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- PDB-6v25: Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp -

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Basic information

Entry
Database: PDB / ID: 6v25
TitleComplex of mutant (K162M) of E. coli L-asparaginase II with L-Asp
Components(L-asparaginase 2) x 2
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / inactive mutant
Function / homology
Function and homology information


L-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, 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, 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.
Similarity search - Domain/homology
ASPARTIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.78 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
C: L-asparaginase 2
B: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,8526
Polymers142,5864
Non-polymers2662
Water26,2301456
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16600 Å2
ΔGint-51 kcal/mol
Surface area40460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)61.530, 123.967, 76.302
Angle α, β, γ (deg.)90.000, 96.800, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 35588.895 Da / Num. of mol.: 2 / 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: Protein L-asparaginase 2 / L-asparaginase II / L-ASNase II / L-asparagine amidohydrolase II


Mass: 35703.980 Da / Num. of mol.: 2 / 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
#3: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1456 / 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: 2.02 Å3/Da / Density % sol: 39.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.2
Details: 0.1 M sodium acetate, pH 5.2, 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: May 22, 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
ReflectionResolution: 1.78→40 Å / Num. obs: 102698 / % possible obs: 94.3 % / Redundancy: 2.8 % / Rmerge(I) obs: 0.024 / Rpim(I) all: 0.017 / Rrim(I) all: 0.029 / Χ2: 0.736 / Net I/σ(I): 21.3 / Num. measured all: 288124
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.78-1.811.90.30933700.8290.2460.3980.65962.3
1.81-1.8420.24839030.8830.190.3150.6571.7
1.84-1.882.20.21743080.9130.1620.2720.63778.9
1.88-1.922.30.18750040.9360.1410.2350.68292.7
1.92-1.962.70.15853570.9530.1140.1960.66998.6
1.96-22.80.13753820.9650.0980.1690.67498.8
2-2.052.80.11553520.9740.0820.1420.6998.9
2.05-2.112.80.09253680.9810.0660.1130.69698.9
2.11-2.172.80.07453780.9860.0520.0910.70199.1
2.17-2.242.90.06354110.9890.0440.0780.75399.2
2.24-2.322.90.05153860.9920.0360.0630.75899.2
2.32-2.422.90.04353710.9930.030.0530.72998.9
2.42-2.532.90.03753820.9940.0260.0460.77499.1
2.53-2.662.90.03154250.9950.0210.0380.79499
2.66-2.832.90.02653830.9960.0180.0310.79898.7
2.83-3.0430.0253410.9970.0140.0250.81598
3.04-3.3530.01753210.9980.0120.0210.84497.8
3.35-3.833.10.01553810.9980.010.0180.85297.8
3.83-4.833.10.01453660.9980.0090.0160.72398.4
4.83-403.50.01455090.9980.0090.0170.6599.1

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Processing

Software
NameVersionClassification
REFMAC5.8.0253refinement
HKL-2000data reduction
HKL-2000data scaling
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3eca

3eca


Resolution: 1.78→24.61 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.952 / WRfactor Rfree: 0.179 / WRfactor Rwork: 0.1263 / FOM work R set: 0.8695 / SU B: 2.699 / SU ML: 0.083 / SU R Cruickshank DPI: 0.114 / SU Rfree: 0.1194 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.114 / ESU R Free: 0.119 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1928 2442 2.4 %RANDOM
Rwork0.136 ---
obs0.1374 98474 93.02 %-
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 max: 120.4 Å2 / Biso mean: 29.504 Å2 / Biso min: 8 Å2
Baniso -1Baniso -2Baniso -3
1-0.02 Å20 Å20.09 Å2
2---0.06 Å20 Å2
3---0.02 Å2
Refinement stepCycle: final / Resolution: 1.78→24.61 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9653 0 18 1484 11155
Biso mean--28.8 39.6 -
Num. residues----1289
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0139849
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179088
X-RAY DIFFRACTIONr_angle_refined_deg2.2221.6513431
X-RAY DIFFRACTIONr_angle_other_deg2.1071.58521139
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.22251288
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.23724.819442
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.121151592
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9011532
X-RAY DIFFRACTIONr_chiral_restr0.1150.21376
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.0211174
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021805
LS refinement shellResolution: 1.78→1.826 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.284 103 -
Rwork0.205 4408 -
all-4511 -
obs--56.58 %

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