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- PDB-8rua: Crystal structure of Rhizobium etli L-asparaginase ReAV C135A mutant -

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Basic information

Entry
Database: PDB / ID: 8rua
TitleCrystal structure of Rhizobium etli L-asparaginase ReAV C135A mutant
ComponentsL-asparaginase II protein
KeywordsHYDROLASE / Rhizobium etli / amidohydrolases / l-asparaginases / zinc-binding proteins / site-directed mutagenesis
Function / homologyL-asparaginase II / L-asparaginase II / metal ion binding / DI(HYDROXYETHYL)ETHER / L-asparaginase II protein
Function and homology information
Biological speciesRhizobium etli (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.71 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)80,84724
Polymers79,0462
Non-polymers1,80122
Water7,927440
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8030 Å2
ΔGint12 kcal/mol
Surface area26560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.064, 91.178, 106.049
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein L-asparaginase II protein


Mass: 39522.805 Da / Num. of mol.: 2 / Mutation: C135A
Source method: isolated from a genetically manipulated source
Details: point mutation C135A / 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 , 6 types, 462 molecules

#2: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 440 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.39 Å3/Da / Density % sol: 48.48 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 30% PEG 4000, 0.2 M Li2SO4, 0.1 M Tris pH 8.5

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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: 0.9763 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 25, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.71→78.06 Å / Num. obs: 82131 / % possible obs: 99.8 % / Redundancy: 12.2 % / Biso Wilson estimate: 25.16 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.122 / Rrim(I) all: 0.128 / Net I/σ(I): 11.89
Reflection shellResolution: 1.71→1.81 Å / Rmerge(I) obs: 1.883 / Mean I/σ(I) obs: 1.54 / Num. unique obs: 13024 / CC1/2: 0.747 / Rrim(I) all: 1.97

