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- PDB-6nx9: ECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 7 -

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Basic information

Entry
Database: PDB / ID: 6nx9
TitleECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE 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 / IMIDAZOLE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.97 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 16, 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
C: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,76512
Polymers142,1794
Non-polymers5868
Water18,7901043
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)142,55810
Polymers142,1794
Non-polymers3786
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area14690 Å2
ΔGint-56 kcal/mol
Surface area42320 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)142,97214
Polymers142,1794
Non-polymers79310
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area15910 Å2
ΔGint-63 kcal/mol
Surface area41460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)152.009, 62.539, 143.239
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-674-

HOH

21A-736-

HOH

31B-647-

HOH

41C-675-

HOH

51D-780-

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-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2 / Feature type: SUBJECT OF INVESTIGATION
#4: 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1043 / 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: 2.03 Å3/Da / Density % sol: 39.52 %
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. Before data collection, crystal was transferred the precipitant containing L-Asn at concentration 20 mM. Subsequently, for the data collection, crystal was briefly transferred to cryo-protecting solution, which had the same composition as precipitant (with L-Asn), 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: Apr 12, 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: 1.97→40 Å / Num. obs: 81136 / % possible obs: 96.6 % / Redundancy: 3.3 % / Rmerge(I) obs: 0.095 / Rpim(I) all: 0.061 / Rrim(I) all: 0.113 / Χ2: 0.827 / Net I/σ(I): 7.1 / Num. measured all: 263837
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.97-22.60.51139000.6740.3650.6310.88392.1
2-2.0430.49138650.6990.3220.5890.86193.9
2.04-2.083.10.40339300.7740.260.4820.88194
2.08-2.123.20.36339190.8120.2330.4330.88694.8
2.12-2.173.20.30239630.8560.1930.3590.83193.9
2.17-2.223.30.26739180.8760.1690.3170.82594.2
2.22-2.273.40.23139520.9040.1460.2740.79694.9
2.27-2.343.40.19539990.9230.1230.2310.79495.5
2.34-2.43.40.17540290.9380.1110.2080.78596.4
2.4-2.483.40.1641240.950.1010.1890.79798.1
2.48-2.573.40.1441050.9580.0890.1660.77598.3
2.57-2.673.40.13140730.9590.0830.1550.77898.1
2.67-2.83.30.1141190.9710.070.1310.76998.2
2.8-2.943.30.09241540.9770.0590.110.75698.4
2.94-3.133.30.07441090.9840.0470.0880.78798.1
3.13-3.373.30.05841440.9880.0370.0690.80898.9
3.37-3.713.30.04541720.9910.0290.0540.89498.5
3.71-4.243.20.03641540.9940.0230.0430.9698.4
4.24-5.343.10.0342090.9950.0190.0360.92798.8
5.34-403.30.02842980.9970.0180.0330.81798.3

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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: 1.97→26.55 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.923 / SU B: 4.546 / SU ML: 0.127 / SU R Cruickshank DPI: 0.1735 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.174 / ESU R Free: 0.167
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2257 2654 3.3 %RANDOM
Rwork0.1595 ---
obs0.1617 78318 96.3 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 95.87 Å2 / Biso mean: 25.501 Å2 / Biso min: 11.83 Å2
Baniso -1Baniso -2Baniso -3
1-0.08 Å2-0 Å2-0.01 Å2
2---0.3 Å2-0 Å2
3---0.14 Å2
Refinement stepCycle: final / Resolution: 1.97→26.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9410 0 39 1049 10498
Biso mean--41.55 34.22 -
Num. residues----1256
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0139631
X-RAY DIFFRACTIONr_bond_other_d0.0010.0178902
X-RAY DIFFRACTIONr_angle_refined_deg2.291.64713134
X-RAY DIFFRACTIONr_angle_other_deg1.5861.57920696
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.39951260
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.35624.734433
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.445151550
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.0791532
X-RAY DIFFRACTIONr_chiral_restr0.1130.21351
X-RAY DIFFRACTIONr_gen_planes_refined0.0150.0210899
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021769
LS refinement shellResolution: 1.971→2.022 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.353 177 -
Rwork0.27 5327 -
all-5504 -
obs--89.06 %

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