PDB-7r5c: Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-29...

ID or keywords:

Entry

Database: PDB / ID: 7r5c

Title

Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-29 (G206C, R207S, D210L, S211V)

Components

(Isoaspartyl peptidase) x 2

Keywords

HYDROLASE / L-asparaginase / mutation

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: 2.2 Å

Authors

Barciszewski, J. / Imiolczyk, B. / Loch, J.I. / 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	Feb 10, 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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Download

PDBx/mmCIF format	7r5c.cif.gz	207.1 KB	Display	PDBx/mmCIF format
PDB format	pdb7r5c.ent.gz		Display	PDB format
PDBx/mmJSON format	7r5c.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	7r5c_validation.pdf.gz	469.6 KB	Display	wwPDB validaton report
Full document	7r5c_full_validation.pdf.gz	473 KB	Display
Data in XML	7r5c_validation.xml.gz	22.3 KB	Display
Data in CIF	7r5c_validation.cif.gz	31 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/r5/7r5c ftp://data.pdbj.org/pub/pdb/validation_reports/r5/7r5c	HTTPS FTP

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Related structure data

Related structure data	7qq8C 7qsfC 7qtcC 7qvrC 7qy6C 7qymC 7qyxC 7r1gC 2zalS 2rig 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/WEFSC9 / Data set type: diffraction image data / Metadata reference: 10.18150/WEFSC9

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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

CCC: Isoaspartyl peptidase

DDD: Isoaspartyl peptidase

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)	51.789, 77.864, 148.363
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	ARG	ARG	AAA	A	4 - 157	4 - 157
2	2	1	ALA	ALA	ARG	ARG	CCC	C	4 - 157	4 - 157
3	3	2	THR	THR	TYR	TYR	BBB	B	179 - 311	1 - 133
4	4	2	THR	THR	TYR	TYR	DDD	D	179 - 311	1 - 133

NCS ensembles :

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

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Protein , 2 types, 4 molecules AAACCCBBBDDD

#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 (E. coli) / References: UniProt: P37595, beta-aspartyl-peptidase
#2: Protein	Isoaspartyl peptidase / Beta-aspartyl-peptidase / EcAIII / Isoaspartyl dipeptidase Mass: 14414.244 Da / Num. of mol.: 2 / Mutation: G206C, R207S, D210L, S211V Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / References: UniProt: P37595, beta-aspartyl-peptidase

#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 (E. coli) / References: UniProt: P37595, beta-aspartyl-peptidase

#2: Protein

Isoaspartyl peptidase / Beta-aspartyl-peptidase / EcAIII / Isoaspartyl dipeptidase

Mass: 14414.244 Da / Num. of mol.: 2 / Mutation: G206C, R207S, D210L, S211V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Escherichia coli (E. coli) / Strain: K12 / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host:

Escherichia coli (E. coli) / References: UniProt: P37595, beta-aspartyl-peptidase

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Non-polymers , 4 types, 111 molecules

#3: Chemical	ChemComp-NA / SODIUM ION Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Chemical	ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₄H₁₂NO₃ / Comment: pH buffer*YM
#5: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#6: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 107 / Source method: isolated from a natural source / Formula: H₂O

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Details

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: 2.24 Å³/Da / Density % sol: 45.02 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7 / Details: 0.1 M PCTP buffer pH 7.0, 25 % w/v PEG 1500

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: ROTATING ANODE / Type: RIGAKU / Wavelength: 1.54178 Å
Detector	Type: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: Jan 13, 2022
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.54178 Å / Relative weight: 1
Reflection	Resolution: 2.2→21.48 Å / Num. obs: 30380 / % possible obs: 97.6 % / Redundancy: 4.5 % / CC_1/2: 0.992 / R_merge(I) obs: 0.124 / R_rim(I) all: 0.14 / Net I/σ(I): 6.4
Reflection shell	Resolution: 2.2→2.27 Å / R_merge(I) obs: 0.496 / Mean I/σ(I) obs: 1.9 / Num. unique obs: 2652 / CC_1/2: 0.902 / R_rim(I) all: 0.555 / % possible all: 99.2

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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: 2.2→21.48 Å / Cor.coef. F_o:F_c: 0.92 / Cor.coef. F_o:F_c free: 0.869 / SU B: 19.292 / SU ML: 0.235 / Cross valid method: FREE R-VALUE / ESU R: 0.336 / ESU R Free: 0.26

	R_factor	Num. reflection	% reflection
R_free	0.3009	1039	3.423 %
R_work	0.2494	29315	-
all	0.251	-	-
obs	-	30354	97.015 %

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: 37.081 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.425 Å²	0 Å²	0 Å²
2-	-	2.441 Å²	-0 Å²
3-	-	-	-2.866 Å²

Refinement step

Cycle: LAST / Resolution: 2.2→21.48 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4193	0	14	107	4314