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.71→45.84 Å / SU ML: 0.2213 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 25.9295
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Hydrogen atoms were added at riding position.
RfactorNum. reflection% reflection
Rfree0.2242 1024 1.25 %
Rwork0.2017 81018 -
obs0.2019 82042 99.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 30.58 Å2
Refinement stepCycle: LAST / Resolution: 1.71→45.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5312 0 116 440 5868
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01025625
X-RAY DIFFRACTIONf_angle_d1.06657588
X-RAY DIFFRACTIONf_chiral_restr0.0627838
X-RAY DIFFRACTIONf_plane_restr0.00981001
X-RAY DIFFRACTIONf_dihedral_angle_d14.52952107
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.71-1.80.32381430.298811326X-RAY DIFFRACTION98.89
1.8-1.910.2551460.22811480X-RAY DIFFRACTION99.95
1.91-2.060.26331460.20311519X-RAY DIFFRACTION99.91
2.06-2.270.24681440.18511496X-RAY DIFFRACTION99.85
2.27-2.60.22091470.187211575X-RAY DIFFRACTION99.86
2.6-3.270.22111470.203211642X-RAY DIFFRACTION99.77
3.27-45.840.20161510.19711980X-RAY DIFFRACTION99.65
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)
14.65153798535-6.044913855021.951738025977.88397362863-2.434803359981.05627379961-0.06669080858460.006218312960490.1918612427590.1269027962420.04744580694480.0781311861365-0.1022535411130.0464661458210.0253909497420.179981659889-0.02226857503820.002173390366350.1646644115550.006691367068670.17989327103425.50928037016.4030788195133.3352263696
21.0347938886-0.114977705452-1.007872490991.386846163910.7777178787511.52195179026-0.04920877635550.0419804068858-0.0583251486108-0.1945900430020.0587026100432-0.2200700544180.08591608330110.270809885321-0.0128057406180.2124016125810.0422919016240.03941282519140.2389541541160.04162162260260.22862671337739.5421516693-12.400985964717.7404016435
33.17909265742-1.119323126231.668590556184.17207177472-2.519840410862.437692036290.09670718592520.149546392704-0.0626126219349-0.193322032883-0.00759707836830.214191218694-0.00548722644137-0.0567483207094-0.09246199227360.2865708042890.05052300421460.02374272316730.223350932872-0.01361805910080.21954652052130.7526320909-16.12804670167.20752671894
41.874009945020.5075662426140.1809110657851.85847815621.020836728961.172113602260.06392540420640.0233237845155-0.02127714287690.0141091218701-0.15342306325-0.2053202312010.2658327111560.1826497074790.08441392641730.2325879769330.07621498374730.01422345192360.2076798739230.05843011783760.21797845352438.0625568879-17.333850628427.5724044929
53.36013184387-1.215253466020.2500959510244.542457393960.2181370471681.25669280541-0.0362530271673-0.168366666957-0.0241184252353-0.158450013753-0.00300433284991-0.3551747337530.01148394639590.2972411958310.03917575340820.1847298010420.01225792087360.07982690207710.2889906556430.04375942471110.25370611195245.8577151792-5.4767013635418.3948509037
65.714263674840.1046953121610.4941461194732.818940226440.455585769422.244210247460.0232946690987-0.1902486135320.1008370862510.02958166315430.00222705208334-0.212086836978-0.02349356389910.179548218817-0.02098861768130.15332270901-0.0003203306528890.02985157812220.1646863834280.03391363535420.16205278637138.54744347051.4521958796929.4835098457
72.25684914978-0.1527246666610.01637984125931.89874178409-0.9204874192822.760732612420.01132274148610.06048205236060.3215176042040.0157250599971-0.0180503196245-0.1948006494840.02099288946450.168650092840.03138917011280.196994981433-0.0184000333689-0.002551970612470.1599000759670.02466478116610.33738283719932.877439985912.768653332625.5198645257
82.26359224522-0.867389531-0.1064189443463.088845471370.103231655981.15380870701-0.0290565332439-0.152927106651-0.1210046848320.320339508572-0.06079960777520.04456921034810.0161628667255-0.1231610414010.09996011292010.172364456894-0.01815593010930.007085265760080.184326917418-0.01377824217180.1545596180119.269416203710.20157242732836.2659301956
92.39719185578-0.739251455458-1.271662632112.220267390010.4226314991491.532412738270.1639051783340.2243422814170.0642599972609-0.33959812336-0.0999854202732-0.195094075048-0.01364790396020.151327361377-0.06147655859570.2481769496470.06698800162270.009149249481750.2208867770.01652919228180.197440877464.7191556945419.85066317829.25008925101
101.44576742085-0.1311961553430.4818075922941.59686181423-0.2931818646650.8947162487180.0251885171077-0.07855169619210.109839225533-0.0329372879531-0.04500995346150.1217869765-0.0568062489792-0.2322574517250.02558863499320.2124471340030.05044134969420.007769054047980.226164279755-0.01305267401010.242789396417-2.3695032177919.355325670626.1360183913
111.33393564053-0.7057951084550.1141949284571.86777577094-0.3746524432381.425502374-0.01221514626110.0346334515966-0.17798334569-0.1782363166320.006069091449230.2725436982580.200929291976-0.1777405355730.01673103592670.167492873417-0.020174670606-0.01161455980650.163884142889-0.01150733512610.218978941931-0.6535802211441.1353181000923.1674758157
123.94910634599-2.32405911019-3.038613487742.780116469442.815893971313.223644172520.03020607328440.0890184112676-0.1869003609580.117331447334-0.2407086837030.0328086684247-0.16264293716-0.3745322643430.2642510358240.2702113085780.00516859491256-0.02010314943760.1752703470080.03326118584990.30839360595310.4544628025-7.6611018067833.3185213138
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 4 through 28 )AA4 - 281 - 25
22chain 'A' and (resid 29 through 97 )AA29 - 9726 - 94
33chain 'A' and (resid 98 through 161 )AA98 - 16195 - 158
44chain 'A' and (resid 162 through 217 )AA162 - 217159 - 214
55chain 'A' and (resid 218 through 265 )AA218 - 265215 - 262
66chain 'A' and (resid 266 through 314 )AA266 - 314263 - 311
77chain 'A' and (resid 315 through 353 )AA315 - 353312 - 350
88chain 'B' and (resid -3 through 46 )BN-3 - 461 - 50
99chain 'B' and (resid 47 through 161 )BN47 - 16151 - 165
1010chain 'B' and (resid 162 through 237 )BN162 - 237166 - 241
1111chain 'B' and (resid 238 through 329 )BN238 - 329242 - 333
1212chain 'B' and (resid 330 through 367 )BN330 - 367334 - 367

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