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- PDB-8rue: Crystal structure of Rhizobium etli L-asparaginase ReAV H139A mutant -

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Basic information

Entry
Database: PDB / ID: 8rue
TitleCrystal structure of Rhizobium etli L-asparaginase ReAV H139A mutant
ComponentsL-asparaginase II proteinAsparaginase
KeywordsHYDROLASE / Rhizobium etli / amidohydrolases / l-asparaginases / zinc-binding proteins / site-directed mutagenesis
Function / homologyL-asparaginase II / L-asparaginase II / ISOPROPYL ALCOHOL / DI(HYDROXYETHYL)ETHER / L-asparaginase II protein
Function and homology information
Biological speciesRhizobium etli (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsPokrywka, K. / Grzechowiak, M. / Sliwiak, J. / Worsztynowicz, P. / Loch, J.I. / Ruszkowski, M. / Gilski, M. / Jaskolski, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2020/37/B/NZ1/03250 Poland
Citation
Journal: Front Chem / Year: 2024
Title: Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.
Authors: Pokrywka, K. / Grzechowiak, M. / Sliwiak, J. / Worsztynowicz, P. / Loch, J.I. / Ruszkowski, M. / Gilski, M. / Jaskolski, M.
#1: Journal: IUCrJ / Year: 2021
Title: Structural and biophysical aspects of l-asparaginases: a growing family with amazing diversity.
Authors: Loch, J.I. / Jaskolski, M.
#2: Journal: Nat Commun / Year: 2021
Title: Crystal structures of the elusive Rhizobium etli L-asparaginase reveal a peculiar active site.
Authors: Loch, J.I. / Imiolczyk, B. / Sliwiak, J. / Wantuch, A. / Bejger, M. / Gilski, M. / Jaskolski, M.
#3: Journal: Acta Crystallogr D Struct Biol / Year: 2023
Title: Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.
Authors: Loch, J.I. / Worsztynowicz, P. / Sliwiak, J. / Grzechowiak, M. / Imiolczyk, B. / Pokrywka, K. / Chwastyk, M. / Gilski, M. / Jaskolski, M.
History
DepositionJan 30, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: L-asparaginase II protein
B: L-asparaginase II protein
C: L-asparaginase II protein
D: L-asparaginase II protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)160,43939
Polymers157,9514
Non-polymers2,48835
Water26,6441479
1
A: L-asparaginase II protein
B: L-asparaginase II protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,39722
Polymers78,9762
Non-polymers1,42220
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6390 Å2
ΔGint-64 kcal/mol
Surface area26200 Å2
MethodPISA
2
C: L-asparaginase II protein
D: L-asparaginase II protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,04217
Polymers78,9762
Non-polymers1,06615
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5760 Å2
ΔGint-60 kcal/mol
Surface area25270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)77.906, 91.472, 114.471
Angle α, β, γ (deg.)90.000, 96.980, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
L-asparaginase II protein / Asparaginase


Mass: 39487.801 Da / Num. of mol.: 4 / Mutation: H139A
Source method: isolated from a genetically manipulated source
Details: point mutation H139A / Source: (gene. exp.) Rhizobium etli (bacteria) / Gene: ansA, RHE_PE00350 / Production host: Escherichia coli (E. coli) / References: UniProt: Q2K0Z2

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Non-polymers , 8 types, 1514 molecules

#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL / Isopropyl alcohol


Mass: 60.095 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#8: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1479 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.56 Å3/Da / Density % sol: 52.01 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 9
Details: 22% PEG 3350, 0.2 M MgCl2, 0.1 M Bicine pH 9.0, 1% v/v isopropanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9196 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 16, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9196 Å / Relative weight: 1
ReflectionResolution: 1.4→77.33 Å / Num. obs: 309393 / % possible obs: 99.2 % / Redundancy: 7 % / Biso Wilson estimate: 15.29 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.088 / Rrim(I) all: 0.095 / Net I/σ(I): 12.68
Reflection shellResolution: 1.4→1.49 Å / Rmerge(I) obs: 1.322 / Mean I/σ(I) obs: 1.46 / Num. unique obs: 49331 / CC1/2: 0.608 / Rrim(I) all: 1.427

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→37.87 Å / SU ML: 0.1514 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 17.7345
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Hydrogen atoms were added at riding position. Anisotropic atomic displacement parameters were used.
RfactorNum. reflection% reflection
Rfree0.1758 1544 0.5 %
Rwork0.1414 307768 -
obs0.1416 309312 99.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 21.17 Å2
Refinement stepCycle: LAST / Resolution: 1.4→37.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10326 0 148 1479 11953
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009411090
X-RAY DIFFRACTIONf_angle_d1.068815041
X-RAY DIFFRACTIONf_chiral_restr0.09021679
X-RAY DIFFRACTIONf_plane_restr0.01151991
X-RAY DIFFRACTIONf_dihedral_angle_d12.91954211
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4-1.450.27331370.250727269X-RAY DIFFRACTION96.93
1.45-1.50.19861400.205927963X-RAY DIFFRACTION99.53
1.5-1.560.22191410.173328003X-RAY DIFFRACTION99.48
1.56-1.630.2051390.148127953X-RAY DIFFRACTION99.44
1.63-1.710.15971410.134327968X-RAY DIFFRACTION99.46
1.71-1.820.17391400.130727968X-RAY DIFFRACTION99.47
1.82-1.960.1531410.123627909X-RAY DIFFRACTION99.04
1.96-2.160.18091420.120628223X-RAY DIFFRACTION99.96
2.16-2.470.1691410.129428109X-RAY DIFFRACTION99.71
2.47-3.120.16891410.144928166X-RAY DIFFRACTION99.42
3.12-37.870.16951410.138828237X-RAY DIFFRACTION98.78

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