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- PDB-8ecd: E. coli L-asparaginase II mutant (V27T) in complex with L-Asp -

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Basic information

Entry
Database: PDB / ID: 8ecd
TitleE. coli L-asparaginase II mutant (V27T) in complex with L-Asp
ComponentsL-asparaginase 2
KeywordsHYDROLASE / 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, 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, 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.
Similarity search - Domain/homology
ASPARTIC ACID / CITRIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.62 Å
AuthorsStrzelczyk, P. / Wlodawer, A. / Lubkowski, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Febs Lett. / Year: 2022
Title: The E. coli L-asparaginase V27T mutant: structural and functional characterization and comparison with theoretical predictions.
Authors: Strzelczyk, P. / Zhang, D. / Wlodawer, A. / Lubkowski, J.
History
DepositionSep 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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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)145,12229
Polymers142,8164
Non-polymers2,30625
Water28,6261589
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)144,27320
Polymers142,8164
Non-polymers1,45716
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area18680 Å2
ΔGint-37 kcal/mol
Surface area42120 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)145,97138
Polymers142,8164
Non-polymers3,15534
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area24330 Å2
ΔGint0 kcal/mol
Surface area41510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.559, 62.781, 141.052
Angle α, β, γ (deg.)90.000, 117.620, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-738-

HOH

21B-735-

HOH

31C-785-

HOH

41D-1275-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: HIS / Beg label comp-ID: HIS / End auth comp-ID: TYR / End label comp-ID: TYR / Refine code: _ / Auth seq-ID: -3 - 326 / Label seq-ID: 5 - 334

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11AA
21BB
12AA
22CC
13AA
23DD
14BB
24CC
15BB
25DD
16CC
26DD

NCS ensembles :
ID
1
2
3
4
5
6

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
L-asparaginase 2 / L-asparaginase II / L-ASNase II / L-asparagine amidohydrolase II


Mass: 35703.938 Da / Num. of mol.: 4 / Mutation: V27T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: ansB, b2957, JW2924 / Plasmid: pET22b(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P00805, asparaginase

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Non-polymers , 5 types, 1614 molecules

#2: Chemical
ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#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: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1589 / 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: 2.08 Å3/Da / Density % sol: 40.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.2 M Ammonium citrate tribasic pH 7.0, 19% w/v Polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Sep 25, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.62→50 Å / Num. obs: 145830 / % possible obs: 97.9 % / Redundancy: 4.4 % / CC1/2: 0.994 / Rmerge(I) obs: 0.076 / Rpim(I) all: 0.04 / Rrim(I) all: 0.086 / Net I/σ(I): 18.12
Reflection shellResolution: 1.62→1.65 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.562 / Mean I/σ(I) obs: 2.04 / Num. unique obs: 6727 / CC1/2: 0.71 / Rpim(I) all: 0.334 / Rrim(I) all: 0.659 / % possible all: 90.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3ECA

3eca


Resolution: 1.62→39.22 Å / Cor.coef. Fo:Fc: 0.979 / Cor.coef. Fo:Fc free: 0.965 / SU B: 1.591 / SU ML: 0.053 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.073 / ESU R Free: 0.078 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1643 2134 1.5 %RANDOM
Rwork0.1268 ---
obs0.1274 140778 95.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 106.75 Å2 / Biso mean: 24.805 Å2 / Biso min: 13.5 Å2
Baniso -1Baniso -2Baniso -3
1--0.09 Å2-0 Å20.05 Å2
2---0.26 Å2-0 Å2
3---0.19 Å2
Refinement stepCycle: final / Resolution: 1.62→39.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9808 0 182 1589 11579
Biso mean--36.96 36.97 -
Num. residues----1309
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.01310153
X-RAY DIFFRACTIONr_bond_other_d0.0010.0169646
X-RAY DIFFRACTIONr_angle_refined_deg2.0871.64813794
X-RAY DIFFRACTIONr_angle_other_deg1.5771.71422202
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.47951310
X-RAY DIFFRACTIONr_dihedral_angle_2_deg41.31225.863423
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.797151624
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4681532
X-RAY DIFFRACTIONr_chiral_restr0.1240.21400
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.0211658
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022102
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A98600.08
12B98600.08
21A102680.07
22C102680.07
31A101110.09
32D101110.09
41B99200.07
42C99200.07
51B99590.06
52D99590.06
61C101450.08
62D101450.08
LS refinement shellResolution: 1.62→1.662 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.22 128 -
Rwork0.199 8476 -
all-8604 -
obs--78.37 %

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