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- PDB-6v24: Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp... -

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Basic information

Entry
Database: PDB / ID: 6v24
TitleComplex of mutant (K162M) of E. coli L-asparaginase II with L-Asp. Covalent acyl-enzyme intermediate.
Components(L-asparaginase 2) x 2
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / catalytically deficient 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: 1.9 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 / Derived calculations / Category: citation / citation_author / struct_conn
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)142,8526
Polymers142,5864
Non-polymers2662
Water18,2131011
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17200 Å2
ΔGint-56 kcal/mol
Surface area39790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.331, 126.117, 76.192
Angle α, β, γ (deg.)90.00, 96.32, 90.00
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
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1011 / 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: 2.07 Å3/Da / Density % sol: 40.67 %
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: Jan 20, 2019 / 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: 1.9→40 Å / Num. obs: 90928 / % possible obs: 98.5 % / Redundancy: 3.4 % / Rmerge(I) obs: 0.062 / Rpim(I) all: 0.038 / Rrim(I) all: 0.073 / Χ2: 0.793 / Net I/σ(I): 8.8 / Num. measured all: 312393
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.9-1.932.60.45842210.6160.3240.5650.77492.1
1.93-1.972.80.39943650.7480.2720.4860.76494.6
1.97-2.0130.37444360.7940.2480.4510.76696.2
2.01-2.053.10.35844790.8180.2320.4290.83197.9
2.05-2.093.20.30545540.8570.1960.3640.81998.6
2.09-2.143.20.26345260.8810.1720.3160.84598.7
2.14-2.193.10.22645220.9120.1510.2740.83298.4
2.19-2.253.40.19145930.9410.120.2270.81699.6
2.25-2.323.80.17545880.960.1030.2040.83699.8
2.32-2.393.80.14646080.9730.0850.170.8199.8
2.39-2.483.80.12945810.9770.0760.1510.8499.8
2.48-2.583.70.10745960.9830.0640.1250.81899.8
2.58-2.73.70.09245880.9880.0550.1070.78999.7
2.7-2.843.60.07446100.990.0450.0870.82199.5
2.84-3.023.40.05545640.9930.0340.0660.76899.4
3.02-3.253.20.04245800.9950.0270.050.7799
3.25-3.583.90.03446080.9970.0190.0390.78199.8
3.58-4.093.90.02646240.9980.0150.030.79199.8
4.09-5.153.80.02146400.9980.0120.0240.72599.8
5.15-403.60.01746450.9980.010.020.65598.7

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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, monomer A

3eca


Resolution: 1.9→39.58 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.95 / SU B: 3.005 / SU ML: 0.086 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.141 / ESU R Free: 0.132 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING
RfactorNum. reflection% reflectionSelection details
Rfree0.19 4455 5 %RANDOM
Rwork0.143 ---
obs0.145 85330 97.6 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 22.09 Å2
Baniso -1Baniso -2Baniso -3
1-0.15 Å20 Å2-0.06 Å2
2---0.07 Å20 Å2
3----0.07 Å2
Refinement stepCycle: LAST / Resolution: 1.9→39.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9786 0 18 1011 10815
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0139974
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179202
X-RAY DIFFRACTIONr_angle_refined_deg2.0611.65213581
X-RAY DIFFRACTIONr_angle_other_deg2.7281.58721400
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.21151303
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.92524.831443
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.68151609
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.0541532
X-RAY DIFFRACTIONr_chiral_restr0.0990.21393
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.0211285
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021819
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.9→1.95 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.311 303 -
Rwork0.284 5654 -
obs--87.69 %

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