PDB-7qyx: Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-24...

ID or keywords:

Entry

Database: PDB / ID: 7qyx

Title

Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-24 (R207A, D210S, S211T)

Components

Beta-aspartyl-peptidase
Isoaspartyl peptidase

Keywords

HYDROLASE / L-asparaginase / mutation

Function / homology

Function and homology information

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

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.85 Å

Authors

Loch, J.I. / Klonecka, A. / Kadziolka, K. / Bonarek, P. / Barciszewski, J. / Imiolczyk, B. / Brzezinski, 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 29, 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	7qyx.cif.gz	199.7 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	7qyx_validation.pdf.gz	447.6 KB	Display	wwPDB validaton report
Full document	7qyx_full_validation.pdf.gz	448.8 KB	Display
Data in XML	7qyx_validation.xml.gz	22.9 KB	Display
Data in CIF	7qyx_validation.cif.gz	32.7 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/qy/7qyx ftp://data.pdbj.org/pub/pdb/validation_reports/qy/7qyx	HTTPS FTP

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Related structure data

Related structure data	7qq8C 7qsfC 7qtcC 7qvrC 7qy6C 7qymC 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: 10.18150/6OXPLO / 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: Beta-aspartyl-peptidase

CCC: Isoaspartyl peptidase

DDD: Beta-aspartyl-peptidase

hetero molecules

66.8 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)	50.258, 74.141, 147.466
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:

Refine code: 1

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	SER	SER	AAA	A	4 - 145	4 - 145
2	1	1	ALA	ALA	SER	SER	CCC	C	4 - 145	4 - 145
3	2	2	THR	THR	TYR	TYR	BBB	B	179 - 311	1 - 133
4	2	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

#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) / Strain (production host): BL 21 Gold / References: UniProt: P37595, beta-aspartyl-peptidase
#2: Protein	Beta-aspartyl-peptidase / Putative L-asparaginase Mass: 14328.045 Da / Num. of mol.: 2 / Mutation: R207A, D210S, S211T Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) Gene: iaaA, BG821_004768, BMC79_000629, D3822_09905, FJQ51_03270, GQW68_09230, NCTC9044_04106, NCTC9702_03859 Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold References: UniProt: A0A3A6SJA6, beta-aspartyl-peptidase, asparaginase
#3: Chemical	ChemComp-NA / SODIUM ION Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#5: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 208 / 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: 2.06 Å³/Da / Density % sol: 40.29 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 Details: 20% PEG 6K; 15% PEG 400; 0.2M MgCl2 in 0.1 Tris-HCl pH 8.5

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.7749 Å
Detector	Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 21, 2021
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.7749 Å / Relative weight: 1
Reflection	Resolution: 1.85→52.335 Å / Num. obs: 48066 / % possible obs: 98.4 % / Redundancy: 13.1 % / CC_1/2: 0.999 / R_merge(I) obs: 0.119 / R_rim(I) all: 0.118 / Net I/σ(I): 15.16
Reflection shell	Resolution: 1.85→1.95 Å / Redundancy: 12.4 % / R_merge(I) obs: 2.141 / Mean I/σ(I) obs: 1.75 / Num. unique obs: 7553 / CC_1/2: 0.789 / R_rim(I) all: 1.852 / % possible all: 97.7

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
XDS		data reduction
XDS		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 2ZAL

2zal

Resolution: 1.85→52.335 Å / Cor.coef. F_o:F_c: 0.961 / Cor.coef. F_o:F_c free: 0.94 / SU B: 8.13 / SU ML: 0.112 / Cross valid method: FREE R-VALUE / ESU R: 0.14 / ESU R Free: 0.134
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.2385	1031	2.151 %
R_work	0.1984	46910	-
all	0.199	-	-
obs	-	47941	99.948 %

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: 32.827 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	4.174 Å²	0 Å²	0 Å²
2-	-	0.061 Å²	-0 Å²
3-	-	-	-4.235 Å²

Refinement step

Cycle: LAST / Resolution: 1.85→52.335 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4063	0	5	208	4276

