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- PDB-6v27: Complex of double mutant (T89V,K162T) of E. coli L-asparaginase I... -

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Basic information

Entry
Database: PDB / ID: 6v27
TitleComplex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asp
ComponentsL-asparaginase 2Asparaginase
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
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
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.3 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: L-asparaginase 2
B: L-asparaginase 2
C: L-asparaginase 2
D: L-asparaginase 2


Theoretical massNumber of molelcules
Total (without water)142,6884
Polymers142,6884
Non-polymers00
Water11,025612
1
A: L-asparaginase 2
B: L-asparaginase 2

A: L-asparaginase 2
B: L-asparaginase 2


Theoretical massNumber of molelcules
Total (without water)142,6884
Polymers142,6884
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area16200 Å2
ΔGint-50 kcal/mol
Surface area39100 Å2
MethodPISA
2
C: L-asparaginase 2
D: L-asparaginase 2

C: L-asparaginase 2
D: L-asparaginase 2


Theoretical massNumber of molelcules
Total (without water)142,6884
Polymers142,6884
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area16670 Å2
ΔGint-51 kcal/mol
Surface area39730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.302, 62.412, 141.753
Angle α, β, γ (deg.)90.000, 117.850, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-497-

HOH

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Components

#1: Protein
L-asparaginase 2 / Asparaginase / 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
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: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 612 / 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: 2.07 Å3/Da / Density % sol: 40.68 %
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 10-20 sec in solution containing 40% (w/v) PEG3350, 5 mM L-L-Asp, 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: Feb 17, 2019 / 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: 2.3→40 Å / Num. obs: 51978 / % possible obs: 98.9 % / Redundancy: 2.7 % / Rmerge(I) obs: 0.073 / Rpim(I) all: 0.053 / Rrim(I) all: 0.091 / Χ2: 0.903 / Net I/σ(I): 9.9 / Num. measured all: 138795
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.3-2.342.50.51325870.7220.3770.640.95498.3
2.34-2.382.60.46225500.7490.3390.5750.94699
2.38-2.432.70.43725790.7840.3190.5440.93398.9
2.43-2.482.70.35725960.8540.2590.4430.92898.9
2.48-2.532.70.37125620.8180.2680.4590.93698.8
2.53-2.592.70.29225930.8880.2110.3620.93499.1
2.59-2.662.70.25625800.9090.1850.3170.90899.1
2.66-2.732.70.24826090.9090.1780.3070.94298.5
2.73-2.812.70.20225710.9350.1460.250.94798.8
2.81-2.92.70.16725670.9570.1210.2070.92899.2
2.9-32.70.13325930.9730.0960.1650.93898.9
3-3.122.70.11625970.9750.0850.1441.00298.9
3.12-3.262.70.08626080.9860.0620.1060.95299.1
3.26-3.442.70.06926120.990.0510.0860.9799.4
3.44-3.652.70.05326100.9940.0380.0650.94999.2
3.65-3.932.70.04126050.9950.030.0510.90899.1
3.93-4.332.60.03326190.9970.0240.0410.87898.9
4.33-4.952.60.02825940.9970.0210.0360.8298.8
4.95-6.242.70.02826710.9970.020.0350.69499.4
6.24-402.90.02226750.9980.0150.0270.64897.5

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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: 2.3→26.5 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.908 / SU B: 7.623 / SU ML: 0.184 / SU R Cruickshank DPI: 0.4248 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.425 / ESU R Free: 0.271
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2513 2481 4.9 %RANDOM
Rwork0.16 ---
obs0.1643 48626 97.57 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 119.39 Å2 / Biso mean: 35.574 Å2 / Biso min: 14.66 Å2
Baniso -1Baniso -2Baniso -3
1-0.26 Å2-0 Å2-0.01 Å2
2---0.45 Å2-0 Å2
3---0.13 Å2
Refinement stepCycle: final / Resolution: 2.3→26.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9517 0 0 613 10130
Biso mean---34.84 -
Num. residues----1270
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0139693
X-RAY DIFFRACTIONr_bond_other_d0.0010.0178964
X-RAY DIFFRACTIONr_angle_refined_deg2.071.65313227
X-RAY DIFFRACTIONr_angle_other_deg1.4221.58620853
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.82151275
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.87524.825429
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.445151563
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4031532
X-RAY DIFFRACTIONr_chiral_restr0.0940.21363
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0210995
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021766
LS refinement shellResolution: 2.3→2.36 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.326 166 -
Rwork0.237 3253 -
all-3419 -
obs--88.83 %

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