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- PDB-6v2g: Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp... -

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Basic information

Entry
Database: PDB / ID: 6v2g
TitleComplex of mutant (K162M) of E. coli L-asparaginase II with L-Asp. Covalent acyl-enzyme intermediate and tetrahedral intermediate.
ComponentsL-asparaginase 2
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / catalytically deficient 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 / cytosol
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
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 / 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: Advisory / Data collection ...Advisory / 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 / pdbx_unobs_or_zero_occ_residues
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


Theoretical massNumber of molelcules
Total (without water)142,8164
Polymers142,8164
Non-polymers00
Water19,9971110
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17150 Å2
ΔGint-52 kcal/mol
Surface area40000 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.097, 125.971, 76.376
Angle α, β, γ (deg.)90.00, 96.70, 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: 35703.980 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1110 / 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: 40.54 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 0.17 M sodium-citrate pH 6 and 18% (w/v) PEG3350, 5 mM L-Asp

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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: Apr 5, 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: 90100 / % possible obs: 97.4 % / Redundancy: 3.2 % / Rmerge(I) obs: 0.044 / Rpim(I) all: 0.029 / Rrim(I) all: 0.053 / Χ2: 0.871 / Net I/σ(I): 13.7 / Num. measured all: 289481
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.9-1.932.30.52333600.4840.3930.6580.97972.6
1.93-1.972.50.43637430.7620.3190.5440.93481.7
1.97-2.012.70.3945490.7990.2790.4820.90898.3
2.01-2.0530.34545500.830.2380.4210.95599
2.05-2.093.10.27245580.890.1850.3310.92799.1
2.09-2.143.10.22946130.9220.1540.2770.94899.4
2.14-2.193.20.19645720.9450.130.2370.93699.5
2.19-2.253.30.16346130.960.1070.1960.94699.7
2.25-2.323.30.14446280.970.0930.1720.9399.8
2.32-2.393.40.12246050.980.0780.1450.96499.8
2.39-2.483.40.10446190.9850.0660.1240.94199.9
2.48-2.583.40.08745860.990.0550.1030.93100
2.58-2.73.40.07646260.9920.0480.090.923100
2.7-2.843.40.05846500.9940.0370.0690.87899.9
2.84-3.023.40.04646080.9960.0290.0540.89899.8
3.02-3.253.40.03546040.9970.0220.0410.90499.7
3.25-3.583.40.02446060.9980.0150.0290.79699.5
3.58-4.093.40.01846540.9990.0120.0220.728100
4.09-5.153.30.01546680.9990.010.0180.585100
5.15-403.60.01546880.9990.0090.0170.58599.3

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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→24.93 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.941 / SU B: 3.884 / SU ML: 0.109 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.146 / ESU R Free: 0.143 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING
RfactorNum. reflection% reflectionSelection details
Rfree0.207 2673 3 %RANDOM
Rwork0.149 ---
obs0.151 85750 96.5 %-
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 mean: 32.28 Å2
Baniso -1Baniso -2Baniso -3
1--0.22 Å20 Å20.25 Å2
2--0.07 Å20 Å2
3---0.09 Å2
Refinement stepCycle: LAST / Resolution: 1.9→24.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9804 0 0 1110 10914
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0139874
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179110
X-RAY DIFFRACTIONr_angle_refined_deg2.2851.65813455
X-RAY DIFFRACTIONr_angle_other_deg2.0921.59221193
X-RAY DIFFRACTIONr_dihedral_angle_1_deg17.7625.3411318
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.93224.883426
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.136151570
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.7141532
X-RAY DIFFRACTIONr_chiral_restr0.1180.21384
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.0211999
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021793
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
Rfree0.272 140 -
Rwork0.227 4422 -
obs--67.36 %

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