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- PDB-7r1g: Structure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-38... -

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Basic information

Entry
Database: PDB / ID: 7r1g
TitleStructure of E.coli Class 2 L-asparaginase EcAIII, mutant RDM1-38 (R207C, D210S, S211V)
Components
  • Beta-aspartyl-peptidase
  • Isoaspartyl peptidase
KeywordsHYDROLASE / L-asparaginase / Ntn-hydrolase / EcAIII
Function / homology
Function and homology information


beta-aspartyl-peptidase / asparaginase / asparaginase activity / beta-aspartyl-peptidase activity / protein autoprocessing / hydrolase activity
Similarity search - Function
Peptidase T2, asparaginase 2 / Asparaginase / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Beta-aspartyl-peptidase / Isoaspartyl peptidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsLoch, J.I. / Kadziolka, K. / Jaskolski, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2020/38/E/NZ1/00035 Poland
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase.
Authors: Loch, J.I. / Klonecka, A. / Kadziolka, K. / Bonarek, P. / Barciszewski, J. / Imiolczyk, B. / Brzezinski, K. / Gilski, M. / Jaskolski, M.
History
DepositionFeb 2, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Isoaspartyl peptidase
BBB: Beta-aspartyl-peptidase
CCC: Isoaspartyl peptidase
DDD: Beta-aspartyl-peptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,7906
Polymers66,7444
Non-polymers462
Water3,063170
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14100 Å2
ΔGint-112 kcal/mol
Surface area20570 Å2
Unit cell
Length a, b, c (Å)49.815, 74.691, 147.358
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Isoaspartyl peptidase / Beta-aspartyl-peptidase / EcAIII / Isoaspartyl dipeptidase


Mass: 19013.773 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: iaaA, spt, ybiK, b0828, JW0812 / Plasmid: pET11d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 GOLD / References: UniProt: P37595, beta-aspartyl-peptidase
#2: Protein Beta-aspartyl-peptidase / Isoaspartyl peptidase / Putative L-asparaginase


Mass: 14358.139 Da / Num. of mol.: 2 / Mutation: R207C, D210S, S211V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: iaaA, iaaA_1, A6583_11900, A9819_04170, AM340_25825, AT845_002782, B6R12_001137, B6R15_002170, B7C15_001855, B9T59_03785, BHS87_04410, BJJ90_17725, BK272_22150, BON86_21650, BON92_22175, BUE82_ ...Gene: iaaA, iaaA_1, A6583_11900, A9819_04170, AM340_25825, AT845_002782, B6R12_001137, B6R15_002170, B7C15_001855, B9T59_03785, BHS87_04410, BJJ90_17725, BK272_22150, BON86_21650, BON92_22175, BUE82_16375, BvCmsKSP076_04930, C2M16_01470, C2U48_05880, C3F40_08425, CDL37_06080, CG831_000717, CIG67_24035, CQP61_18640, CR539_08595, D3Y67_16925, D9J11_12335, D9K17_05660, DNQ45_21365, E2123_17525, E2135_02495, ELT49_19670, ELT58_16270, ExPECSC038_02859, F3N40_09450, F6U69_05970, F9400_10180, F9V07_02875, F9X20_012640, F9X20_12740, FDM60_08630, FGG80_18755, FQ021_11565, FQE77_03270, G4A38_09815, G7635_000303, GBE29_09760, GF699_09605, GJ11_04600, GKF52_09360, GKG12_09800, GQE42_02190, GQE87_02220, GQM13_17755, GRQ19_05905, H0O72_02540, HH795_002282, HIN64_003880, HJO75_000955, HJS37_002784, HMJ82_04325, HNC36_00665, HNC38_00260, HNC59_02050, HNC99_06385, HND24_01780, HNO08_00970, HNY50_16000, HNY54_13200, HV109_15925, HZ71_000057, NCTC10429_05732, NCTC8621_03538, NCTC9706_00685, NCTC9969_03483, SAMEA3472064_00385, SAMEA3752557_02250
Plasmid: pET11d / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0K4KR53, beta-aspartyl-peptidase, asparaginase
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 170 / Source method: isolated from a natural source / Formula: H2O
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: 2.05 Å3/Da / Density % sol: 40.11 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 30% PEG 4000, 0.2 M MgCl2 in 0.1M Tris-HCl pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: Agilent SuperNova / Wavelength: 1.54056 Å
DetectorType: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: May 28, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54056 Å / Relative weight: 1
ReflectionResolution: 1.95→21.131 Å / Num. obs: 38942 / % possible obs: 95.3 % / Redundancy: 2.7 % / CC1/2: 0.991 / Rmerge(I) obs: 0.099 / Rrim(I) all: 0.121 / Net I/σ(I): 7
Reflection shellResolution: 1.95→2 Å / Rmerge(I) obs: 0.361 / Mean I/σ(I) obs: 2.1 / Num. unique obs: 2596 / CC1/2: 0.741 / Rrim(I) all: 0.464

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
CrysalisProdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2ZAL

