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- PDB-8bp9: Structure of E. coli Class 2 L-asparaginase EcAIII, mutant M200W ... -

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Basic information

Entry
Database: PDB / ID: 8bp9
TitleStructure of E. coli Class 2 L-asparaginase EcAIII, mutant M200W (crystal M200W#2)
Components
  • Isoaspartyl peptidase subunit alpha
  • Isoaspartyl peptidase subunit beta
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 (E. coli)
Escherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsSciuk, A. / 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 16, 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)67,16910
Polymers66,9944
Non-polymers1756
Water4,792266
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14660 Å2
ΔGint-135 kcal/mol
Surface area20530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)50.974, 74.475, 146.787
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#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 (E. coli) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold / References: UniProt: P37595
#2: Protein Isoaspartyl peptidase subunit beta


Mass: 14483.160 Da / Num. of mol.: 2 / Mutation: M200W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold / References: UniProt: P37595
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / 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.: 266 / Source method: isolated from a natural source / Formula: H2O
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: 2.08 Å3/Da / Density % sol: 40.85 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 20-30% PEG4000, 10-15%PEG400,0.2M MgCl2 in 100 mM Tris-HCl pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: Agilent SuperNova / Wavelength: 1.5418 Å
DetectorType: AGILENT ATLAS CCD / Detector: CCD / Date: Oct 25, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.7→73.4 Å / Num. obs: 61686 / % possible obs: 99.1 % / Redundancy: 4 % / CC1/2: 0.995 / Rmerge(I) obs: 0.082 / Rrim(I) all: 0.094 / Net I/σ(I): 9.9
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 3.1 % / Rmerge(I) obs: 0.378 / Mean I/σ(I) obs: 2.5 / Num. unique obs: 3153 / CC1/2: 0.749 / Rrim(I) all: 0.455 / % possible all: 97.8

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Processing

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

2zal


Resolution: 1.7→73.4 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.917 / SU B: 5.195 / SU ML: 0.084 / Cross valid method: FREE R-VALUE / ESU R: 0.11 / ESU R Free: 0.108
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2284 1056 1.715 %
Rwork0.1933 60525 -
all0.194 --
obs-61581 98.781 %
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: 11.731 Å2
Baniso -1Baniso -2Baniso -3
1--0.327 Å2-0 Å20 Å2
2--0.46 Å2-0 Å2
3----0.133 Å2
Refinement stepCycle: LAST / Resolution: 1.7→73.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4253 0 6 266 4525
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0134432
X-RAY DIFFRACTIONr_bond_other_d0.0030.0154205
X-RAY DIFFRACTIONr_angle_refined_deg1.5981.6316031
X-RAY DIFFRACTIONr_angle_other_deg1.5311.5739661
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9535607
X-RAY DIFFRACTIONr_dihedral_angle_2_deg28.42422.153209
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.78315698
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.4811530
X-RAY DIFFRACTIONr_chiral_restr0.0740.2588
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.025227
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02981
X-RAY DIFFRACTIONr_nbd_refined0.2150.2955
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1880.24225
X-RAY DIFFRACTIONr_nbtor_refined0.1640.22217
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0830.22080
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1570.2244
X-RAY DIFFRACTIONr_metal_ion_refined0.2580.213
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1980.213
X-RAY DIFFRACTIONr_nbd_other0.2770.262
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.2140.221
X-RAY DIFFRACTIONr_mcbond_it0.7370.942398
X-RAY DIFFRACTIONr_mcbond_other0.7370.942397
X-RAY DIFFRACTIONr_mcangle_it1.3031.4073012
X-RAY DIFFRACTIONr_mcangle_other1.3031.4083013
X-RAY DIFFRACTIONr_scbond_it0.9461.1342034
X-RAY DIFFRACTIONr_scbond_other0.9461.1352035
X-RAY DIFFRACTIONr_scangle_it1.5611.6373018
X-RAY DIFFRACTIONr_scangle_other1.5611.6383019
X-RAY DIFFRACTIONr_lrange_it3.08311.9024973
X-RAY DIFFRACTIONr_lrange_other2.95411.7564917
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
1.7-1.7440.311800.30543380.30545150.8240.82397.85160.287
1.744-1.7920.324870.26543010.26744310.8380.86599.02960.246
1.792-1.8440.277710.23742220.23843180.8710.89599.4210.215
1.844-1.9010.255640.2341040.23141880.8970.999.52240.21
1.901-1.9630.264940.22339990.22441050.8930.90599.70770.207
1.963-2.0320.283640.21538470.21639210.8780.91799.7450.201
2.032-2.1080.267590.21237230.21337890.9080.92899.81530.199
2.108-2.1940.246650.20736040.20836790.9190.93699.72820.198
2.194-2.2920.26540.18534580.18635240.9140.94399.65950.176
2.292-2.4040.225590.18132920.18233680.9250.94199.49520.173
2.404-2.5330.176530.18931600.18832270.9520.93899.56620.179
2.533-2.6870.205440.17829920.17830550.9370.94799.37810.172
2.687-2.8720.223520.17928050.1828760.9270.94599.33940.173
2.872-3.1020.217420.18226060.18326830.9410.94698.69550.179
3.102-3.3970.175390.17524150.17524990.9660.95798.19930.177
3.397-3.7970.155410.15721310.15722440.9670.96996.79140.162
3.797-4.3830.186250.13719150.13820190.9670.97796.08720.144
4.383-5.3630.222310.15316300.15417170.9590.97696.73850.163
5.363-7.5640.142190.19113010.1913670.9640.95896.56180.193
7.564-73.3930.274130.1816820.1838150.930.96185.27610.19
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)
10.2147-0.06760.02280.1270.01260.2096-0.02720.0377-0.0293-0.009-0.0066-0.01590.0020.01570.03380.0082-0.00810.00830.0133-0.00160.0195-3.3494-4.32212.6772
20.22180.0144-0.15760.1226-0.04440.19460.01530.02710.00590.0024-0.02510.0081-0.0051-0.04070.00980.0017-0.00090.00160.0205-0.00840.0222-17.8812-0.332816.9421
30.07620.0257-0.0460.30550.07790.0938-0.00560.01090.02380.04890.0076-0.05260.0189-0.0116-0.0020.0090-0.01210.0027-0.0030.0475-2.9334.604646.0986
40.0099-0.0098-0.01840.2627-0.03890.2707-0.01-0.00120.0130.023-0.0060.01650.0647-0.03860.0160.0195-0.01010.0070.0079-0.01130.032-16.7864-1.265842.8901
Refinement TLS group
IDRefine-IDRefine TLS-IDSelectionAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1ALLAAA4 - 158
2X-RAY DIFFRACTION2ALLBBB179 - 313
3X-RAY DIFFRACTION3ALLCCC2 - 160
4X-RAY DIFFRACTION4ALLDDD179 - 312

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