PDB-7qtc: Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-3 ...

ID or keywords:

Entry

Database: PDB / ID: 7qtc

Title

Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-3 (G206H, R207T, D210P, S211Q)

Components

Isoaspartyl peptidase
Isoaspartyl peptidase subunit beta

Keywords

HYDROLASE / L-asparaginase / Ntn-hydrolase / EcAIII

Function / homology

Function and homology information

beta-aspartyl-peptidase / asparaginase activity / beta-aspartyl-peptidase activity / protein autoprocessing / hydrolase activity / cytoplasm
Similarity search - Function

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

MOLECULAR REPLACEMENT / Resolution: 2.55 Å

Authors

Loch, J.I. / Kadziolka, K. / Jaskolski, M.

Funding support

Poland, 1items

Citation

Journal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase.
Authors: Loch, J.I. / Klonecka, A. / Kadziolka, K. / Bonarek, P. / Barciszewski, J. / Imiolczyk, B. / Brzezinski, K. / Gilski, M. / Jaskolski, M.

History

Deposition	Jan 14, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Jul 13, 2022	Provider: repository / Type: Initial release
Revision 1.1	Jan 31, 2024	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7qtc.cif.gz	203.2 KB	Display	PDBx/mmCIF format
PDB format	pdb7qtc.ent.gz		Display	PDB format
PDBx/mmJSON format	7qtc.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	7qtc_validation.pdf.gz	408.3 KB	Display	wwPDB validaton report
Full document	7qtc_full_validation.pdf.gz	412.4 KB	Display
Data in XML	7qtc_validation.xml.gz	12.5 KB	Display
Data in CIF	7qtc_validation.cif.gz	19.1 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/qt/7qtc ftp://data.pdbj.org/pub/pdb/validation_reports/qt/7qtc	HTTPS FTP

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Related structure data

Related structure data	7qq8C 7qsfC 7qvrC 7qy6C 7qymC 7qyxC 7r1gC 7r5cC 2zalS 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
Experimental dataset #1	Data reference: 10.18150/R8VJ7V / Data set type: diffraction image data

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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

BBB: Isoaspartyl peptidase subunit beta

CCC: Isoaspartyl peptidase

DDD: Isoaspartyl peptidase subunit beta

hetero molecules

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

1

Idetical with deposited unit
defined by author&software
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	51.353, 75.039, 149.032
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	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	ALA	ALA	PHE	PHE	AAA	A	4 - 144	4 - 144
2	2	1	ALA	ALA	PHE	PHE	CCC	C	4 - 144	4 - 144
3	3	2	THR	THR	GLU	GLU	BBB	B	179 - 313	1 - 135
4	4	2	THR	THR	GLU	GLU	DDD	D	179 - 313	1 - 135

NCS ensembles :

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

#1: Protein	Isoaspartyl peptidase / Beta-aspartyl-peptidase / EcAIII / Isoaspartyl dipeptidase 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 BL21 (bacteria) / Variant (production host): Gold / References: UniProt: P37595, beta-aspartyl-peptidase
#2: Protein	Isoaspartyl peptidase subunit beta Mass: 14476.230 Da / Num. of mol.: 2 / Mutation: G206H, R207T, D210P, S211Q 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 BL21 (bacteria) / Variant (production host): Gold / References: UniProt: P37595
#3: Chemical	ChemComp-NA / SODIUM ION Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 142 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	N

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.14 Å³/Da / Density % sol: 42.62 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 20% PEG 6000, 0.2 M MgCl2 in 0.1M 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: Agilent SuperNova / Wavelength: 1.54056 Å
Detector	Type: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: Jul 14, 2020
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.54056 Å / Relative weight: 1
Reflection	Resolution: 2.55→20.36 Å / Num. obs: 17974 / % possible obs: 92.7 % / Redundancy: 2.8 % / CC_1/2: 0.983 / R_merge(I) obs: 0.119 / R_rim(I) all: 0.143 / Net I/σ(I): 7.2
Reflection shell	Resolution: 2.55→2.66 Å / Redundancy: 2.5 % / R_merge(I) obs: 0.676 / Mean I/σ(I) obs: 2.1 / Num. unique obs: 2215 / CC_1/2: 0.604 / R_rim(I) all: 0.83 / % possible all: 95.3

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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
Starting model: 2ZAL

2zal

Resolution: 2.55→20.358 Å / Cor.coef. F_o:F_c: 0.934 / Cor.coef. F_o:F_c free: 0.893 / SU B: 23.841 / SU ML: 0.268 / Cross valid method: FREE R-VALUE / ESU R: 2.323 / ESU R Free: 0.33

	R_factor	Num. reflection	% reflection
R_free	0.2483	1010	5.632 %
R_work	0.2023	16922	-
all	0.205	-	-
obs	-	17932	92.044 %

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: 33.407 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.204 Å²	0 Å²	0 Å²
2-	-	1.543 Å²	-0 Å²
3-	-	-	-1.747 Å²

Refinement step

Cycle: LAST / Resolution: 2.55→20.358 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4022	0	2	142	4166

