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- PDB-6v28: Complex of double mutant (T89V,K162T) of E. coli L-asparaginase I... -

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Basic information

Entry
Database: PDB / ID: 6v28
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, 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
IMIDAZOLE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.95 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)143,47013
Polymers142,6884
Non-polymers7839
Water22,0321223
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)143,0108
Polymers142,6884
Non-polymers3224
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area18110 Å2
ΔGint-45 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)143,93118
Polymers142,6884
Non-polymers1,24314
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area20650 Å2
ΔGint-57 kcal/mol
Surface area40170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.342, 62.538, 141.299
Angle α, β, γ (deg.)90.000, 117.680, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11D-708-

HOH

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Components

#1: Protein
L-asparaginase 2 / 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
Details: Residue 21 (AEI) was modified. It forms the covalent bond with the substrate (L-Asp)
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-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H5N2
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1223 / 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: 40.72 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 40% (w/v) PEG3350, 10 mM L-Asn, and 0.17 M sodium citrate

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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: Jun 13, 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: 1.95→40 Å / Num. obs: 84238 / % possible obs: 98.3 % / Redundancy: 3.1 % / CC1/2: 0.912 / Rmerge(I) obs: 0.066 / Rpim(I) all: 0.044 / Χ2: 0.892 / Net I/σ(I): 15.7
Reflection shellResolution: 1.95→1.98 Å / Rmerge(I) obs: 0.514 / Mean I/σ(I) obs: 1.72 / Num. unique obs: 3808 / CC1/2: 0.671 / Rpim(I) all: 0.423 / Χ2: 1.012

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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: 1.95→27.9 Å / Cor.coef. Fo:Fc: 0.971 / Cor.coef. Fo:Fc free: 0.952 / SU B: 6.888 / SU ML: 0.104 / SU R Cruickshank DPI: 0.1595 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.16 / ESU R Free: 0.139
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1863 4018 4.8 %RANDOM
Rwork0.1418 ---
obs0.144 79550 97.63 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 112.67 Å2 / Biso mean: 26.443 Å2 / Biso min: 10.47 Å2
Baniso -1Baniso -2Baniso -3
1--0.24 Å2-0 Å20.28 Å2
2---0.45 Å2-0 Å2
3---0.26 Å2
Refinement stepCycle: final / Resolution: 1.95→27.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9768 0 56 1223 11047
Biso mean--47.32 33.36 -
Num. residues----1306
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0210054
X-RAY DIFFRACTIONr_bond_other_d0.0020.029293
X-RAY DIFFRACTIONr_angle_refined_deg1.8641.96513713
X-RAY DIFFRACTIONr_angle_other_deg1.079321632
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.62751330
X-RAY DIFFRACTIONr_dihedral_angle_2_deg42.00526.058411
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.94151620
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4841532
X-RAY DIFFRACTIONr_chiral_restr0.1180.21651
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02111357
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021807
LS refinement shellResolution: 1.95→2.001 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.318 266 -
Rwork0.275 5116 -
all-5382 -
obs--86.03 %
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)
11.29140.10230.16450.7677-0.12721.05660.0718-0.2327-0.06780.1451-0.00270.11520.1141-0.1472-0.06920.1886-0.0295-0.01370.13780.02530.0435-1.86818.26219.332
21.48890.56350.222.1553-0.51811.74460.05140.0582-0.1465-0.0879-0.0359-0.42790.17570.4195-0.01550.13160.0491-0.01420.1949-0.02990.12424.61721.4839.306
31.19260.25230.57180.9683-0.22751.7483-0.01650.25960.0525-0.159-0.0086-0.2415-0.13670.37390.0250.2037-0.05540.04030.1940.02330.155317.37744.852-8.616
41.58860.17450.37141.6098-0.71231.7419-0.0585-0.1470.21660.2465-0.0066-0.1483-0.26320.18050.0650.1829-0.0364-0.03350.1191-0.05340.09217.4341.40119.735
50.95410.20150.1730.7853-0.09340.7842-0.02080.18670.0639-0.17390.0016-0.0945-0.01350.06950.01920.0609-0.00050.00470.04140.02490.0521-17.06144.89651.153
61.15230.11090.29031.2611-0.53891.08180.0056-0.17780.09950.1865-0.0195-0.0937-0.0930.04890.01390.0618-0.0029-0.02780.0389-0.02250.0352-12.50841.55379.318
71.51050.03660.11421.0624-0.3460.62880-0.24950.00480.2397-0.00430.0473-0.0135-0.07210.00430.1141-0.00920.00210.05460.00510.0198-31.70618.04181.957
81.3120.36850.03091.0801-0.31870.67230.0050.0145-0.090.0027-0.0089-0.17370.10110.11650.00390.03790.0231-0.01340.0234-0.00560.0535-7.00721.57367.867
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 200
2X-RAY DIFFRACTION2A210 - 326
3X-RAY DIFFRACTION3B1 - 200
4X-RAY DIFFRACTION4B210 - 326
5X-RAY DIFFRACTION5C0 - 200
6X-RAY DIFFRACTION6C210 - 326
7X-RAY DIFFRACTION7D0 - 200
8X-RAY DIFFRACTION8D210 - 326

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