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- PDB-2gez: Crystal structure of potassium-independent plant asparaginase -

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Basic information

Entry
Database: PDB / ID: 2gez
TitleCrystal structure of potassium-independent plant asparaginase
Components
  • L-asparaginase alpha subunit
  • L-asparaginase beta subunit
KeywordsHYDROLASE / isoaspartyl aminopeptidase / L-asparaginase / Ntn-hydrolase / autoproteolysis / taspase / sodium binding
Function / homology
Function and homology information


beta-aspartyl-peptidase / hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides / beta-aspartyl-peptidase activity / proteolysis
Similarity search - Function
(Glycosyl)asparaginase / Peptidase T2, asparaginase 2 / Asparaginase / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Isoaspartyl peptidase/L-asparaginase
Similarity search - Component
Biological speciesLupinus luteus (yellow lupine)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsMichalska, K. / Bujacz, G. / Jaskolski, M.
Citation
Journal: J.Mol.Biol. / Year: 2006
Title: Crystal structure of plant asparaginase.
Authors: Michalska, K. / Bujacz, G. / Jaskolski, M.
#1: Journal: Eur.J.Biochem. / Year: 2004
Title: Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.
Authors: Borek, D. / Michalska, K. / Brzezinski, K. / Kisiel, A. / Podkowinski, J. / Bonthron, T.D. / Krowarsch, D. / Otlewski, J. / Jaskolski, M.
#2: Journal: J.Biol.Chem. / Year: 2005
Title: Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate.
Authors: Michalska, K. / Brzezinski, K. / Jaskolski, M.
#3: Journal: Acta Crystallogr.,Sect.D / Year: 2000
Title: Crystallization and preliminary crystallographic studies of a new L-asparaginase encoded by the Escherichia coli genome.
Authors: Borek, D. / Jaskolski, M.
#4: Journal: Acta Crystallogr.,Sect.D / Year: 2004
Title: Structure of the isoaspartyl peptidase with L-asparaginase activity from Escherichia coli.
Authors: Prahl, A. / Pazgier, M. / Hejazi, M. / Lockau, W. / Lubkowski, J.
#5: Journal: Nature / Year: 1995
Title: A protein catalytic framework with an N-terminal nucleophile is capable of self-activation.
Authors: Brannigan, J.A. / Dodson, G. / Duggleby, H.J. / Moody, P.C. / Smith, J.L. / Tomchick, D.R. / Murzin, A.G.
#6: Journal: Nat.Struct.Biol. / Year: 1995
Title: Three-dimensional structure of human lysosomal aspartylglucosaminidase.
Authors: Oinonen, C. / Tikkanen, R. / Rouvinen, J. / Peltonen, L.
#7: Journal: J.Biol.Chem. / Year: 1998
Title: Crystal structures of Flavobacterium glycosylasparaginase. An N-terminal nucleophile hydrolase activated by intramolecular proteolysis.
Authors: Guo, H.C. / Xu, Q. / Buckley, D. / Guan, C.
#8: Journal: Structure / Year: 2005
Title: Crystal structure of human Taspase1, a crucial protease regulating the function of MLL.
Authors: Khan, J.A. / Dunn, B.M. / Tong, L.
History
DepositionMar 21, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 25, 2006Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Advisory / Derived calculations / Version format compliance
Revision 1.3Aug 30, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_source / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: L-asparaginase alpha subunit
B: L-asparaginase beta subunit
C: L-asparaginase alpha subunit
D: L-asparaginase beta subunit
E: L-asparaginase alpha subunit
F: L-asparaginase beta subunit
G: L-asparaginase alpha subunit
H: L-asparaginase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,43818
Polymers139,1338
Non-polymers30510
Water2,018112
1
A: L-asparaginase alpha subunit
B: L-asparaginase beta subunit
C: L-asparaginase alpha subunit
D: L-asparaginase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,7199
Polymers69,5674
Non-polymers1525
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14800 Å2
ΔGint-145 kcal/mol
Surface area21190 Å2
MethodPISA, PQS
2
E: L-asparaginase alpha subunit
F: L-asparaginase beta subunit
G: L-asparaginase alpha subunit
H: L-asparaginase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,7199
Polymers69,5674
Non-polymers1525
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14850 Å2
ΔGint-147 kcal/mol
Surface area21040 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)47.68, 60.20, 114.63
Angle α, β, γ (deg.)100.6, 92.9, 113.4
Int Tables number1
Space group name H-MP1
DetailsThe asymmetric unit contains two biological assemblies. Each of them is an (alpha/beta)2 heterotetramer or a dimer of (alpha/beta) heterodimers. Subunits alpha (chains A, C, E, G) and beta (chains B, D, F, H) are, respectively, the N- and C-terminal products of autoproteolytic cleavage of a precursor.

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Components

#1: Protein
L-asparaginase alpha subunit / L-asparagine amidohydrolase


Mass: 21165.877 Da / Num. of mol.: 4 / Fragment: N-terminal subunit (residues 1-192)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lupinus luteus (yellow lupine) / Strain: cv. Ventus / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Codon Plus RIP / References: UniProt: Q9ZSD6, asparaginase
#2: Protein
L-asparaginase beta subunit / L-asparagine amidohydrolase


