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- PDB-8bkf: Structure of E. coli Class 2 L-asparaginase EcAIII, mutant M200T ... -

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Basic information

Entry
Database: PDB / ID: 8bkf
TitleStructure of E. coli Class 2 L-asparaginase EcAIII, mutant M200T (crystal M200T#o)
Components(Isoaspartyl peptidase subunit ...) x 2
KeywordsHYDROLASE / L-asparaginase / isoaspartylpeptidase / mutation / Ntn-hydrolase
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 K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.221 Å
AuthorsSciuk, A. / Ruszkowski, M. / Jaskolski, M. / Loch, J.I.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2020/38/E/NZ1/00035 Poland
CitationJournal: Protein Sci. / Year: 2023
Title: The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.
Authors: Janicki, M. / Sciuk, A. / Zielezinski, A. / Ruszkowski, M. / Ludwikow, A. / Karlowski, W.M. / Jaskolski, M. / Loch, J.I.
History
DepositionNov 9, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 7, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID
Revision 1.2Feb 7, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Isoaspartyl peptidase subunit alpha
BBB: Isoaspartyl peptidase subunit beta
CCC: Isoaspartyl peptidase subunit alpha
DDD: Isoaspartyl peptidase subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,03611
Polymers66,8244
Non-polymers2127
Water10,701594
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14910 Å2
ΔGint-152 kcal/mol
Surface area20630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.852, 75.886, 147.762
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Isoaspartyl peptidase subunit ... , 2 types, 4 molecules AAACCCBBBDDD

#1: Protein Isoaspartyl peptidase subunit alpha


Mass: 19013.773 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 GOLD / References: UniProt: P37595
#2: Protein Isoaspartyl peptidase subunit beta


Mass: 14398.055 Da / Num. of mol.: 2 / Mutation: M200T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 GOLD / References: UniProt: P37595

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Non-polymers , 4 types, 601 molecules

#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.: 4 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 594 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.09 Å3/Da / Density % sol: 41.18 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 20-30% PEG4000,10-15% PEG400, 0,2 M magnesium chloride in 100mM Tris-HCl pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 5, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.22→73.89 Å / Num. obs: 166404 / % possible obs: 99.9 % / Redundancy: 12.7 % / CC1/2: 0.999 / Rmerge(I) obs: 0.072 / Rrim(I) all: 0.075 / Net I/σ(I): 16.4
Reflection shellResolution: 1.22→1.24 Å / Redundancy: 12.1 % / Rmerge(I) obs: 1.696 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 8052 / CC1/2: 0.596 / Rrim(I) all: 1.771 / % possible all: 98.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2ZAL

2zal


Resolution: 1.221→73.89 Å / Cor.coef. Fo:Fc: 0.983 / Cor.coef. Fo:Fc free: 0.978 / SU B: 1.142 / SU ML: 0.022 / Cross valid method: NONE / ESU R: 0.032 / ESU R Free: 0.032
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1502 1018 0.612 %
Rwork0.1284 165275 -
all0.129 --
obs-166293 99.88 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 16.927 Å2
Baniso -1Baniso -2Baniso -3
1-0.664 Å20 Å20 Å2
2--0.012 Å2-0 Å2
3----0.676 Å2
Refinement stepCycle: LAST / Resolution: 1.221→73.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4257 0 7 594 4858
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0134570
X-RAY DIFFRACTIONr_bond_other_d0.0010.0154343
X-RAY DIFFRACTIONr_angle_refined_deg1.2951.6326246
X-RAY DIFFRACTIONr_angle_other_deg1.471.5710001
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2985640
X-RAY DIFFRACTIONr_dihedral_angle_2_deg27.53922.105209
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.72615726
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.4151530
X-RAY DIFFRACTIONr_chiral_restr0.0640.2608
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.025437
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021007
X-RAY DIFFRACTIONr_nbd_refined0.210.2900
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1680.24252
X-RAY DIFFRACTIONr_nbtor_refined0.1550.22227
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0810.21984
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1260.2369
X-RAY DIFFRACTIONr_metal_ion_refined0.1030.29
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.20.216
X-RAY DIFFRACTIONr_nbd_other0.210.254
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1290.247
X-RAY DIFFRACTIONr_mcbond_it0.8861.5242473
X-RAY DIFFRACTIONr_mcbond_other0.8711.5232472
X-RAY DIFFRACTIONr_mcangle_it1.1942.2923130
X-RAY DIFFRACTIONr_mcangle_other1.1942.2933131
X-RAY DIFFRACTIONr_scbond_it1.2261.8452097
X-RAY DIFFRACTIONr_scbond_other1.2241.842095
X-RAY DIFFRACTIONr_scangle_it1.5312.6613112
X-RAY DIFFRACTIONr_scangle_other1.5312.6613112
X-RAY DIFFRACTIONr_lrange_it2.58520.3885144
X-RAY DIFFRACTIONr_lrange_other2.5120.1245109
X-RAY DIFFRACTIONr_rigid_bond_restr0.72538913
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.221-1.2530.268790.237119730.237121770.8650.87998.97350.219
1.253-1.2870.213780.203118060.203118840.9250.9141000.183
1.287-1.3250.2780.184114390.184115170.9190.9311000.162
1.325-1.3660.179850.163111690.163112540.9520.9461000.139
1.366-1.410.154550.145108370.145108920.9590.9591000.121
1.41-1.460.173580.123105380.123105960.9670.9691000.102
1.46-1.5150.158460.108101370.108101830.9690.9771000.089
1.515-1.5770.11500.09697380.09697880.9820.9831000.079
1.577-1.6470.122650.09193700.09194350.980.9851000.078
1.647-1.7270.126550.09889630.09890180.9820.9851000.085
1.727-1.820.137490.09985150.09985660.9780.98599.97670.088
1.82-1.9310.134490.10181200.10181700.9770.98399.98780.092
1.931-2.0640.148500.10576080.10576580.9760.9841000.099
2.064-2.2290.142370.11271250.11271620.9810.9821000.109
2.229-2.4420.13380.11365670.11366060.9830.9899.98490.113
2.442-2.730.105420.12559640.12460080.980.97799.96670.128
2.73-3.1510.179380.13552960.13553340.9660.9741000.143
3.151-3.8580.171350.13644980.13645340.9690.97899.97790.151
3.858-5.4480.129210.13135680.13135890.9840.9821000.152
5.448-73.8810.178100.19920450.19920850.9540.96598.56110.222

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