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- PDB-6nxa: ECAII(D90T,K162T) MUTANT AT PH 7 -

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Basic information

Entry
Database: PDB / ID: 6nxa
TitleECAII(D90T,K162T) MUTANT AT PH 7
ComponentsL-asparaginase 2
KeywordsHYDROLASE / inactive mutant / hydrolysis of L-asparagine
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
ACETIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.93 Å
AuthorsLubkowski, J. / Wlodawer, A.
Citation
Journal: Sci Rep / Year: 2019
Title: Opportunistic complexes of E. coli L-asparaginases with citrate anions.
Authors: Lubkowski, J. / Chan, W. / Wlodawer, A.
#1: Journal: J. Mol. Biol. / Year: 2007
Title: Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L-asparaginase I.
Authors: Yun, M.K. / Nourse, A. / White, S.W. / Rock, C.O. / Heath, R.J.
History
DepositionFeb 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Database references / Category: citation / citation_author
Item: _citation.page_last / _citation.pdbx_database_id_DOI ..._citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 19, 2020Group: Structure summary / Category: struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)142,69611
Polymers142,1794
Non-polymers5167
Water21,2761181
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)142,60410
Polymers142,1794
Non-polymers4246
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area14950 Å2
ΔGint-60 kcal/mol
Surface area41930 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)142,78812
Polymers142,1794
Non-polymers6098
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area15360 Å2
ΔGint-57 kcal/mol
Surface area41140 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.885, 62.509, 143.160
Angle α, β, γ (deg.)90.000, 118.190, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-687-

HOH

21A-779-

HOH

31B-747-

HOH

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Components

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


Mass: 35544.820 Da / Num. of mol.: 4 / Mutation: K162T
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 / Cell line (production host): JC2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (DE3) / Variant (production host): ansA, ansB, iaaA triple knockout / References: UniProt: P00805, asparaginase
#2: Chemical
ChemComp-ACY / ACETIC ACID


Mass: 60.052 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H4O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1181 / Source method: isolated from a natural source / Formula: H2O

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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: 2.11 Å3/Da / Density % sol: 41.61 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Protein, at the concentration 15 mg/ml in 50 mM HEPES buffer pH 7 and 150 mM sodium chloride was mixed with equivolume solution of precipitant that contained, 17% (w/v) PEG3350 and 0.17 M ...Details: Protein, at the concentration 15 mg/ml in 50 mM HEPES buffer pH 7 and 150 mM sodium chloride was mixed with equivolume solution of precipitant that contained, 17% (w/v) PEG3350 and 0.17 M ammonium citrate pH 7. Resulting droplets were equilibrated against the precipitant. For the data collection, crystal was briefly transferred to cryo-protecting solution, which had the same composition as precipitant, except concentration of PEG3350 was increased to 35 % (v/w/) and 10% (v/v), and also contained 15% of glycerol
Temp details: incubator-controlled

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Stream of liquid nitrogen / 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: Jul 2, 2018 / Details: Multilayer 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: 1.93→40 Å / Num. obs: 86035 / % possible obs: 96.1 % / Redundancy: 3.3 % / Rmerge(I) obs: 0.1 / Rpim(I) all: 0.064 / Rrim(I) all: 0.119 / Χ2: 0.923 / Net I/σ(I): 7.8 / Num. measured all: 284693
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.93-1.962.50.51235280.6460.3590.6280.98379
1.96-22.80.47840830.7090.3250.580.95591.7
2-2.043.20.46241310.7410.2950.5490.95393.4
2.04-2.083.20.39441650.7910.250.4680.94693.5
2.08-2.123.30.33941760.8330.2140.4020.95394.2
2.12-2.173.30.28842320.8730.1810.3410.95694
2.17-2.233.40.25741700.8920.1610.3040.96194
2.23-2.293.40.22742950.9160.1430.2690.96596.3
2.29-2.363.40.18943060.9410.1190.2240.94197.6
2.36-2.433.50.17543640.9510.110.2070.97597.5
2.43-2.523.50.15343790.9590.0960.1810.9597.8
2.52-2.623.50.1443820.9650.0880.1660.95198.2
2.62-2.743.50.12244270.9740.0770.1450.94798.6
2.74-2.883.40.10543980.9790.0670.1250.93198.9
2.88-3.063.40.08444280.9860.0540.10.92199.2
3.06-3.33.40.06744660.9910.0430.080.90998.9
3.3-3.633.40.05444700.9930.0350.0640.92299.7
3.63-4.163.30.0445050.9960.0260.0480.87399.7
4.16-5.243.20.03345390.9970.0210.040.82599.8
5.24-403.50.02945910.9980.0180.0340.70899.1

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.8.0238refinement
PDB_EXTRACT3.24data extraction
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.93→26.54 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.914 / SU B: 4.329 / SU ML: 0.122 / SU R Cruickshank DPI: 0.1547 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.155 / ESU R Free: 0.161
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2274 2649 3.1 %RANDOM
Rwork0.1524 ---
obs0.1547 83055 95.95 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 114.3 Å2 / Biso mean: 33.522 Å2 / Biso min: 15.22 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å2-0 Å20.04 Å2
2---0.4 Å2-0 Å2
3---0.17 Å2
Refinement stepCycle: final / Resolution: 1.93→26.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9410 0 34 1184 10628
Biso mean--53.18 43.18 -
Num. residues----1256
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0139625
X-RAY DIFFRACTIONr_bond_other_d0.0010.0178898
X-RAY DIFFRACTIONr_angle_refined_deg2.3171.64713129
X-RAY DIFFRACTIONr_angle_other_deg1.5691.57920691
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.38651262
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.89124.745432
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.285151551
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.5381532
X-RAY DIFFRACTIONr_chiral_restr0.110.21352
X-RAY DIFFRACTIONr_gen_planes_refined0.0150.0210885
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021763
LS refinement shellResolution: 1.93→1.98 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.3 130 -
Rwork0.224 5222 -
all-5352 -
obs--81.67 %

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