Mass: 13617.478 Da / Num. of mol.: 4 / Fragment: C-terminal subunit (residues 193-325)
Source method: isolated from a genetically manipulated source
Details: Subunits alpha (chains A, C, E, G) and beta (chains B, D, F, H) are, respectively, the N- and C-terminal products of autoproteolytic cleavage of a precursor
Source: (gene. exp.) Lupinus luteus (yellow lupine) / Strain: cv. Ventus / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Codon Plus RIP / References: UniProt: Q9ZSD6, asparaginase
#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
#4: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 112 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.11 Å3/Da / Density % sol: 41.77 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 20% PEG 4000, 100 mM HEPES, 200 mM MgCl2, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 292.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: X11 / Wavelength: 0.81 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Mar 5, 2004
RadiationMonochromator: Triangular monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.81 Å / Relative weight: 1
ReflectionResolution: 2.6→25 Å / Num. all: 34621 / Num. obs: 34621 / % possible obs: 99 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 2.8 % / Biso Wilson estimate: 40.9 Å2 / Rmerge(I) obs: 0.073 / Net I/σ(I): 14.3
Reflection shellResolution: 2.6→2.69 Å / Redundancy: 2.5 % / Rmerge(I) obs: 0.152 / Mean I/σ(I) obs: 6.1 / Num. unique all: 3371 / % possible all: 96.9

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Processing

Software
NameVersionClassification
REFMAC5.2.0005refinement
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1K2X

1k2x


Resolution: 2.6→25 Å / Cor.coef. Fo:Fc: 0.933 / Cor.coef. Fo:Fc free: 0.883 / SU B: 22.964 / SU ML: 0.234 / TLS residual ADP flag: LIKELY RESIDUAL / Isotropic thermal model: isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.349 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.254 1741 5 %RANDOM
Rwork0.189 ---
all0.192 34621 --
obs0.192 32880 98.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 25.11 Å2
Baniso -1Baniso -2Baniso -3
1--1.13 Å21.3 Å20.02 Å2
2--4.16 Å20.19 Å2
3----1.92 Å2
Refinement stepCycle: LAST / Resolution: 2.6→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8693 0 10 112 8815
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0228823
X-RAY DIFFRACTIONr_bond_other_d0.028244
X-RAY DIFFRACTIONr_angle_refined_deg1.5841.96711962
X-RAY DIFFRACTIONr_angle_other_deg319153
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.95651171
X-RAY DIFFRACTIONr_dihedral_angle_2_deg24.663341
X-RAY DIFFRACTIONr_dihedral_angle_3_deg151474
X-RAY DIFFRACTIONr_dihedral_angle_4_deg1550
X-RAY DIFFRACTIONr_chiral_restr0.0850.21415
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.029931
X-RAY DIFFRACTIONr_gen_planes_other0.021635
X-RAY DIFFRACTIONr_nbd_refined0.2250.22014
X-RAY DIFFRACTIONr_nbd_other0.28942
X-RAY DIFFRACTIONr_nbtor_refined0.1780.24340
X-RAY DIFFRACTIONr_nbtor_other0.25154
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1570.2283
X-RAY DIFFRACTIONr_metal_ion_refined0.0660.28
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.212
X-RAY DIFFRACTIONr_symmetry_vdw_other0.266
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.21
X-RAY DIFFRACTIONr_mcbond_it0.4771.55815
X-RAY DIFFRACTIONr_mcbond_other1.52450
X-RAY DIFFRACTIONr_mcangle_it0.87929312
X-RAY DIFFRACTIONr_scbond_it1.47933059
X-RAY DIFFRACTIONr_scangle_it2.5384.52650
LS refinement shellHighest resolution: 2.6 Å / Num. reflection Rwork: 4685 / Total num. of bins used: 10
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°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)
12.6966-0.4412-0.8651.55550.52981.9216-0.02540.47740.4306-0.26630.0412-0.0507-0.31870.1716-0.0159-0.0068-0.0720.0046-0.05650.05280.00739.750711.1189-10.71
22.0609-0.387-0.16751.71330.44751.8755-0.04920.314-0.18-0.03480.01280.13060.1880.01330.0365-0.0515-0.04120.00220.0512-0.017-0.08795.387-3.4236-13.8382
31.0970.6393-0.15921.8701-0.35911.5110.0236-0.21140.08910.14350.0140.0127-0.02380.0562-0.0376-0.09770.06280.0318-0.102-0.0298-0.0638-10.44291.289314.7101
42.2398-0.0775-0.14221.26450.51721.6128-0.1251-0.0554-0.3559-0.02560.0475-0.12990.25440.12520.0776-0.03990.02340.0253-0.12630.0096-0.0596-4.0657-11.2889.2378
51.378-0.53150.28641.8407-0.16251.42550.01830.1831-0.1371-0.12310.03250.05050.0883-0.0202-0.0508-0.1017-0.0342-0.016-0.1286-0.0293-0.0635-10.551325.624237.4893
62.1706-0.29740.23081.27070.69572.0304-0.1260.07670.3416-0.00670.026-0.0712-0.33090.15740.1-0.0635-0.0311-0.0259-0.14820.0067-0.0406-4.082138.270942.7524
72.45140.73330.66641.6060.18742.41460.0848-0.4256-0.40830.19640.009-0.05150.29560.1487-0.0938-0.03530.06470.0028-0.06850.052-0.00549.806216.133862.9469
82.4531.09930.73471.58980.431.977-0.0303-0.40830.15880.0987-0.02040.1162-0.1782-0.06820.0507-0.03670.0372-0.0031-0.0176-0.0272-0.08635.449730.717165.9204
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA2 - 1595 - 162
2X-RAY DIFFRACTION2BB193 - 3251 - 133
3X-RAY DIFFRACTION3CC2 - 1675 - 170
4X-RAY DIFFRACTION4DD193 - 3251 - 133
5X-RAY DIFFRACTION5EE2 - 1665 - 169
6X-RAY DIFFRACTION6FF193 - 3251 - 133
7X-RAY DIFFRACTION7GG2 - 1595 - 162
8X-RAY DIFFRACTION8HH193 - 3251 - 133

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