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- PDB-6pa9: E. coli L-asparaginase II mutant (T12V) in complex with L-Asn at ... -

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Basic information

Entry
Database: PDB / ID: 6pa9
TitleE. coli L-asparaginase II mutant (T12V) 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


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
ASPARAGINE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.88 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 1.3Nov 20, 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,7565
Polymers71,4002
Non-polymers3563
Water8,755486
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)143,51310
Polymers142,8004
Non-polymers7136
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,-y,z1
Buried area16170 Å2
ΔGint-56 kcal/mol
Surface area40090 Å2
MethodPISA
Unit cell
Length a, b, c (Å)61.066, 68.912, 130.969
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-749-

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.: 2 / Mutation: T12V
Source method: isolated from a genetically manipulated source
Details: Expressed variant contains 8 additional N-terminal residues MDHHHHHH (affinity tag) and mutation (K162M) 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.: 2 / Source method: obtained synthetically / Formula: C4H8N2O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 486 / 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: 1.93 Å3/Da / Density % sol: 36.26 %
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, 5 mM L-Asn

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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: Jul 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: 1.88→40 Å / Num. obs: 42994 / % possible obs: 94 % / Redundancy: 5.6 % / Rmerge(I) obs: 0.044 / Rpim(I) all: 0.02 / Rrim(I) all: 0.049 / Χ2: 0.901 / Net I/σ(I): 14.1 / Num. measured all: 240226
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.88-1.913.80.61415140.7910.3330.7020.90967.1
1.91-1.954.10.52617000.8150.2710.5950.9676.6
1.95-1.984.30.44819850.8720.230.5060.91388.1
1.98-2.0350.40321110.9040.1920.4480.89393.3
2.03-2.075.20.34820810.9290.1640.3860.90892.7
2.07-2.125.30.26621070.9630.1240.2950.92193.5
2.12-2.175.40.21621000.9760.10.2390.92493.2
2.17-2.235.50.19321580.9760.0890.2130.92195.5
2.23-2.295.50.1522230.9840.0690.1650.94397
2.29-2.375.70.12521780.990.0570.1370.91796.9
2.37-2.455.80.10722070.9940.0490.1180.91297.3
2.45-2.555.80.0922420.9950.040.0990.89497.8
2.55-2.6760.07522200.9960.0330.0820.86698.2
2.67-2.816.10.06322420.9970.0270.0690.84398.6
2.81-2.986.20.04922630.9980.0210.0530.82599
2.98-3.216.20.03722850.9990.0160.040.81898.6
3.21-3.546.20.02722870.9990.0120.0290.80299.6
3.54-4.056.10.02231410.0090.0220.8899.4
4.05-5.15.90.017232910.0070.0181.02798.6
5.1-406.20.017244810.0070.0190.99398.4

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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: 1.88→40 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.944 / WRfactor Rfree: 0.1947 / WRfactor Rwork: 0.1442 / FOM work R set: 0.8473 / SU B: 6.873 / SU ML: 0.108 / SU R Cruickshank DPI: 0.1652 / SU Rfree: 0.1522 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.165 / ESU R Free: 0.152 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2157 2081 4.9 %RANDOM
Rwork0.1621 ---
obs0.1647 40674 93.52 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 87.68 Å2 / Biso mean: 28.436 Å2 / Biso min: 9.64 Å2
Baniso -1Baniso -2Baniso -3
1--0.53 Å2-0 Å2-0 Å2
2--0.15 Å2-0 Å2
3---0.38 Å2
Refinement stepCycle: final / Resolution: 1.88→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4772 0 24 486 5282
Biso mean--23.62 27.7 -
Num. residues----638
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0210.024880
X-RAY DIFFRACTIONr_bond_other_d0.0020.024523
X-RAY DIFFRACTIONr_angle_refined_deg2.0011.9586652
X-RAY DIFFRACTIONr_angle_other_deg1.11310518
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5415639
X-RAY DIFFRACTIONr_dihedral_angle_2_deg41.09826.078204
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.7115793
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4141516
X-RAY DIFFRACTIONr_chiral_restr0.1260.2800
X-RAY DIFFRACTIONr_gen_planes_refined0.010.0215499
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02874
LS refinement shellResolution: 1.881→1.93 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.287 117 -
Rwork0.279 2042 -
all-2159 -
obs--65.27 %
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)
11.64550.2696-0.34721.77410.07581.72150.04060.01770.41770.2171-0.021-0.3128-0.25940.0304-0.01970.0701-0.0092-0.0520.0330.01170.1959-16.06916.115-43.084
22.8090.69210.11471.8883-0.10660.692-0.07150.4075-0.2005-0.18110.0859-0.22650.11140.0613-0.01440.03390.00710.01660.1288-0.04560.0378-20.796-6.67-58.237
31.759-0.8037-0.31072.0194-0.09251.6545-0.1143-0.2576-0.32510.63610.0764-0.3370.18880.21290.03790.35570.0473-0.16170.10910.05790.2087-15.968-16.415-22.685
42.4293-1.1303-0.222.49150.17431.9385-0.1726-0.65640.27771.15750.2252-0.3356-0.12330.2579-0.05260.80660.0397-0.16340.3072-0.08380.0596-20.3516.046-7.167
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 209
2X-RAY DIFFRACTION2A210 - 326
3X-RAY DIFFRACTION3B1 - 209
4X-RAY DIFFRACTION4B210 - 326

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