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- PDB-6paa: E. coli L-asparaginase II mutant (T12V) in complex with L-Asp at ... -

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Basic information

Entry
Database: PDB / ID: 6paa
TitleE. coli L-asparaginase II mutant (T12V) in complex with L-Asp at pH 5.5
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
ASPARTIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.85 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 1.3Oct 30, 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)143,3328
Polymers142,8004
Non-polymers5324
Water19,3661075
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)143,3328
Polymers142,8004
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area14700 Å2
ΔGint-50 kcal/mol
Surface area40440 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)143,3328
Polymers142,8004
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area14610 Å2
ΔGint-52 kcal/mol
Surface area40730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.837, 62.472, 143.373
Angle α, β, γ (deg.)90.000, 118.110, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-621-

HOH

21B-644-

HOH

31C-687-

HOH

41D-646-

HOH

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Components

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


Mass: 35699.988 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Expressed variant contains 8 additional N-terminal residues MDHHHHHH (affinity tag) and mutation (T12V) 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-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1075 / 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: 2.1 Å3/Da / Density % sol: 41.43 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: Crystals were grown 0.17 M NH4-citrate, pH 5.5, 17-18% PEG3350, 5 mM L-Asp

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Data collection

DiffractionMean temperature: 100 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: Nov 5, 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: 92363 / % possible obs: 91 % / Redundancy: 3 % / Rmerge(I) obs: 0.053 / Rpim(I) all: 0.035 / Rrim(I) all: 0.064 / Χ2: 0.914 / Net I/σ(I): 14.9 / Num. measured all: 277746
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.85-1.881.90.39427870.7260.350.5290.97955.1
1.88-1.9220.3732100.760.3110.4860.98663.6
1.92-1.952.20.33537290.8040.2710.4330.96573.4
1.95-1.992.30.32643770.8570.2520.4140.93487.7
1.99-2.042.60.32546730.8550.2350.4040.93293.3
2.04-2.082.70.27147560.8930.1910.3330.94793.4
2.08-2.142.80.22646930.930.1570.2770.9693.5
2.14-2.192.90.18746470.9490.1280.2270.95992.3
2.19-2.2630.15446040.9580.1040.1860.93991.3
2.26-2.333.20.13446740.9710.0880.1610.97492
2.33-2.413.20.10748540.980.070.1290.95895.4
2.41-2.513.20.09149060.9870.0590.1080.93697.6
2.51-2.623.30.07949760.990.0510.0940.93498.1
2.62-2.763.30.06750030.9920.0430.080.93298.8
2.76-2.943.40.05450470.9940.0340.0640.89599.1
2.94-3.163.40.04350300.9960.0280.0510.85699
3.16-3.483.30.03550420.9970.0220.0410.78699.4
3.48-3.983.30.03150890.9960.020.0370.75499.8
3.98-5.023.20.0351150.9970.0190.0350.76399.3
5.02-403.50.03951510.9970.0240.0461.08597.9

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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: 1.85→40 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.914 / WRfactor Rfree: 0.2174 / WRfactor Rwork: 0.1605 / FOM work R set: 0.8327 / SU B: 3.836 / SU ML: 0.113 / SU R Cruickshank DPI: 0.1616 / SU Rfree: 0.1557 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.162 / ESU R Free: 0.156 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2286 4403 4.8 %RANDOM
Rwork0.1699 ---
obs0.1727 86417 89.51 %-
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: 93.45 Å2 / Biso mean: 26.299 Å2 / Biso min: 11.02 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å2-0 Å20.01 Å2
2---0.49 Å2-0 Å2
3---0.33 Å2
Refinement stepCycle: final / Resolution: 1.85→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9323 0 36 1075 10434
Biso mean--27.36 33.05 -
Num. residues----1243
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.029547
X-RAY DIFFRACTIONr_bond_other_d0.0020.028858
X-RAY DIFFRACTIONr_angle_refined_deg1.8931.95913022
X-RAY DIFFRACTIONr_angle_other_deg1.092320605
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.76951253
X-RAY DIFFRACTIONr_dihedral_angle_2_deg41.53625.99399
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.276151560
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.771532
X-RAY DIFFRACTIONr_chiral_restr0.1190.21567
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02110748
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021712
LS refinement shellResolution: 1.85→1.898 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.307 153 -
Rwork0.253 3542 -
all-3695 -
obs--49.35 %

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