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- PDB-6v29: Complex of double mutant (T89V,K162T) of E. coli L-asparaginase I... -

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Basic information

Entry
Database: PDB / ID: 6v29
TitleComplex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asp
ComponentsL-asparaginase 2
KeywordsHYDROLASE / L-asparagine hydrolase / anti-cancer drug / inactive mutant
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 / cytosol
Similarity search - Function
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, 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.
Similarity search - Domain/homology
ASPARTIC ACID / 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 / refine_hist
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine_hist.d_res_low

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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)143,31214
Polymers142,2274
Non-polymers1,08510
Water18,3391018
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)143,12812
Polymers142,2274
Non-polymers9018
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area15990 Å2
ΔGint-56 kcal/mol
Surface area40980 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)143,49616
Polymers142,2274
Non-polymers1,26912
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area16970 Å2
ΔGint-56 kcal/mol
Surface area40280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.616, 62.430, 143.066
Angle α, β, γ (deg.)90.000, 118.090, 90.000
Int Tables number5
Space group name H-MC121

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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-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: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1018 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 293 K / Method: vapor diffusion, hanging drop / pH: 6.2
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 10-20 sec in solution containing 40% (w/v) PEG3350, 5 mM L-L-Asp, and 0.17 M sodium citrate (pH 6.2)

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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: May 22, 2018
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: 79279 / % possible obs: 99.1 % / Redundancy: 3.2 % / Rmerge(I) obs: 0.06 / Rpim(I) all: 0.039 / Rrim(I) all: 0.072 / Χ2: 0.879 / Net I/σ(I): 10.7 / Num. measured all: 256387
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.032.90.52638660.7170.3580.6390.9698.3
2.03-2.072.90.41439140.8040.280.5010.96598.5
2.07-2.1130.36739110.8330.2460.4440.95998.4
2.11-2.153.10.29839460.90.1960.3590.96898.6
2.15-2.23.10.26538670.9080.1730.3180.96598.3
2.2-2.253.20.23939210.9240.1550.2860.94799
2.25-2.313.20.20339880.9420.1310.2430.96899.2
2.31-2.373.30.1739160.9550.1090.2030.95799.3
2.37-2.443.30.16339500.9660.1040.1940.96999.3
2.44-2.523.40.14139660.970.090.1680.9699.2
2.52-2.613.30.12539920.9770.080.1490.97199.5
2.61-2.713.40.10439660.9840.0660.1240.9399.3
2.71-2.843.40.08539790.9890.0540.1010.9199.4
2.84-2.993.40.06939310.9930.0430.0810.90799.5
2.99-3.173.30.05240150.9950.0330.0620.91499.5
3.17-3.423.40.03739830.9970.0240.0440.84199.6
3.42-3.763.30.02539910.9980.0160.0290.76499.7
3.76-4.313.30.01740330.9990.0110.020.66399.8
4.31-5.423.20.01440450.9990.0090.0160.59299.5
5.42-403.50.011409910.0070.0130.5698.8

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.8.0158refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3eca

3eca


Resolution: 2→26 Å / Cor.coef. Fo:Fc: 0.972 / Cor.coef. Fo:Fc free: 0.945 / SU B: 4.024 / SU ML: 0.109 / SU R Cruickshank DPI: 0.162 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.162 / ESU R Free: 0.155
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2037 4004 5.1 %RANDOM
Rwork0.1424 ---
obs0.1454 75132 98.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 117.12 Å2 / Biso mean: 30.012 Å2 / Biso min: 12.73 Å2
Baniso -1Baniso -2Baniso -3
1-0.12 Å2-0 Å20.14 Å2
2---0.42 Å20 Å2
3---0.09 Å2
Refinement stepCycle: final / Resolution: 2→26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9295 0 72 1018 10385
Biso mean--48.45 36.85 -
Num. residues----1239
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.029524
X-RAY DIFFRACTIONr_bond_other_d0.0020.028808
X-RAY DIFFRACTIONr_angle_refined_deg1.9221.9612981
X-RAY DIFFRACTIONr_angle_other_deg1.087320479
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.54551239
X-RAY DIFFRACTIONr_dihedral_angle_2_deg42.54526400
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.778151536
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.1311532
X-RAY DIFFRACTIONr_chiral_restr0.120.21568
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02110689
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021703
LS refinement shellResolution: 2.001→2.052 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.29 280 -
Rwork0.249 5336 -
all-5616 -
obs--96.41 %

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