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.009	0.013	4091
X-RAY DIFFRACTION	r_bond_other_d	0.007	0.015	3906
X-RAY DIFFRACTION	r_angle_refined_deg	1.585	1.629	5551
X-RAY DIFFRACTION	r_angle_other_deg	1.39	1.57	8968
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.093	5	554
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	30.155	22.581	186
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	15.067	15	639
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	17.894	15	24
X-RAY DIFFRACTION	r_chiral_restr	0.065	0.2	547
X-RAY DIFFRACTION	r_gen_planes_refined	0.006	0.02	4754
X-RAY DIFFRACTION	r_gen_planes_other	0.003	0.02	878
X-RAY DIFFRACTION	r_nbd_refined	0.205	0.2	902
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.197	0.2	3954
X-RAY DIFFRACTION	r_nbtor_refined	0.157	0.2	1969
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.08	0.2	1913
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.152	0.2	138
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.044	0.2	2
X-RAY DIFFRACTION	r_metal_ion_refined	0.187	0.2	5
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.325	0.2	20
X-RAY DIFFRACTION	r_nbd_other	0.326	0.2	48
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.399	0.2	2
X-RAY DIFFRACTION	r_mcbond_it	1.468	2.364	2219
X-RAY DIFFRACTION	r_mcbond_other	1.467	2.362	2218
X-RAY DIFFRACTION	r_mcangle_it	2.611	3.536	2766
X-RAY DIFFRACTION	r_mcangle_other	2.61	3.538	2767
X-RAY DIFFRACTION	r_scbond_it	1.365	2.643	1872
X-RAY DIFFRACTION	r_scbond_other	1.363	2.64	1870
X-RAY DIFFRACTION	r_scangle_it	2.374	3.866	2783
X-RAY DIFFRACTION	r_scangle_other	2.373	3.866	2783
X-RAY DIFFRACTION	r_lrange_it	5.949	43.913	16300
X-RAY DIFFRACTION	r_lrange_other	5.925	43.944	16259
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.133	0.05	3932
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.126	0.05	3526

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	0.13294	0.05007
1	2	CCC	X-RAY DIFFRACTION	Local ncs	0.13294	0.05007
2	3	BBB	X-RAY DIFFRACTION	Local ncs	0.12641	0.05007
2	4	DDD	X-RAY DIFFRACTION	Local ncs	0.12641	0.05007

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 (%)
2.55-2.615	0.351	75	0.319	1239	X-RAY DIFFRACTION	94.7368
2.615-2.686	0.277	70	0.291	1239	X-RAY DIFFRACTION	95.2
2.686-2.762	0.355	50	0.284	1186	X-RAY DIFFRACTION	94.3511
2.762-2.845	0.43	59	0.273	1139	X-RAY DIFFRACTION	92.9403
2.845-2.937	0.31	58	0.258	1113	X-RAY DIFFRACTION	94.0562
2.937-3.038	0.311	68	0.253	1060	X-RAY DIFFRACTION	92.7632
3.038-3.15	0.273	68	0.231	1015	X-RAY DIFFRACTION	93.2014
3.15-3.275	0.283	62	0.218	978	X-RAY DIFFRACTION	91.954
3.275-3.417	0.243	67	0.209	918	X-RAY DIFFRACTION	90.0366
3.417-3.578	0.264	52	0.189	911	X-RAY DIFFRACTION	93.2236
3.578-3.765	0.259	49	0.222	821	X-RAY DIFFRACTION	87.0871
3.765-3.985	0.244	60	0.179	810	X-RAY DIFFRACTION	90.8142
3.985-4.249	0.204	47	0.149	761	X-RAY DIFFRACTION	91.61
4.249-4.573	0.148	44	0.127	712	X-RAY DIFFRACTION	90
4.573-4.984	0.157	39	0.131	676	X-RAY DIFFRACTION	91.0828
4.984-5.531	0.22	51	0.151	600	X-RAY DIFFRACTION	90.795
5.531-6.31	0.207	35	0.174	567	X-RAY DIFFRACTION	92.4731
6.31-7.549	0.244	22	0.158	499	X-RAY DIFFRACTION	91.2434
7.549-10.01	0.192	18	0.131	392	X-RAY DIFFRACTION	89.9123
10.01-20.358	0.21	16	0.212	286	X-RAY DIFFRACTION	92.9231

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	0.6493	-0.3369	-0.346	0.4862	-0.2436	0.9241	0.03	0.0494	0.1526	-0.064	0.028	-0.0133	0.1304	-0.0617	-0.058	0.0478	0.0044	0.0466	0.1515	0.043	0.1354	4.4	7.62	27.961
2	0.4083	-0.1925	0.1931	0.7424	-0.4947	0.8887	0.0382	-0.079	0.0803	-0.0797	-0.1379	-0.0454	0.1969	0.1531	0.0997	0.0468	0.033	0.026	0.1751	0.0288	0.0373	16.953	-0.166	31.206
3	0.7725	0.4208	0.5289	0.3903	0.1483	1.4806	-0.1892	-0.0973	-0.0101	-0.0447	-0.0111	0.051	-0.0135	-0.0613	0.2003	0.1003	0.0734	0.059	0.1509	-0.0035	0.1014	3.373	-5.65	61.099
4	0.777	0.1321	-0.0103	0.0797	0.1847	0.8989	-0.2174	-0.0058	0.0113	-0.0387	0.0236	0.0459	-0.0689	0.2323	0.1938	0.0928	0.0144	-0.0306	0.2198	0.0081	0.0584	17.381	-0.195	57.634

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	AAA	2 - 145
2	X-RAY DIFFRACTION	2	ALL	BBB	179 - 313
3	X-RAY DIFFRACTION	3	ALL	CCC	4 - 145
4	X-RAY DIFFRACTION	4	ALL	DDD	179 - 313

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