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- PDB-2zal: Crystal structure of E. coli isoaspartyl aminopeptidase/L-asparag... -

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Basic information

Entry
Database: PDB / ID: 2zal
TitleCrystal structure of E. coli isoaspartyl aminopeptidase/L-asparaginase in complex with L-aspartate
Components(L-asparaginaseAsparaginase) x 2
KeywordsHYDROLASE / isoaspartyl peptidase / asparaginase / Ntn-hydrolase / autoproteolysis / L-aspartate/calcium cluster
Function / homology
Function and homology information


beta-aspartyl-peptidase / asparaginase activity / beta-aspartyl-peptidase activity / protein autoprocessing / hydrolase activity / cytoplasm
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
ASPARTIC ACID / Isoaspartyl peptidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsMichalska, K. / Brzezinski, K. / Jaskolski, M.
Citation
Journal: J.Biol.Chem. / Year: 2005
Title: Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate
Authors: Michalska, K. / Brzezinski, K. / Jaskolski, M.
#1: 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.
#2: 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.E. / Smith, J.L. / Tomchick, D.R. / Murzin, A.G.
#3: Journal: Protein Sci. / Year: 1998
Title: Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum
Authors: Xuan, J. / Tarentino, A.L. / Grimwood, B.G. / Plummer Jr., T.H. / Cui, T. / Guan, C. / Van Roey, P.
#4: Journal: Nat.Struct.Biol. / Year: 1995
Title: Three-dimensional structure of human lysosomal aspartylglucosaminidase
Authors: Oinonen, C. / Tikkanen, R. / Rouvinen, J. / Peltonen, L.
#5: 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.
#6: Journal: Cell(Cambridge,Mass.) / Year: 1999
Title: Structural insights into the mechanism of intramolecular proteolysis
Authors: Xu, Q. / Buckley, D. / Guan, C. / Guo, H.-C.
#7: Journal: Biochem.J. / Year: 2004
Title: Autoproteolytic activation of human aspartylglucosaminidase
Authors: Saarela, J. / Oinonen, C. / Jalanko, A. / Rouvinen, J. / Peltonen, L.
#8: 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.
History
DepositionOct 7, 2007Deposition site: PDBJ / Processing site: PDBJ
SupersessionOct 30, 2007ID: 1SEO
Revision 1.0Oct 30, 2007Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Source and taxonomy / Version format compliance
Revision 1.2May 23, 2018Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.3Nov 1, 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: L-asparaginase
B: L-asparaginase
C: L-asparaginase
D: L-asparaginase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,96019
Polymers61,7684
Non-polymers1,19215
Water4,468248
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.89, 77.28, 147.53
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein L-asparaginase / Asparaginase / L-ASPARAGINASE SUBUNIT ALPHA / L-asparagine amidohydrolase


Mass: 17071.527 Da / Num. of mol.: 2 / Fragment: N-terminal subunit (alpha), UNP residues 2-161
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12
Description: The N-terminal methionine has been removed by an intracellular aminopeptidase (E. coli). During expression and purification, the protein undergoes autoproteolytic cleavage into alpha and ...Description: The N-terminal methionine has been removed by an intracellular aminopeptidase (E. coli). During expression and purification, the protein undergoes autoproteolytic cleavage into alpha and beta subunits. The residues 162-178 were not present in the crystallized material due to partial, non-specific degradation at the c-terminus. The number of missing residues was determined by mass spectrometry
Gene: ybiK (iaaA) / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
References: UniProt: P37595, beta-aspartyl-peptidase, asparaginase
#2: Protein L-asparaginase / Asparaginase / L-ASPARAGINASE SUBUNIT BETA / L-asparagine amidohydrolase


Mass: 13812.512 Da / Num. of mol.: 2 / Fragment: C-terminal subunit (beta), UNP residues 179-315
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12
Description: During expression and purification, the protein undergoes autoproteolytic cleavage into alpha and beta subunits. The residues 316-321 were not present in the crystallized material due to ...Description: During expression and purification, the protein undergoes autoproteolytic cleavage into alpha and beta subunits. The residues 316-321 were not present in the crystallized material due to partial, non-specific degradation at the C-terminus. The number of missing residues was determined by mass spectrometry
Gene: ybiK (iaaA) / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
References: UniProt: P37595, beta-aspartyl-peptidase, asparaginase

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Non-polymers , 6 types, 263 molecules

#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#5: Chemical
ChemComp-ASP / ASPARTIC ACID / Aspartic acid


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C4H7NO4
#6: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 248 / 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: 2.3 Å3/Da / Density % sol: 46.41 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 100mM Tris/HCl, 80mM calcium chloride, 100mM sodium aspartate, 17% PEG 4000, 13% PEG 400, pH 8.5, VAPOR DIFFUSION, HANGING DROP, temperature 292K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I711 / Wavelength: 1.095 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Apr 28, 2002
RadiationMonochromator: Si single crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.095 Å / Relative weight: 1
ReflectionResolution: 1.9→20 Å / Num. all: 43572 / Num. obs: 43572 / % possible obs: 95.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 3.5 % / Biso Wilson estimate: 18.67 Å2 / Rmerge(I) obs: 0.064 / Net I/σ(I): 15
Reflection shellResolution: 1.9→1.97 Å / Redundancy: 3 % / Rmerge(I) obs: 0.256 / Mean I/σ(I) obs: 2.2 / Num. unique all: 4085 / % possible all: 91.3

