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- PDB-6pa8: ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 7.0 -

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Basic information

Entry
Database: PDB / ID: 6pa8
TitleECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 7.0
ComponentsL-asparaginase 2
KeywordsHYDROLASE / inactive mutant / hydrolysis of L-asparagine
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, 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
ASPARAGINE / CITRATE ANION / IMIDAZOLE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.9 Å
AuthorsLubkowski, J. / Wlodawer, A.
CitationJournal: Protein Sci. / Year: 2019
Title: Geometric considerations support the double-displacement catalytic mechanism of l-asparaginase.
Authors: Lubkowski, J. / Wlodawer, A.
History
DepositionJun 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 2, 2019Group: Data collection / Database references / Category: citation_author / Item: _citation_author.identifier_ORCID
Revision 1.3Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entry_details.has_protein_modification

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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)143,75214
Polymers142,6884
Non-polymers1,06410
Water19,7801098
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)143,58714
Polymers142,6884
Non-polymers89910
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area17070 Å2
ΔGint-60 kcal/mol
Surface area41470 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)143,91714
Polymers142,6884
Non-polymers1,22910
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area18400 Å2
ΔGint-46 kcal/mol
Surface area41740 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.793, 62.746, 140.966
Angle α, β, γ (deg.)90.000, 117.710, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-662-

HOH

21B-678-

HOH

31C-693-

HOH

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Components

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Protein , 1 types, 4 molecules ABCD

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


Mass: 35671.914 Da / Num. of mol.: 4 / Mutation: T89V, K162T
Source method: isolated from a genetically manipulated source
Details: Expressed variant contains 8 additional N-terminal residues MDHHHHHH (affinity tag) and two mutations (T89V and K162T) in mature protein
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ansB, b2957, JW2924 / Plasmid: pET22b(+)
Details (production host): contains secretion sequence pelB leader
Cell (production host): bacteria / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): JC2 strain lacking in ansA / References: UniProt: P00805, asparaginase

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Non-polymers , 6 types, 1108 molecules

#2: Chemical
ChemComp-ASN / ASPARAGINE


Type: L-peptide linking / Mass: 132.118 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H8N2O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H5N2
#6: Chemical ChemComp-FLC / CITRATE ANION


Mass: 189.100 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H5O7
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1098 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: 40.94 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Crystals were grown from 0.17 M NH4-citrate, pH 7.0, 17-18% PEG3350, then soaked in equivalent solution containing L-Asn at concentration 0.5 mM

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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 R 4M / Detector: PIXEL / Date: Sep 12, 2018 / Details: X-ray mirrors VariMax HF
RadiationMonochromator: Multilayer X-ray mirrors VariMax HF / Protocol: 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: 88385 / % possible obs: 95.3 % / Redundancy: 3 % / Rmerge(I) obs: 0.044 / Rpim(I) all: 0.029 / Rrim(I) all: 0.053 / Χ2: 0.854 / Net I/σ(I): 13.6 / Num. measured all: 262551
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.9-1.9320.42535980.7360.3410.5480.90778.5
1.93-1.972.20.35439340.8040.2720.4490.9285.3
1.97-2.012.50.32643630.8420.2340.4040.91293.8
2.01-2.052.70.30144190.8760.2050.3660.89796.3
2.05-2.092.70.24743910.9120.1660.2990.90395.4
2.09-2.142.80.20643600.9440.1370.2490.93494.9
2.14-2.192.80.16843330.9620.1110.2020.95193.9
2.19-2.252.90.1543340.9650.10.1810.95894.2
2.25-2.3230.12743420.9730.0840.1530.96393.4
2.32-2.3930.10743550.9810.070.1290.95894.5
2.39-2.4830.09844140.9850.0640.1180.92394.8
2.48-2.583.10.08144300.9880.0530.0970.90396.2
2.58-2.73.20.06945050.9910.0450.0830.85597.1
2.7-2.843.20.05645610.9930.0360.0670.84398.4
2.84-3.023.30.04545980.9950.0290.0530.84499.3
3.02-3.253.30.03446290.9970.0220.040.78499.6
3.25-3.583.30.02646700.9980.0160.030.76999.9
3.58-4.093.30.0246650.9980.0130.0240.788100
4.09-5.153.20.01747160.9990.0110.020.728100
5.15-403.50.01647680.9990.010.0190.6299.3

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
REFMAC5.8.0158refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.9→40 Å / Cor.coef. Fo:Fc: 0.968 / Cor.coef. Fo:Fc free: 0.948 / WRfactor Rfree: 0.1736 / WRfactor Rwork: 0.1314 / FOM work R set: 0.8692 / SU B: 3.328 / SU ML: 0.095 / SU R Cruickshank DPI: 0.1507 / SU Rfree: 0.1379 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.151 / ESU R Free: 0.138 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1941 4394 5 %RANDOM
Rwork0.1465 ---
obs0.1489 83883 95.12 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 92.48 Å2 / Biso mean: 22.727 Å2 / Biso min: 10.58 Å2
Baniso -1Baniso -2Baniso -3
1-0.04 Å20 Å20.07 Å2
2---0.18 Å20 Å2
3---0.04 Å2
Refinement stepCycle: final / Resolution: 1.9→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9760 0 72 1098 10930
Biso mean--30.11 29.69 -
Num. residues----1305
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.01910024
X-RAY DIFFRACTIONr_bond_other_d0.0020.029219
X-RAY DIFFRACTIONr_angle_refined_deg1.8521.95513670
X-RAY DIFFRACTIONr_angle_other_deg1.065321442
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.47351314
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.93125.929420
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.898151608
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.5521532
X-RAY DIFFRACTIONr_chiral_restr0.1220.21636
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02111330
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021815
LS refinement shellResolution: 1.9→1.949 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.309 258 -
Rwork0.26 5102 -
all-5360 -
obs--78.57 %

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