (E. coli) / Strain (production host): BLG21 GOLD / References: UniProt: P37595

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Non-polymers , 4 types, 190 molecules

#4: Chemical	ChemComp-NA / SODIUM ION Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#5: Chemical	ChemComp-MG / MAGNESIUM ION Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H₂O

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Details

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: 2.04 Å³/Da / Density % sol: 39.6 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 20 PEG4000, 10% PEG400, 0.2 M MgCl2 in 100 mM Tris-HCl pH 8.5

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SEALED TUBE / Type: RIGAKU PhotonJet-S / Wavelength: 1.54184 Å
Detector	Type: RIGAKU HyPix-3000 / Detector: PIXEL / Date: Oct 31, 2022
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.54184 Å / Relative weight: 1
Reflection	Resolution: 1.9→19.3 Å / Num. obs: 42775 / % possible obs: 97.6 % / Redundancy: 3.1 % / CC_1/2: 0.949 / R_merge(I) obs: 0.148 / R_rim(I) all: 0.177 / Net I/σ(I): 18
Reflection shell	Resolution: 1.9→1.9 Å / R_merge(I) obs: 0.283 / Mean I/σ(I) obs: 3.2 / Num. unique obs: 2856 / CC_1/2: 0.865 / R_rim(I) all: 0.344 / % possible all: 97.8

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
CrysalisPro		data reduction
Aimless		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.9→19.3 Å / Cor.coef. F_o:F_c: 0.913 / Cor.coef. F_o:F_c free: 0.87 / SU B: 10.656 / SU ML: 0.153 / Cross valid method: FREE R-VALUE / ESU R: 0.193 / ESU R Free: 0.174

	R_factor	Num. reflection	% reflection
R_free	0.2752	1022	2.391 %
R_work	0.2319	41713	-
all	0.233	-	-
obs	-	42735	97.114 %

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: 21.496 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-1.906 Å²	-0 Å²	-0 Å²
2-	-	-0.017 Å²	0 Å²
3-	-	-	1.924 Å²

Refinement step

Cycle: LAST / Resolution: 1.9→19.3 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4214	0	5	185	4404

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.01	0.013	4304
X-RAY DIFFRACTION	r_bond_other_d	0.003	0.015	4132
X-RAY DIFFRACTION	r_angle_refined_deg	1.553	1.633	5838
X-RAY DIFFRACTION	r_angle_other_deg	1.38	1.575	9482
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.492	5	583
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	30.11	22.1	200
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.531	15	685
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	20.23	15	29
X-RAY DIFFRACTION	r_chiral_restr	0.071	0.2	573
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	5002
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	937
X-RAY DIFFRACTION	r_nbd_refined	0.201	0.2	994
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.194	0.2	4425
X-RAY DIFFRACTION	r_nbtor_refined	0.159	0.2	2114
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.085	0.2	2097
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.164	0.2	214
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.054	0.2	3
X-RAY DIFFRACTION	r_metal_ion_refined	0.221	0.2	7
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.196	0.2	15
X-RAY DIFFRACTION	r_nbd_other	0.216	0.2	52
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.21	0.2	10
X-RAY DIFFRACTION	r_mcbond_it	0.979	1.832	2323
X-RAY DIFFRACTION	r_mcbond_other	0.978	1.831	2322
X-RAY DIFFRACTION	r_mcangle_it	1.617	2.737	2900
X-RAY DIFFRACTION	r_mcangle_other	1.617	2.738	2901
X-RAY DIFFRACTION	r_scbond_it	0.938	2.042	1981
X-RAY DIFFRACTION	r_scbond_other	0.937	2.039	1979
X-RAY DIFFRACTION	r_scangle_it	1.55	3	2935
X-RAY DIFFRACTION	r_scangle_other	1.55	3	2935
X-RAY DIFFRACTION	r_lrange_it	3.221	22.672	4807
X-RAY DIFFRACTION	r_lrange_other	3.14	22.639	4788
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.099	0.05	4365
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.098	0.05	3828