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Processing

Software
NameVersionClassification
REFMAC5.1.24refinement
MAR345dtbdata collection
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1K2X

1k2x


Resolution: 1.9→20 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.955 / SU B: 2.746 / SU ML: 0.08 / TLS residual ADP flag: LIKELY RESIDUAL / Isotropic thermal model: Isotropic / Cross valid method: R-free / σ(F): 0 / σ(I): 0 / ESU R: 0.135 / ESU R Free: 0.121 / Stereochemistry target values: Engh & Huber
Details: MAXIMUM LIKELIHOOD TARGET. THE REFINEMENT INCLUDED TLS PARAMETERS. THE RESIDUES 158-161 FROM CHAIN C (SUBUNIT ALPHA) AS WELL AS THE RESIDUES 314-315 FROM THE CHAINS B AND D (SUBUNITS BETA) ...Details: MAXIMUM LIKELIHOOD TARGET. THE REFINEMENT INCLUDED TLS PARAMETERS. THE RESIDUES 158-161 FROM CHAIN C (SUBUNIT ALPHA) AS WELL AS THE RESIDUES 314-315 FROM THE CHAINS B AND D (SUBUNITS BETA) WERE NOT MODELED DUE TO POOR ELECTRON DENSITY. CHAIN A IS COMPLETE. IN EACH OF THE TWO ACTIVE SITES, A CLEARLY VISIBLE PRODUCT OF THE ENZYMATIC REACTION, L-ASPARTATE, IS MODELED. IN INTERSTITIAL POSITIONS, THE STRUCTURE CONTAINS A BIG 3L-ASP/5CA COORDINATION COMPLEX.
RfactorNum. reflection% reflectionSelection details
Rfree0.188 1372 3.1 %Random
Rwork0.157 ---
all0.158 42200 --
obs0.158 42200 95.71 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 13.27 Å2
Baniso -1Baniso -2Baniso -3
1-1.05 Å20 Å20 Å2
2---0.36 Å20 Å2
3----0.69 Å2
Refinement stepCycle: LAST / Resolution: 1.9→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4259 0 69 248 4576
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0214397
X-RAY DIFFRACTIONr_bond_other_d0.0020.024087
X-RAY DIFFRACTIONr_angle_refined_deg1.341.9735948
X-RAY DIFFRACTIONr_angle_other_deg0.8339450
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.925590
X-RAY DIFFRACTIONr_chiral_restr0.080.2687
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.025025
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02856
X-RAY DIFFRACTIONr_nbd_refined0.2150.2848
X-RAY DIFFRACTIONr_nbd_other0.2430.24982
X-RAY DIFFRACTIONr_nbtor_other0.0850.22671
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1440.2208
X-RAY DIFFRACTIONr_metal_ion_refined0.1080.216
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.3210.227
X-RAY DIFFRACTIONr_symmetry_vdw_other0.3110.253
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1350.210
X-RAY DIFFRACTIONr_mcbond_it0.6721.52918
X-RAY DIFFRACTIONr_mcangle_it1.2524599
X-RAY DIFFRACTIONr_scbond_it2.2931479
X-RAY DIFFRACTIONr_scangle_it3.914.51345
LS refinement shellResolution: 1.904→2.006 Å / Total num. of bins used: 10 /
RfactorNum. reflection
Rfree0.235 159
Rwork0.201 5774
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)
11.3726-0.26360.2911.5176-0.2391.3085-0.0226-0.09920.03990.1150.00170.059-0.0814-0.11240.0210.0145-0.00440.0270.0380.00120.0530.7234-1.333477.3544
21.2746-0.1488-0.17311.4810.22250.7348-0.0043-0.0620.03020.1247-0.0118-0.1616-0.10810.15590.0160.0561-0.00490.00120.0332-0.00410.053316.1174-1.463276.0772
31.6526-0.2077-0.92870.7140.0253.4344-0.02740.23530.0836-0.13190.01210.07510.0106-0.27740.01540.0836-0.0129-0.00770.07230.03660.07427.78288.799445.6619
41.1674-0.10390.34911.1990.03971.7906-0.04790.1183-0.0913-0.09890.016-0.11820.17030.20610.03190.0688-0.00020.03040.0963-0.00970.068919.5465-0.587849.9628
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA2 - 1611 - 160
2X-RAY DIFFRACTION2BB179 - 3131 - 135
3X-RAY DIFFRACTION3CC2 - 1571 - 156
4X-RAY DIFFRACTION4DD179 - 3131 - 135

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