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- PDB-6nx7: ECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 5.6 -

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Basic information

Entry
Database: PDB / ID: 6nx7
TitleECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 5.6
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
ACETIC ACID / CITRIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 2.15 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
Revision 1.3Oct 23, 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

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,3424
Polymers71,0902
Non-polymers2522
Water5,603311
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)142,6848
Polymers142,1794
Non-polymers5044
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_555x-y,-y,-z+2/31
Buried area14550 Å2
ΔGint-60 kcal/mol
Surface area41590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)126.199, 126.199, 89.439
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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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.: 2 / 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.: 1 / Source method: obtained synthetically / Formula: C2H4O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 311 / Source method: isolated from a natural source / Formula: H2O
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: 2.89 Å3/Da / Density % sol: 57.44 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
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, 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, 0.17 M ammonium citrate pH 5.6, and 20 mM L-Asn. Resulting droplets were equilibrated against the precipitant. For the data collection, crystal was mounted in a quartz capillary
PH range: 5.9-6 / Temp details: incubator-controlled

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Data collection

DiffractionMean temperature: 298 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: Jun 10, 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: 2.15→40 Å / Num. obs: 44670 / % possible obs: 99.3 % / Redundancy: 3.8 % / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.059 / Rrim(I) all: 0.118 / Χ2: 0.837 / Net I/σ(I): 7.4 / Num. measured all: 168511
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.15-2.193.50.63222310.6720.3850.7440.89699.5
2.19-2.233.60.58421860.7460.3490.6840.90499.5
2.23-2.273.70.54822110.7710.3260.6410.90899.4
2.27-2.323.80.47322250.8010.2770.5510.87599.5
2.32-2.373.80.422010.8440.2320.4650.89699.4
2.37-2.423.90.3422280.8960.1940.3930.82999.2
2.42-2.483.90.31422180.9050.1790.3630.8199.4
2.48-2.553.90.27421970.9220.1560.3170.77299.2
2.55-2.623.90.24822460.9260.1420.2870.79299.4
2.62-2.713.90.21822060.9410.1240.2520.78199.5
2.71-2.813.80.18122360.9560.1030.2090.80399.4
2.81-2.923.80.15422170.9660.0880.1780.78399.5
2.92-3.053.80.11622060.9770.0660.1350.79799.3
3.05-3.213.80.09622510.9850.0540.110.95799.4
3.21-3.413.80.07322420.9890.0410.0841.11699.5
3.41-3.683.80.06122520.9920.0340.070.79799.9
3.68-4.053.80.04722740.9950.0270.0550.696100
4.05-4.633.70.04122360.9950.0230.0470.79498.5
4.63-5.833.70.03722340.9950.0210.0430.85797.5
5.83-403.70.02623730.9960.0150.030.69698.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0158refinement
HKL-3000data reduction
HKL-3000data scaling
PDB_EXTRACT3.24data extraction
REFMAC5.8.0158phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 2.15→40 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.956 / WRfactor Rfree: 0.157 / WRfactor Rwork: 0.1117 / FOM work R set: 0.8673 / SU B: 4.226 / SU ML: 0.102 / SU R Cruickshank DPI: 0.14 / SU Rfree: 0.1393 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.14 / ESU R Free: 0.139 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1828 2205 5 %RANDOM
Rwork0.1324 ---
obs0.1348 42085 98.44 %-
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: 141.67 Å2 / Biso mean: 31.95 Å2 / Biso min: 12.47 Å2
Baniso -1Baniso -2Baniso -3
1--0.01 Å2-0.01 Å2-0 Å2
2---0.01 Å20 Å2
3---0.04 Å2
Refinement stepCycle: final / Resolution: 2.15→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4670 0 17 314 5001
Biso mean--51.7 37.49 -
Num. residues----624
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.024829
X-RAY DIFFRACTIONr_bond_other_d0.0020.024445
X-RAY DIFFRACTIONr_angle_refined_deg1.9671.9576602
X-RAY DIFFRACTIONr_angle_other_deg1.059310350
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4595642
X-RAY DIFFRACTIONr_dihedral_angle_2_deg42.55425.941202
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.61815782
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.0131516
X-RAY DIFFRACTIONr_chiral_restr0.1160.2794
X-RAY DIFFRACTIONr_gen_planes_refined0.010.0215466
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02870
LS refinement shellResolution: 2.15→2.206 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.25 151 -
Rwork0.216 2926 -
all-3077 -
obs--93.95 %

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