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.011	0.013	4140
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.015	3956
X-RAY DIFFRACTION	r_angle_refined_deg	1.568	1.629	5620
X-RAY DIFFRACTION	r_angle_other_deg	1.484	1.569	9083
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.889	5	566
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	27.211	22.419	186
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.892	15	651
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	14.485	15	25
X-RAY DIFFRACTION	r_chiral_restr	0.075	0.2	558
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	4817
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	893
X-RAY DIFFRACTION	r_nbd_refined	0.208	0.2	849
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.178	0.2	3931
X-RAY DIFFRACTION	r_nbtor_refined	0.163	0.2	2054
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.088	0.2	1920
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.147	0.2	188
X-RAY DIFFRACTION	r_metal_ion_refined	0.104	0.2	5
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.196	0.2	13
X-RAY DIFFRACTION	r_nbd_other	0.236	0.2	40
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.218	0.2	10
X-RAY DIFFRACTION	r_mcbond_it	1.779	2.696	2252
X-RAY DIFFRACTION	r_mcbond_other	1.767	2.694	2251
X-RAY DIFFRACTION	r_mcangle_it	2.631	4.033	2810
X-RAY DIFFRACTION	r_mcangle_other	2.633	4.035	2811
X-RAY DIFFRACTION	r_scbond_it	2.439	3.122	1888
X-RAY DIFFRACTION	r_scbond_other	2.434	3.118	1886
X-RAY DIFFRACTION	r_scangle_it	3.808	4.538	2806
X-RAY DIFFRACTION	r_scangle_other	3.806	4.538	2806
X-RAY DIFFRACTION	r_lrange_it	5.559	33.791	4515
X-RAY DIFFRACTION	r_lrange_other	5.542	33.627	4490
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.062	0.05	4123
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.051	0.05	3667

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	0.06231	0.05008
1	2	CCC	X-RAY DIFFRACTION	Local ncs	0.06231	0.05008
2	3	BBB	X-RAY DIFFRACTION	Local ncs	0.05109	0.0501
2	4	DDD	X-RAY DIFFRACTION	Local ncs	0.05109	0.0501

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 (%)
1.85-1.898	0.347	52	0.353	3454	X-RAY DIFFRACTION	99.943
1.898-1.95	0.343	76	0.321	3334	X-RAY DIFFRACTION	99.9414
1.95-2.006	0.279	69	0.282	3241	X-RAY DIFFRACTION	99.9094
2.006-2.068	0.26	63	0.24	3136	X-RAY DIFFRACTION	100
2.068-2.136	0.229	77	0.223	3048	X-RAY DIFFRACTION	100
2.136-2.211	0.206	74	0.199	2951	X-RAY DIFFRACTION	100
2.211-2.294	0.201	76	0.183	2858	X-RAY DIFFRACTION	99.9319
2.294-2.388	0.247	54	0.174	2758	X-RAY DIFFRACTION	99.9645
2.388-2.494	0.2	55	0.168	2663	X-RAY DIFFRACTION	99.9265
2.494-2.615	0.259	53	0.182	2523	X-RAY DIFFRACTION	99.9612
2.615-2.757	0.248	48	0.184	2423	X-RAY DIFFRACTION	100
2.757-2.924	0.237	69	0.179	2273	X-RAY DIFFRACTION	100
2.924-3.125	0.282	45	0.197	2181	X-RAY DIFFRACTION	99.9102
3.125-3.375	0.305	56	0.195	2017	X-RAY DIFFRACTION	99.9518
3.375-3.696	0.166	36	0.17	1857	X-RAY DIFFRACTION	99.9472
3.696-4.132	0.205	34	0.175	1717	X-RAY DIFFRACTION	99.9429
4.132-4.768	0.265	28	0.172	1513	X-RAY DIFFRACTION	100
4.768-5.834	0.231	27	0.199	1298	X-RAY DIFFRACTION	99.9246
5.834-8.224	0.174	22	0.2	1044	X-RAY DIFFRACTION	100
8.224-52.335	0.263	17	0.224	609	X-RAY DIFFRACTION	99.3651

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	0.1735	0.0876	0.0191	0.0911	-0.1462	0.6661	-0.0399	-0.0523	-0.0192	-0.017	-0.0021	0.0026	0.0252	-0.059	0.042	0.0241	0.0322	0.0146	0.0634	0.0002	0.0914	2.8901	-5.1134	61.1042
2	0.0552	-0.0544	-0.1352	0.2872	0.0408	0.3727	0.0002	-0.0414	-0.009	0.0048	-0.0175	0.0039	-0.0139	0.107	0.0172	0.0111	0.0203	-0.0082	0.1006	0.013	0.09	17.3698	0.1036	58.098
3	0.0889	-0.0692	-0.1062	0.2082	-0.1226	0.4262	-0.0004	0.0275	0.0188	-0.0157	-0.0099	-0.0007	0.0225	-0.0511	0.0103	0.0021	-0.0063	0.0015	0.0736	0.0011	0.106	4.7851	6.7546	28.6379
4	0.0117	0.0275	0.0247	0.2219	-0.016	0.385	0.0019	0.0034	0.0279	-0.0366	-0.0332	-0.0013	0.0825	0.0784	0.0313	0.0237	0.0176	0.0157	0.0758	0.0144	0.1061	18.083	-0.4223	31.8847

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	AAA	4 - 152
2	X-RAY DIFFRACTION	2	ALL	BBB	179 - 312
3	X-RAY DIFFRACTION	3	ALL	CCC	3 - 146
4	X-RAY DIFFRACTION	4	ALL	DDD	179 - 313

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