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- PDB-3c17: Hexagonal Crystal Structure of Precursor E. coli Isoaspartyl Pept... -

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Entry
Database: PDB / ID: 3c17
TitleHexagonal Crystal Structure of Precursor E. coli Isoaspartyl Peptidase/l-Asparaginase (ECAIII) with Active-site T179A mutation
ComponentsL-asparaginase precursor
KeywordsHYDROLASE / ISOASPARTYL PEPTIDASE / ASPARAGINASE / NTN-HYDROLASE / AUTOPROTEOLYSIS / PRECURSOR
Function / homologybeta-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 speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsMichalska, K. / Hernandez-Santoyo, A. / Jaskolski, M.
Citation
Journal: J.Biol.Chem. / Year: 2008
Title: The Mechanism of Autocatalytic Activation of Plant-type L-Asparaginases
Authors: Michalska, K. / Hernandez-Santoyo, A. / Jaskolski, M.
#1: Journal: To be Published
Title: Crystal packing of plant-type L-asparaginase from Escherichia coli
Authors: Michalska, K. / Borek, D. / Hernandez-Santoyo, A. / 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: J.Mol.Biol. / Year: 2006
Title: Crystal structure of plant asparaginase
Authors: Michalska, K. / Bujacz, G. / Jaskolski, M.
#5: 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.
#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: J.Biol.Chem. / Year: 1998
Title: Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase
Authors: Guan, C. / Liu, Y. / Shao, Y. / Cui, T. / Liao, W. / Ewel, A. / Whitaker, R. / Paulus, H.
#8: Journal: J.Biol.Chem. / Year: 1998
Title: Activation and oligomerization of aspartylglucosaminidase
Authors: Saarela, J. / Laine, M. / Tikkanen, R. / Oinonen, C. / Jalanko, A. / Rouvinen, J. / Peltonen, L.
#9: Journal: Structure / Year: 2003
Title: A dual role for an aspartic acid in glycosylasparaginase autoproteolysis
Authors: Qian, X. / Guan, C. / Guo, H.-C.
#10: 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.
History
DepositionJan 22, 2008Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Apr 1, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Source and taxonomy / Version format compliance
Revision 1.2Nov 10, 2021Group: Data collection / Database references / Derived calculations
Category: database_2 / diffrn_source ...database_2 / diffrn_source / pdbx_struct_conn_angle / struct_conn / 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 / _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_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_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_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 / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Nov 1, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: L-asparaginase precursor
B: L-asparaginase precursor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,38130
Polymers66,5252
Non-polymers85628
Water7,819434
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6810 Å2
ΔGint-289.4 kcal/mol
Surface area20140 Å2
MethodPISA
Unit cell
Length a, b, c (Å)149.4700, 149.4700, 214.3500
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122

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Components

#1: Protein L-asparaginase precursor / L-asparagine amidohydrolase


Mass: 33262.676 Da / Num. of mol.: 2 / Mutation: T179A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12
Description: THE N-TERMINAL METHIONINE WAS REMOVED BY E. COLI INTRACELLULAR AMINOPEPTIDASE
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: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Na
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 434 / 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: 5.2 Å3/Da / Density % sol: 76.35 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 4.3M NaCl, 0.1M HEPES, pH 7.5, VAPOR DIFFUSION, HANGING DROP, temperature 292K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: X12 / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Feb 3, 2007
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.95→47.67 Å / Num. all: 98405 / Num. obs: 98405 / % possible obs: 96.6 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 5.9 % / Biso Wilson estimate: 27.3 Å2 / Rmerge(I) obs: 0.107 / Net I/σ(I): 14.3
Reflection shellResolution: 1.95→2.06 Å / Redundancy: 5.2 % / Rmerge(I) obs: 0.76 / Mean I/σ(I) obs: 2.2 / Num. unique all: 14515 / % possible all: 98.5

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
MAR345dtbdata collection
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1K2X

