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- PDB-6nx8: ECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 6.2 -

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Basic information

Entry
Database: PDB / ID: 6nx8
TitleECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 6.2
ComponentsL-asparaginase 2Asparaginase
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 / CITRIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.85 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,3424
Polymers71,0902
Non-polymers2522
Water7,566420
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-61 kcal/mol
Surface area41460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)126.280, 126.280, 89.686
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 / Asparaginase / L-asparaginase II / L-ASNase II / L-asparagine amidohydrolase II


Mass: 35544.820 Da / Num. of mol.: 2 / Mutation: D90T, 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 / 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 / Citric acid


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 420 / 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: 2.9 Å3/Da / Density % sol: 57.64 %
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 6.2, 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: 6.2-6.3 / Temp details: incubator-controlled

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Data collection

DiffractionMean temperature: 298 K / Ambient temp details: room temperature / 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: May 11, 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: 1.85→40 Å / Num. obs: 70498 / % possible obs: 99.9 % / Redundancy: 4.7 % / Rmerge(I) obs: 0.071 / Rpim(I) all: 0.036 / Rrim(I) all: 0.08 / Χ2: 0.866 / Net I/σ(I): 12.2 / Num. measured all: 333956
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.85-1.883.90.7534800.6720.420.8640.9299.7
1.88-1.924.20.66335100.7370.3650.760.917100
1.92-1.954.40.57334910.8030.3090.6540.945100
1.95-1.994.50.49734870.8570.2610.5630.927100
1.99-2.044.70.45835200.8950.2350.5160.935100
2.04-2.084.80.36334840.9230.1840.4080.922100
2.08-2.144.80.30434880.9480.1530.3410.991100
2.14-2.194.80.24935120.9650.1250.280.929100
2.19-2.264.90.2234980.9720.110.2460.923100
2.26-2.334.90.18535110.9780.0920.2070.931100
2.33-2.414.90.15535230.9860.0770.1730.90799.9
2.41-2.514.90.13535000.9880.0670.1510.90899.9
2.51-2.624.90.11135220.9910.0550.1250.87699.9
2.62-2.764.90.09335040.9920.0460.1040.84499.8
2.76-2.944.90.07235450.9940.0360.0810.845100
2.94-3.164.80.05335460.9960.0270.060.818100
3.16-3.484.80.03935370.9970.020.0440.81299.8
3.48-3.984.70.03135740.9980.0160.0350.799100
3.98-5.024.90.02535910.9980.0120.0280.701100
5.02-405.10.01936750.9990.0090.0210.55498.6

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.8.0158refinement
PDB_EXTRACT3.24data extraction
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.85→40 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.972 / SU B: 2.194 / SU ML: 0.062 / SU R Cruickshank DPI: 0.0827 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.083 / ESU R Free: 0.082
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1555 2047 3 %RANDOM
Rwork0.1292 ---
obs0.13 66360 96.95 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 112.92 Å2 / Biso mean: 25.678 Å2 / Biso min: 11.57 Å2
Baniso -1Baniso -2Baniso -3
1--0.07 Å2-0.03 Å20 Å2
2---0.07 Å2-0 Å2
3---0.22 Å2
Refinement stepCycle: final / Resolution: 1.85→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4672 0 17 424 5113
Biso mean--43.3 36.35 -
Num. residues----624
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.024891
X-RAY DIFFRACTIONr_bond_other_d0.0020.024521
X-RAY DIFFRACTIONr_angle_refined_deg1.9381.9586704
X-RAY DIFFRACTIONr_angle_other_deg1.072310544
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0085663
X-RAY DIFFRACTIONr_dihedral_angle_2_deg42.69726.068206
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.88215804
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.5061516
X-RAY DIFFRACTIONr_chiral_restr0.1280.2810
X-RAY DIFFRACTIONr_gen_planes_refined0.010.0215550
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02878
LS refinement shellResolution: 1.85→1.898 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.272 112 -
Rwork0.233 3804 -
all-3916 -
obs--76.19 %

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