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- PDB-6v2a: Complex of double mutant (T89V,K162T) of E. coli L-asparaginase I... -

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Basic information

Entry
Database: PDB / ID: 6v2a
TitleComplex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asn
ComponentsL-asparaginase 2
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / inactive mutant
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 / Resolution: 2 Å
AuthorsLubkowski, J. / Wlodawer, A.
CitationJournal: Biochemistry / Year: 2020
Title: Mechanism of Catalysis by l-Asparaginase.
Authors: Lubkowski, J. / Vanegas, J. / Chan, W.K. / Lorenzi, P.L. / Weinstein, J.N. / Sukharev, S. / Fushman, D. / Rempe, S. / Anishkin, A. / Wlodawer, A.
History
DepositionNov 22, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation / citation_author
Item: _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 10, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _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)142,7568
Polymers142,2274
Non-polymers5284
Water10,791599
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)142,7568
Polymers142,2274
Non-polymers5284
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area16120 Å2
ΔGint-53 kcal/mol
Surface area40010 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)142,7568
Polymers142,2274
Non-polymers5284
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area16970 Å2
ΔGint-55 kcal/mol
Surface area39840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.439, 62.594, 141.659
Angle α, β, γ (deg.)90.000, 117.800, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-591-

HOH

21B-582-

HOH

31C-659-

HOH

41D-664-

HOH

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Components

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


Mass: 35556.828 Da / Num. of mol.: 4 / Mutation: T89V,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 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): JC2 / 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 599 / 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: 2.09 Å3/Da / Density % sol: 41.09 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.7
Details: Crystals grown at 0.17 M sodium citrate (pH 6) and 17-18% (w/v) PEG3350. Soaked for 1-2 minutes (empirically determined for each crystal) in equivalent solution with 0.025% (w/v) ...Details: Crystals grown at 0.17 M sodium citrate (pH 6) and 17-18% (w/v) PEG3350. Soaked for 1-2 minutes (empirically determined for each crystal) in equivalent solution with 0.025% (w/v) glutaraldehyde. Finally transferred and soaked for 3-5 min in solution containing 40% (w/v) PEG3350, 5 mM L-Asn, and 0.17 M sodium citrate (pH 4.7)

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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 X 4M / Detector: PIXEL / Date: Mar 15, 2018 / Details: Multilayer X-ray mirrors VariMax HF
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2→40 Å / Num. obs: 73175 / % possible obs: 92.6 % / Redundancy: 2.8 % / Rmerge(I) obs: 0.039 / Rpim(I) all: 0.027 / Rrim(I) all: 0.048 / Χ2: 0.853 / Net I/σ(I): 15.5 / Num. measured all: 207458
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.032.60.50934210.690.3710.6330.92386.7
2.03-2.072.60.40134190.7870.2910.4980.95187.7
2.07-2.112.70.34234520.840.2490.4250.93988.1
2.11-2.152.70.2835040.8850.2020.3470.93787.8
2.15-2.22.70.24134790.9060.1750.2990.93889.9
2.2-2.252.70.20435240.9310.1460.2520.93390.4
2.25-2.312.70.17236100.9470.1230.2130.96690.9
2.31-2.372.70.14435460.9660.1020.1770.93991.1
2.37-2.442.80.1335240.9750.0920.160.93589.5
2.44-2.522.70.11134970.9770.0780.1360.92188.6
2.52-2.612.80.10235010.9810.0710.1250.93388.6
2.61-2.712.80.08235230.9860.0580.1010.9389.5
2.71-2.842.70.06236050.9920.0440.0770.86591.7
2.84-2.992.70.05137910.9940.0360.0630.88196.3
2.99-3.172.80.03839130.9960.0270.0460.84299
3.17-3.4230.02839470.9970.0190.0340.81799.6
3.42-3.763.10.02139530.9980.0140.0250.76499.6
3.76-4.313.10.01639920.9990.0110.0190.68899.8
4.31-5.423.20.01439110.9990.0090.0170.64797.9
5.42-403.40.01440630.9990.0090.0160.65199

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.8.0253refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3eca

3eca


Resolution: 2→25.98 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.942 / SU B: 4.074 / SU ML: 0.111 / SU R Cruickshank DPI: 0.1978 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.198 / ESU R Free: 0.168
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2088 3444 4.7 %RANDOM
Rwork0.1574 ---
obs0.1598 69457 92.07 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 113.25 Å2 / Biso mean: 29.194 Å2 / Biso min: 11.13 Å2
Baniso -1Baniso -2Baniso -3
1-0.1 Å2-0 Å20 Å2
2---0.52 Å2-0 Å2
3---0.27 Å2
Refinement stepCycle: final / Resolution: 2→25.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9619 0 36 599 10254
Biso mean--24.44 30.33 -
Num. residues----1291
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0139800
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179079
X-RAY DIFFRACTIONr_angle_refined_deg1.8931.64813360
X-RAY DIFFRACTIONr_angle_other_deg1.4571.58221105
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.43751285
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.14124.908436
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.637151587
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.1421532
X-RAY DIFFRACTIONr_chiral_restr0.0890.21372
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0211123
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021785
LS refinement shellResolution: 2.004→2.056 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.281 237 -
Rwork0.261 4453 -
all-4690 -
obs--80.99 %

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