1k2x


Resolution: 1.95→44.51 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.95 / SU B: 5.255 / SU ML: 0.077 / TLS residual ADP flag: LIKELY RESIDUAL / Isotropic thermal model: isotropic / Cross valid method: R-FREE / σ(F): 0 / σ(I): 0 / ESU R: 0.092 / ESU R Free: 0.091 / Stereochemistry target values: Engh & Huber
Details: HYDROGEN ATOMS WERE ADDED AT RIDING POSITIONS. MAXIMUM LIKELIHOOD TARGET. THE REFINEMENT INCLUDED TLS PARAMETERS. THE RESIDUES 160-167 AND 315-321 FROM CHAIN A AS WELL AS 162-167 AND 314-321 ...Details: HYDROGEN ATOMS WERE ADDED AT RIDING POSITIONS. MAXIMUM LIKELIHOOD TARGET. THE REFINEMENT INCLUDED TLS PARAMETERS. THE RESIDUES 160-167 AND 315-321 FROM CHAIN A AS WELL AS 162-167 AND 314-321 FROM CHAIN B WERE NOT MODELED DUE TO POOR ELECTRON DENSITY.
RfactorNum. reflection% reflectionSelection details
Rfree0.203 992 1 %RANDOM
Rwork0.184 ---
obs0.184 97402 95.75 %-
all-97402 --
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: 27.62 Å2
Baniso -1Baniso -2Baniso -3
1--1.66 Å2-0.83 Å20 Å2
2---1.66 Å20 Å2
3---2.49 Å2
Refinement stepCycle: LAST / Resolution: 1.95→44.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4442 0 28 434 4904
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0224577
X-RAY DIFFRACTIONr_bond_other_d0.0010.023026
X-RAY DIFFRACTIONr_angle_refined_deg1.3991.9736204
X-RAY DIFFRACTIONr_angle_other_deg0.94837407
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8765629
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.93924.27185
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.57615754
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.9491532
X-RAY DIFFRACTIONr_chiral_restr0.0830.2711
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.025270
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02892
X-RAY DIFFRACTIONr_nbd_refined0.2270.2976
X-RAY DIFFRACTIONr_nbd_other0.1950.23509
X-RAY DIFFRACTIONr_nbtor_refined0.1740.22259
X-RAY DIFFRACTIONr_nbtor_other0.090.22442
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1380.2354
X-RAY DIFFRACTIONr_metal_ion_refined0.1380.230
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.060.24
X-RAY DIFFRACTIONr_symmetry_vdw_other0.3090.226
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.3240.21
X-RAY DIFFRACTIONr_mcbond_it0.6311.53035
X-RAY DIFFRACTIONr_mcbond_other0.1741.51288
X-RAY DIFFRACTIONr_mcangle_it1.1124798
X-RAY DIFFRACTIONr_scbond_it1.96731567
X-RAY DIFFRACTIONr_scangle_it3.1624.51397
LS refinement shellResolution: 1.95→2.055 Å / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.249 137 -
Rwork0.255 14351 -
obs-14351 98.11 %
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)
11.86020.4992-0.14042.2684-0.81611.35950.0866-0.00890.23840.2178-0.03690.0445-0.1767-0.0396-0.0497-0.0517-0.0469-0.0207-0.1917-0.0131-0.1908-63.089439.593635.3131
21.29340.3068-0.41872.2092-0.7221.22920.04960.0047-0.13810.1106-0.1248-0.4361-0.05710.24640.0751-0.0755-0.0715-0.0572-0.1230.0187-0.1338-51.499829.523935.7534
31.94920.6343-0.11762.4537-0.69111.0994-0.05220.2247-0.122-0.26980.10810.19060.1486-0.132-0.05590.0001-0.091-0.0728-0.1412-0.0351-0.1519-77.78089.302127.8186
42.07020.71430.01471.7415-0.5321.0760.0064-0.0832-0.26750.0664-0.0295-0.11230.19080.05290.0231-0.0079-0.0466-0.0368-0.1879-0.0035-0.1017-67.2186.349938.3755
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA2 - 1591 - 158
2X-RAY DIFFRACTION2AA168 - 314167 - 313
3X-RAY DIFFRACTION3BB2 - 1611 - 160
4X-RAY DIFFRACTION4BB168 - 313167 - 312

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