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- PDB-6pa5: ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 8.3 IN SPACE... -

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Basic information

Entry
Database: PDB / ID: 6pa5
TitleECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 8.3 IN SPACE GROUP P2(1)
ComponentsL-asparaginase 2
KeywordsHYDROLASE / inactive mutant / hydrolysis of L-asparagine
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
ASPARAGINE / IMIDAZOLE / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2 Å
AuthorsLubkowski, J. / Wlodawer, A.
CitationJournal: Protein Sci. / Year: 2019
Title: Geometric considerations support the double-displacement catalytic mechanism of l-asparaginase.
Authors: Lubkowski, J. / Wlodawer, A.
History
DepositionJun 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 2, 2019Group: Data collection / Database references / Category: citation_author / Item: _citation_author.identifier_ORCID

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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
E: L-asparaginase 2
F: L-asparaginase 2
G: L-asparaginase 2
H: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)287,19226
Polymers285,3758
Non-polymers1,81718
Water37,9582107
1
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)143,76915
Polymers142,6884
Non-polymers1,08111
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17290 Å2
ΔGint-38 kcal/mol
Surface area39820 Å2
MethodPISA
2
E: L-asparaginase 2
F: L-asparaginase 2
G: L-asparaginase 2
H: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,42311
Polymers142,6884
Non-polymers7367
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16670 Å2
ΔGint-40 kcal/mol
Surface area39740 Å2
MethodPISA
Unit cell
Length a, b, c (Å)140.530, 62.335, 151.047
Angle α, β, γ (deg.)90.000, 117.680, 90.000
Int Tables number4
Space group name H-MP1211

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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.: 8 / Mutation: T89V, K162T
Source method: isolated from a genetically manipulated source
Details: Expressed variant contains 8 additional N-terminal residues MDHHHHHH (affinity tag) and mutation (T89V,K162T) in mature protein
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ansB, b2957, JW2924 / Plasmid: pET22b(+)
Details (production host): contains secretion sequence pelB leader
Cell (production host): bacteria / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): JC2 strain lacking in ansA / References: UniProt: P00805, asparaginase
#2: Chemical
ChemComp-ASN / ASPARAGINE


Type: L-peptide linking / Mass: 132.118 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C4H8N2O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2107 / 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: 2.05 Å3/Da / Density % sol: 40.09 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.3
Details: Crystals grown in 0.17 M NH4-citrate, pH 7.0, 17-18% PEG3350, then soaked for 1 min in 0.1 M Tris, pH 8.3, 19-PEG3350 with 5 mM of L-Asn

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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 R 4M / Detector: PIXEL / Date: Oct 5, 2018 / Details: Multilayer X-ray mirrors VariMax HF
RadiationMonochromator: Multilayer X-ray mirrors VariMax HF / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.736
11-H, -K, H+L20.264
ReflectionResolution: 2→40 Å / Num. obs: 150541 / % possible obs: 95.8 % / Redundancy: 2.9 % / Rmerge(I) obs: 0.066 / Rpim(I) all: 0.044 / Rrim(I) all: 0.079 / Χ2: 0.868 / Net I/σ(I): 9 / Num. measured all: 442685
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.032.50.4967410.8480.3440.6020.71287.3
2.03-2.072.60.40969460.890.2870.5030.72688.2
2.07-2.112.60.36668410.9070.2540.4470.74788.2
2.11-2.152.60.29869850.9430.2070.3650.79289.2
2.15-2.22.70.27569860.9470.1890.3350.79189.7
2.2-2.252.70.24172270.9570.1660.2940.79492.3
2.25-2.312.70.20474010.9690.140.2490.8194.3
2.31-2.372.80.17974600.9710.1220.2170.84495.9
2.37-2.442.80.16676060.9730.1130.2010.86697.1
2.44-2.522.90.14876680.9780.1010.180.88798
2.52-2.612.90.1378050.9840.0890.1580.91299.1
2.61-2.7130.11777590.9840.0790.1410.95599.2
2.71-2.8430.09677920.9910.0640.1160.94999.4
2.84-2.993.10.08277910.9920.0540.0981.0199.7
2.99-3.173.10.06878420.9920.0450.0821.03599.8
3.17-3.423.20.05179120.9970.0330.0610.98999.9
3.42-3.763.30.04278680.9970.0270.050.95799.9
3.76-4.313.30.03679010.9980.0230.0420.91499.8
4.31-5.423.20.03279050.9980.0210.0390.77199.2
5.42-403.50.03181050.9980.0190.0360.799.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
REFMAC5.8.0158refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→26.21 Å / Cor.coef. Fo:Fc: 0.979 / Cor.coef. Fo:Fc free: 0.957 / SU B: 4.304 / SU ML: 0.119 / SU R Cruickshank DPI: 0.0441 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.044 / ESU R Free: 0.036
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2137 4012 2.7 %RANDOM
Rwork0.1702 ---
obs0.1714 146167 95.46 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 118.98 Å2 / Biso mean: 31.558 Å2 / Biso min: 10.8 Å2
Baniso -1Baniso -2Baniso -3
1-1.36 Å20 Å21.84 Å2
2---2.51 Å2-0 Å2
3---1.15 Å2
Refinement stepCycle: final / Resolution: 2→26.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19092 0 125 2113 21330
Biso mean--40.16 36.66 -
Num. residues----2558
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0219528
X-RAY DIFFRACTIONr_bond_other_d0.0020.0218106
X-RAY DIFFRACTIONr_angle_refined_deg1.9721.9626607
X-RAY DIFFRACTIONr_angle_other_deg1.108342084
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.79952555
X-RAY DIFFRACTIONr_dihedral_angle_2_deg41.43426.079806
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.231153159
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4311564
X-RAY DIFFRACTIONr_chiral_restr0.1270.23204
X-RAY DIFFRACTIONr_gen_planes_refined0.010.02122004
X-RAY DIFFRACTIONr_gen_planes_other0.0020.023525
LS refinement shellResolution: 1.999→2.05 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.339 243 -
Rwork0.292 9516 -
all-9759 -
obs--85.08 %

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