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	0.09871	0.05007
1	2	CCC	X-RAY DIFFRACTION	Local ncs	0.09871	0.05007
2	3	BBB	X-RAY DIFFRACTION	Local ncs	0.09777	0.05009
2	4	DDD	X-RAY DIFFRACTION	Local ncs	0.09777	0.05009

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 (%)
1.9-1.949	0.363	79	0.318	3022	X-RAY DIFFRACTION	97.6078
1.949-2.002	0.378	71	0.275	3031	X-RAY DIFFRACTION	99.7428
2.002-2.059	0.299	81	0.26	2916	X-RAY DIFFRACTION	99.8667
2.059-2.122	0.319	62	0.268	2833	X-RAY DIFFRACTION	99.553
2.122-2.19	0.339	78	0.268	2785	X-RAY DIFFRACTION	99.7909
2.19-2.266	0.28	69	0.242	2682	X-RAY DIFFRACTION	99.4577
2.266-2.351	0.319	63	0.24	2583	X-RAY DIFFRACTION	99.4363
2.351-2.445	0.308	65	0.254	2461	X-RAY DIFFRACTION	99.2534
2.445-2.552	0.289	43	0.25	2415	X-RAY DIFFRACTION	99.2329
2.552-2.674	0.271	59	0.254	2300	X-RAY DIFFRACTION	99.2845
2.674-2.816	0.269	70	0.252	2160	X-RAY DIFFRACTION	98.7162
2.816-2.983	0.335	45	0.239	2043	X-RAY DIFFRACTION	97.4335
2.983-3.183	0.299	34	0.241	1931	X-RAY DIFFRACTION	97.2291
3.183-3.43	0.258	43	0.234	1787	X-RAY DIFFRACTION	96.063
3.43-3.746	0.237	38	0.214	1608	X-RAY DIFFRACTION	93.1522
3.746-4.169	0.278	29	0.191	1419	X-RAY DIFFRACTION	90.3306
4.169-4.776	0.173	27	0.167	1249	X-RAY DIFFRACTION	89.1684
4.776-5.762	0.242	28	0.182	1055	X-RAY DIFFRACTION	87.2683
5.762-7.809	0.198	28	0.175	860	X-RAY DIFFRACTION	88.0079
7.809-19.3	0.096	10	0.173	573	X-RAY DIFFRACTION	85.8616

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	0.2386	-0.0585	0.1789	0.3205	0.0437	0.2093	-0.0221	0.045	-0.0435	-0.032	0.025	-0.0283	-0.0188	0.0516	-0.0029	0.0207	-0.0129	0.007	0.0177	-0.0027	0.0521	-4.1655	-6.1518	27.9168
2	0.0977	-0.0476	-0.0732	0.2993	0.1108	0.2807	-0.0101	0.009	-0.0347	-0.0099	-0.0069	0.0382	-0.0746	-0.0207	0.017	0.0391	0.0013	-0.011	0.0028	0.0045	0.0586	-17.8423	-0.0987	31.9038
3	0.3547	0.2084	-0.2249	0.1871	-0.2226	0.5329	-0.0184	-0.0721	0.021	0.0194	-0.0439	-0.0305	-0.028	0.0881	0.0623	0.0426	0.0012	-0.0168	0.0294	0.0023	0.0324	-1.5711	4.2158	60.0689
4	0.1246	-0.0462	0.2289	0.4372	-0.0448	0.4858	-0.0058	-0.0563	-0.0031	-0.0026	0.0129	0.0153	0.043	-0.0879	-0.0071	0.0501	0.0178	0.0133	0.0309	0.0055	0.0168	-16.4263	-0.1393	57.8879

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	AAA	2 - 157
2	X-RAY DIFFRACTION	2	ALL	BBB	179 - 313
3	X-RAY DIFFRACTION	3	ALL	CCC	4 - 158
4	X-RAY DIFFRACTION	4	ALL	DDD	179 - 313

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Isoaspartyl peptidase subunit ... , 3 types, 4 molecules AAACCCBBBDDD

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Non-polymers , 4 types, 190 molecules

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Isoaspartyl peptidase subunit ... , 3 types, 4 molecules AAACCCBBBDDD

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