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.011	0.013	4273
X-RAY DIFFRACTION	r_bond_other_d	0.006	0.015	4107
X-RAY DIFFRACTION	r_angle_refined_deg	1.622	1.629	5793
X-RAY DIFFRACTION	r_angle_other_deg	1.385	1.569	9418
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.52	5	577
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	29.657	22.103	195
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	16.717	15	678
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	18.578	15	28
X-RAY DIFFRACTION	r_chiral_restr	0.069	0.2	571
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	4941
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	927
X-RAY DIFFRACTION	r_nbd_refined	0.197	0.2	900
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.188	0.2	4105
X-RAY DIFFRACTION	r_nbtor_refined	0.157	0.2	2059
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.084	0.2	2049
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.19	0.2	130
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.063	0.2	1
X-RAY DIFFRACTION	r_metal_ion_refined	0.168	0.2	6
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.091	0.2	6
X-RAY DIFFRACTION	r_nbd_other	0.196	0.2	32
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.25	0.2	3
X-RAY DIFFRACTION	r_mcbond_it	2.134	3.204	2309
X-RAY DIFFRACTION	r_mcbond_other	2.132	3.203	2308
X-RAY DIFFRACTION	r_mcangle_it	3.182	4.793	2879
X-RAY DIFFRACTION	r_mcangle_other	3.183	4.795	2880
X-RAY DIFFRACTION	r_scbond_it	2.277	3.512	1964
X-RAY DIFFRACTION	r_scbond_other	2.277	3.512	1965
X-RAY DIFFRACTION	r_scangle_it	3.432	5.166	2912
X-RAY DIFFRACTION	r_scangle_other	3.432	5.166	2913
X-RAY DIFFRACTION	r_lrange_it	4.961	38.782	4583
X-RAY DIFFRACTION	r_lrange_other	4.956	38.765	4577
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.104	0.05	4387
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.071	0.05	3640

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	0.10408	0.05008
1	2	CCC	X-RAY DIFFRACTION	Local ncs	0.10408	0.05008
2	3	BBB	X-RAY DIFFRACTION	Local ncs	0.07123	0.05009
2	4	DDD	X-RAY DIFFRACTION	Local ncs	0.07123	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 (%)
2.2-2.257	0.301	89	0.313	2151	X-RAY DIFFRACTION	98.809
2.257-2.318	0.387	74	0.309	2096	X-RAY DIFFRACTION	99.3135
2.318-2.384	0.269	61	0.305	2067	X-RAY DIFFRACTION	99.2537
2.384-2.456	0.337	62	0.302	1999	X-RAY DIFFRACTION	98.8015
2.456-2.536	0.362	66	0.313	1896	X-RAY DIFFRACTION	98.2474
2.536-2.624	0.324	77	0.279	1856	X-RAY DIFFRACTION	98.4717
2.624-2.721	0.375	78	0.292	1777	X-RAY DIFFRACTION	97.9409
2.721-2.83	0.361	55	0.284	1726	X-RAY DIFFRACTION	97.8034
2.83-2.954	0.292	58	0.256	1644	X-RAY DIFFRACTION	97.4799
2.954-3.095	0.347	46	0.259	1590	X-RAY DIFFRACTION	97.1496
3.095-3.259	0.326	47	0.272	1534	X-RAY DIFFRACTION	97.6529
3.259-3.451	0.284	55	0.259	1442	X-RAY DIFFRACTION	98.2283
3.451-3.683	0.284	41	0.259	1354	X-RAY DIFFRACTION	97.0772
3.683-3.968	0.254	47	0.228	1240	X-RAY DIFFRACTION	95.8302
3.968-4.332	0.219	36	0.204	1157	X-RAY DIFFRACTION	94.5325
4.332-4.819	0.298	50	0.199	1004	X-RAY DIFFRACTION	92.9453
4.819-5.519	0.274	36	0.211	920	X-RAY DIFFRACTION	92.4565
5.519-6.65	0.348	26	0.232	795	X-RAY DIFFRACTION	92.6637
6.65-8.984	0.278	24	0.199	651	X-RAY DIFFRACTION	93.62
8.984-21.48	0.321	11	0.222	416	X-RAY DIFFRACTION	86.4372

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	0.364	0.2381	0.2018	0.4725	-0.186	0.4974	0.0234	-0.0773	-0.0177	0.0743	-0.0363	0.0196	0.0091	-0.0562	0.0129	0.0917	0.0219	0.0173	0.0294	-0.0089	0.0785	2.9054	-5.1419	60.6831
2	0.4071	-0.2453	-0.2633	0.5126	0.2708	0.7449	0.011	-0.0261	0.0239	0.0432	-0.0134	-0.062	-0.025	0.1524	0.0024	0.0345	-0.0021	-0.0368	0.0504	0.0069	0.0713	17.6517	-1.2868	56.8235
3	0.1526	-0.1824	-0.025	0.6905	-0.4414	0.5447	-0.0074	0.0086	0.0449	-0.0788	0.0538	0.0371	0.0746	-0.0685	-0.0464	0.0464	-0.0195	-0.012	0.0116	0.0071	0.0796	3.0893	6.503	27.6438
4	0.1419	-0.0388	0.2818	0.6356	-0.478	0.8828	0.0416	0.0358	0.0158	-0.1069	-0.0726	-0.0817	0.1889	0.1323	0.0309	0.0706	0.0335	0.0187	0.0229	-0.0057	0.0864	17.0393	-0.1161	30.5857

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	AAA	4 - 157
2	X-RAY DIFFRACTION	1	ALL	AaA	159 - 163
3	X-RAY DIFFRACTION	2	ALL	BBB	179 - 312
4	X-RAY DIFFRACTION	2	ALL	BaB	314 - 317
5	X-RAY DIFFRACTION	3	ALL	CCC	4 - 157
6	X-RAY DIFFRACTION	3	ALL	CaC	159 - 164
7	X-RAY DIFFRACTION	4	ALL	DDD	179 - 313
8	X-RAY DIFFRACTION	4	ALL	DaD	314 - 316

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