2zal


Resolution: 1.95→21.131 Å / Cor.coef. Fo:Fc: 0.886 / Cor.coef. Fo:Fc free: 0.848 / SU B: 11.353 / SU ML: 0.158 / Cross valid method: FREE R-VALUE / ESU R: 0.231 / ESU R Free: 0.193
RfactorNum. reflection% reflection
Rfree0.2832 1020 2.627 %
Rwork0.248 37806 -
all0.249 --
obs-38826 94.765 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 15.092 Å2
Baniso -1Baniso -2Baniso -3
1-2.384 Å20 Å2-0 Å2
2---1.841 Å20 Å2
3----0.543 Å2
Refinement stepCycle: LAST / Resolution: 1.95→21.131 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4180 0 2 170 4352
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0134266
X-RAY DIFFRACTIONr_bond_other_d0.0030.0154096
X-RAY DIFFRACTIONr_angle_refined_deg1.6171.6295788
X-RAY DIFFRACTIONr_angle_other_deg1.381.5699401
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.5285583
X-RAY DIFFRACTIONr_dihedral_angle_2_deg28.59522.228193
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.75315676
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.2451527
X-RAY DIFFRACTIONr_chiral_restr0.070.2571
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.024955
X-RAY DIFFRACTIONr_gen_planes_other0.0040.02923
X-RAY DIFFRACTIONr_nbd_refined0.2170.2950
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1870.24109
X-RAY DIFFRACTIONr_nbtor_refined0.160.22079
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0840.21954
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1740.2195
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0430.24
X-RAY DIFFRACTIONr_metal_ion_refined0.1780.25
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.2680.223
X-RAY DIFFRACTIONr_nbd_other0.3030.246
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.250.29
X-RAY DIFFRACTIONr_mcbond_it0.5761.0742317
X-RAY DIFFRACTIONr_mcbond_other0.5751.0732316
X-RAY DIFFRACTIONr_mcangle_it1.0231.6032890
X-RAY DIFFRACTIONr_mcangle_other1.0241.6052891
X-RAY DIFFRACTIONr_scbond_it0.6191.2091949
X-RAY DIFFRACTIONr_scbond_other0.6181.2061947
X-RAY DIFFRACTIONr_scangle_it1.0151.7692893
X-RAY DIFFRACTIONr_scangle_other1.0151.7692893
X-RAY DIFFRACTIONr_lrange_it3.47913.3564690
X-RAY DIFFRACTIONr_lrange_other3.45113.324677
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.95-20.391750.32726330.32929830.6310.74990.78110.327
2-2.0550.345600.28126090.28228550.8260.84893.48510.281
2.055-2.1130.291580.27125860.27228050.8110.87294.26030.271
2.113-2.1780.377670.27225600.27527440.8190.85895.73620.272
2.178-2.2480.324630.26125000.26226490.8470.86896.75350.261
2.248-2.3260.271650.2424260.24125480.8720.89597.7630.24
2.326-2.4130.251690.22523790.22624890.9030.91398.35280.225
2.413-2.510.282750.22823170.2324090.8770.91199.29430.228
2.51-2.620.316590.24422300.24522990.8580.89599.5650.244
2.62-2.7460.302610.25821270.25922020.8650.88799.36420.258
2.746-2.8920.318540.25920360.2621110.8680.88699.00520.259
2.892-3.0640.264490.25719070.25719860.8920.8998.48940.257
3.064-3.270.265480.2518210.25119050.9030.90998.11020.25
3.27-3.5250.302390.23716290.23917560.8940.9294.98860.237
3.525-3.8510.223400.23414550.23416440.9220.92190.93670.234
3.851-4.2880.232450.22912500.22914920.940.92486.79620.229
4.288-4.9180.189240.21411240.21313380.9490.94485.79970.214
4.918-5.9430.327330.2489770.2511530.8520.91587.59760.248
5.943-8.0910.248210.2187670.2199380.9040.92584.00850.218
8.091-21.1310.27150.2284730.2296190.9240.9578.83680.228
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)
10.0699-0.0169-0.06890.2381-0.14010.2472-0.01040.01610.0439-0.02220.01790.02430.0301-0.0196-0.00750.0042-0.0053-0.00660.05820.00870.08413.93015.001827.7915
20.20120.11240.00590.3005-0.0090.2902-0.01680.0150.04410.00570.0093-0.01680.07280.04830.00750.02220.00990.00240.05660.01020.075317.9879-0.742631.6899
30.17030.04230.08490.0609-0.11540.544-0.028-0.0466-0.0265-0.0030.00060.0099-0.02-0.04680.02740.00750.01930.00430.0606-00.07192.0324-4.911360.6441
40.06880.0049-0.16270.326-0.00430.3850.0152-0.027-0.00740.0265-0.029-0.0134-0.03720.0640.01380.01080.0046-0.01410.07340.00450.061917.12830.010558.066
Refinement TLS group
IDRefine-IDRefine TLS-IDSelectionAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1ALLAAA2 - 159
2X-RAY DIFFRACTION2ALLBBB179 - 313
3X-RAY DIFFRACTION3ALLCCC4 - 158
4X-RAY DIFFRACTION4ALLDDD179 - 313

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