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- PDB-6pa3: E. coli L-asparaginase II double mutant (T89V,K162T) in complex w... -

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Basic information

Entry
Database: PDB / ID: 6pa3
TitleE. coli L-asparaginase II double mutant (T89V,K162T) 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


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 / IMIDAZOLE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.65 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 1.3Aug 19, 2020Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.4Oct 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 / _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)143,44812
Polymers142,6884
Non-polymers7608
Water21,1681175
1
A: L-asparaginase 2
B: L-asparaginase 2
hetero molecules

C: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,44812
Polymers142,6884
Non-polymers7608
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area10340 Å2
ΔGint-46 kcal/mol
Surface area50080 Å2
MethodPISA
Unit cell
Length a, b, c (Å)152.852, 62.990, 141.293
Angle α, β, γ (deg.)90.000, 117.830, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-711-

HOH

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Components

#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
#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-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1175 / Source method: isolated from a natural source / Formula: H2O
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: 2.11 Å3/Da / Density % sol: 41.65 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Crystals were grown in 0.17 M NH4-citrate, pH 7.0, 17-18% PEG3350, 10 mM L-Asn

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Mar 5, 2016
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.65→50 Å / Num. obs: 141828 / % possible obs: 99.7 % / Redundancy: 3.7 % / Rmerge(I) obs: 0.077 / Rpim(I) all: 0.046 / Rrim(I) all: 0.09 / Χ2: 0.991 / Net I/σ(I): 8.6 / Num. measured all: 526021
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.65-1.713.10.431136670.7820.2880.5210.64997
1.71-1.783.70.343141410.8930.2070.4020.684100
1.78-1.863.70.246141710.9390.1480.2880.751100
1.86-1.963.80.173142210.9690.1040.2020.831100
1.96-2.083.80.129141370.980.0770.150.95100
2.08-2.243.80.097142340.9880.0580.1131.064100
2.24-2.463.80.079141600.9910.0470.0921.15100
2.46-2.823.80.067142940.9930.040.0781.158100
2.82-3.553.80.068142760.9930.040.0791.454100
3.55-503.70.054145270.9940.0330.0631.10199.8

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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: 1.65→40 Å / Cor.coef. Fo:Fc: 0.971 / Cor.coef. Fo:Fc free: 0.957 / SU B: 3.764 / SU ML: 0.065 / SU R Cruickshank DPI: 0.0853 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.085 / ESU R Free: 0.087
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1841 2723 1.9 %RANDOM
Rwork0.1493 ---
obs0.15 139105 99.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 106.75 Å2 / Biso mean: 19.651 Å2 / Biso min: 9.16 Å2
Baniso -1Baniso -2Baniso -3
1-1.07 Å20 Å2-0.04 Å2
2---0.77 Å20 Å2
3----0.17 Å2
Refinement stepCycle: final / Resolution: 1.65→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9884 0 52 1175 11111
Biso mean--21.68 27.5 -
Num. residues----1322
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.01910127
X-RAY DIFFRACTIONr_bond_other_d0.0020.029300
X-RAY DIFFRACTIONr_angle_refined_deg1.9091.95213810
X-RAY DIFFRACTIONr_angle_other_deg1.125321629
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.20251330
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.85725.882425
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.069151624
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.6291532
X-RAY DIFFRACTIONr_chiral_restr0.1540.21649
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02111465
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021845
LS refinement shellResolution: 1.652→1.694 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.269 166 -
Rwork0.229 9596 -
all-9762 -
obs--93.6 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.21140.2638-0.02030.7737-0.09670.85960.0295-0.0688-0.12720.0917-0.0545-0.04040.08430.04760.0250.0227-0.0101-0.00290.07590.01490.01468.01919.23715.836
20.68890.00290.22140.7762-0.64981.886-0.00290.05170.09110.0092-0.0672-0.1347-0.15010.20020.07010.028-0.0387-0.0020.08410.01690.051217.69744.2572.024
30.8227-0.00120.24490.5801-0.15751.1568-0.02770.0550.1031-0.0325-0.0107-0.0793-0.06740.0740.03840.0113-0.00290.00960.00760.01120.0381-15.22643.76961.414
41.48570.4237-0.03860.6638-0.20140.78160.0633-0.1182-0.09840.0854-0.0252-0.05160.1164-0.0105-0.03810.05550.0014-0.01260.01310.010.0119-22.26118.7276.703
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 326
2X-RAY DIFFRACTION1A401
3X-RAY DIFFRACTION2B1 - 326
4X-RAY DIFFRACTION2B401
5X-RAY DIFFRACTION3C1 - 326
6X-RAY DIFFRACTION3C401
7X-RAY DIFFRACTION4D1 - 326
8X-RAY DIFFRACTION4D401

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