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- PDB-6v26: Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp -

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Basic information

Entry
Database: PDB / ID: 6v26
TitleComplex of mutant (K162M) of E. coli L-asparaginase II with L-Asp
ComponentsL-asparaginase 2Asparaginase
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / inactive mutant
Function / homology
Function and homology information


asparagine catabolic process / asparaginase / asparaginase activity / outer membrane-bounded periplasmic space / protein homotetramerization / periplasmic space / protein-containing complex / identical protein binding
Similarity search - Function
L-asparaginase, N-terminal domain / Rossmann fold - #40 / L-asparaginase, type II / Asparaginase/glutaminase, active site 1 / Asparaginase / glutaminase active site signature 1. / L-asparaginase, C-terminal / Asparaginase/glutaminase, active site 2 / Asparaginase/glutaminase, C-terminal / Glutaminase/Asparaginase C-terminal domain / Asparaginase / glutaminase active site signature 2. ...L-asparaginase, N-terminal domain / Rossmann fold - #40 / L-asparaginase, type II / Asparaginase/glutaminase, active site 1 / Asparaginase / glutaminase active site signature 1. / L-asparaginase, C-terminal / Asparaginase/glutaminase, active site 2 / Asparaginase/glutaminase, C-terminal / Glutaminase/Asparaginase C-terminal domain / Asparaginase / glutaminase active site signature 2. / Asparaginase / Asparaginase/glutaminase-like / L-asparaginase, N-terminal / Asparaginase/glutaminase-like superfamily / L-asparaginase, N-terminal domain superfamily / Asparaginase, N-terminal / Asparaginase / glutaminase domain profile. / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ASPARTIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsLubkowski, J. / Wlodawer, A.
CitationJournal: Biochemistry / Year: 2020
Title: Mechanism of Catalysis by l-Asparaginase.
Authors: Lubkowski, J. / Vanegas, J. / Chan, W.K. / Lorenzi, P.L. / Weinstein, J.N. / Sukharev, S. / Fushman, D. / Rempe, S. / Anishkin, A. / Wlodawer, A.
History
DepositionNov 22, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation / citation_author
Item: _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 10, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: L-asparaginase 2
B: L-asparaginase 2
C: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,8888
Polymers142,3564
Non-polymers5324
Water22,3931243
1
A: L-asparaginase 2
B: L-asparaginase 2
hetero molecules

A: L-asparaginase 2
B: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,8888
Polymers142,3564
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area16000 Å2
ΔGint-52 kcal/mol
Surface area39690 Å2
MethodPISA
2
C: L-asparaginase 2
D: L-asparaginase 2
hetero molecules

C: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,8888
Polymers142,3564
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area17110 Å2
ΔGint-57 kcal/mol
Surface area39860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.017, 62.287, 140.723
Angle α, β, γ (deg.)90.000, 117.540, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-617-

HOH

21C-682-

HOH

31C-851-

HOH

41D-632-

HOH

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Components

#1: Protein
L-asparaginase 2 / Asparaginase / L-asparaginase II / L-ASNase II / L-asparagine amidohydrolase II


Mass: 35588.895 Da / Num. of mol.: 4 / Mutation: K162M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ansB, b2957, JW2924 / Plasmid: pET-22b(+)
Details (production host): ORF contains a secretion sequence, 'HHHHHH' affinity tag and sequence of doubly mutated mature EcAII
Cell (production host): mesophilic bacteria / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): JC2 / References: UniProt: P00805, asparaginase
#2: Chemical
ChemComp-ASP / ASPARTIC ACID / Aspartic acid


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1243 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.06 Å3/Da / Density % sol: 40.33 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 0.1 M sodium acetate, pH 5.6, 5 mM L-Asp, and 18-20% (w/v) PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Jul 17, 2018 / Details: Multilayer X-ray mirrors VariMax HF
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.863
11-H, -K, H+L20.137
ReflectionResolution: 1.8→40 Å / Num. obs: 99158 / % possible obs: 92.3 % / Redundancy: 3 % / Rmerge(I) obs: 0.073 / Rpim(I) all: 0.048 / Rrim(I) all: 0.088 / Χ2: 0.82 / Net I/σ(I): 11.5 / Num. measured all: 296557
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.8-1.832.20.32433020.7490.2520.4130.43461.5
1.83-1.862.30.32137730.7780.2420.4040.47170.8
1.86-1.92.50.26144500.8480.1940.3270.49782.7
1.9-1.9430.22846920.8730.1550.2770.54787.9
1.94-1.983.10.20347310.8980.1350.2450.58588.9
1.98-2.033.10.18548100.9150.1220.2220.57490.6
2.03-2.083.10.1649750.9260.1060.1930.67192.4
2.08-2.133.10.14550040.9390.0950.1740.77494.2
2.13-2.23.10.1351590.9430.0850.1560.8196.4
2.2-2.273.10.1252410.9490.0780.1440.88697.3
2.27-2.353.10.11351740.9550.0730.1350.96197.5
2.35-2.443.10.10552550.9610.0680.1260.99597.9
2.44-2.553.10.09552700.9630.0610.1131.06898
2.55-2.693.10.09152720.9630.0590.1091.10698
2.69-2.863.10.08452720.9570.0550.1011.13698.4
2.86-3.083.10.08153060.9520.0530.0971.14698.5
3.08-3.3930.06553390.9760.0430.0780.98498.7
3.39-3.8830.05853540.9780.0390.070.87298.9
3.88-4.882.90.05353070.9820.0350.0640.68998.1
4.88-403.10.04854720.9880.0310.0570.52798.2

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.8.0158refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3eca

3eca


Resolution: 1.8→24.95 Å / Cor.coef. Fo:Fc: 0.931 / Cor.coef. Fo:Fc free: 0.927 / SU B: 1.996 / SU ML: 0.068 / SU R Cruickshank DPI: 0.0347 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.035 / ESU R Free: 0.027
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.196 4755 4.8 %RANDOM
Rwork0.1861 ---
obs0.1866 93692 91.1 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 95.5 Å2 / Biso mean: 25.808 Å2 / Biso min: 11.96 Å2
Baniso -1Baniso -2Baniso -3
1--0.75 Å20 Å2-1.43 Å2
2---1.71 Å2-0 Å2
3---2.46 Å2
Refinement stepCycle: final / Resolution: 1.8→24.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9581 0 36 1250 10867
Biso mean--23.44 32.52 -
Num. residues----1284
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.029798
X-RAY DIFFRACTIONr_bond_other_d0.0030.029059
X-RAY DIFFRACTIONr_angle_refined_deg1.961.95913362
X-RAY DIFFRACTIONr_angle_other_deg1.129321077
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.77851292
X-RAY DIFFRACTIONr_dihedral_angle_2_deg42.36226.074405
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.775151595
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.1561532
X-RAY DIFFRACTIONr_chiral_restr0.1190.21605
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02111073
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021761
LS refinement shellResolution: 1.8→1.83 Å

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