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- PDB-12as: ASPARAGINE SYNTHETASE MUTANT C51A, C315A COMPLEXED WITH L-ASPARAG... -

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Basic information

Entry
Database: PDB / ID: 12as
TitleASPARAGINE SYNTHETASE MUTANT C51A, C315A COMPLEXED WITH L-ASPARAGINE AND AMP
ComponentsASPARAGINE SYNTHETASE
KeywordsLIGASE / ASPARAGINE SYNTHETASE / NITROGEN FIXATION
Function / homology
Function and homology information


aspartate-ammonia ligase / aspartate-ammonia ligase activity / L-asparagine biosynthetic process / : / DNA damage response / protein homodimerization activity / ATP binding / identical protein binding / cytosol
Similarity search - Function
Aspartate--ammonia ligase / Aspartate-ammonia ligase / Bira Bifunctional Protein; Domain 2 / BirA Bifunctional Protein; domain 2 / Aminoacyl-tRNA synthetase, class II / Aminoacyl-transfer RNA synthetases class-II family profile. / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / ASPARAGINE / Aspartate--ammonia ligase
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria)
MethodX-RAY DIFFRACTION / Resolution: 2.2 Å
AuthorsNakatsu, T. / Kato, H. / Oda, J.
Citation
Journal: Nat.Struct.Biol. / Year: 1998
Title: Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
Authors: Nakatsu, T. / Kato, H. / Oda, J.
#1: Journal: Acta Crystallogr.,Sect.D / Year: 1996
Title: Crystallization and Preliminary Crystallographic Study of Asparagine Synthetase from Escherichia Coli
Authors: Nakatsu, T. / Kato, H. / Oda, J.
#2: Journal: Biosci.Biotechnol.Biochem. / Year: 1992
Title: Overexpression and Purification of Asparagine Synthetase from Escherichia Coli
Authors: Sugiyama, A. / Kato, H. / Nishioka, T. / Oda, J.
History
DepositionDec 2, 1997Processing site: BNL
Revision 1.0Dec 30, 1998Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 3, 2021Group: Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.process_site / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 2, 2023Group: Refinement description / Category: pdbx_initial_refinement_model
Revision 1.5May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ASPARAGINE SYNTHETASE
B: ASPARAGINE SYNTHETASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,2256
Polymers73,2672
Non-polymers9594
Water3,657203
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5430 Å2
ΔGint-25 kcal/mol
Surface area24890 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.000, 126.130, 52.860
Angle α, β, γ (deg.)90.00, 105.59, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (-1, -0.00022, 0.00086), (-0.00022, -0.87596, -0.48238), (0.00086, -0.48238, 0.87597)
Vector: 64.29259, 68.35215, 17.54653)

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Components

#1: Protein ASPARAGINE SYNTHETASE / L-ASPARTATE\:AMMONIA LIGASE (AMP-FORMING)


Mass: 36633.367 Da / Num. of mol.: 2 / Mutation: C51A, C315A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Species: Escherichia coli / Strain: K-12 / Gene: ASNA / Plasmid: PUNAD37 CYS-FREE / Gene (production host): ASNA / Production host: Escherichia coli (E. coli) / Strain (production host): JM109 / References: UniProt: P00963, aspartate-ammonia ligase
#2: Chemical ChemComp-ASN / ASPARAGINE


Type: L-peptide linking / Mass: 132.118 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H8N2O3 / Details: COMPLEXED WITH ASN AND AMP
#3: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Comment: AMP*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H2O

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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: 2.3 Å3/Da / Density % sol: 47 %
Crystal growpH: 7.5
Details: PROTEIN CRYSTALLIZED FROM 45% SATURATED AMMONIUM SULFATE, 22 MM ASPARAGINE, 88 MM MGCL2, 10 %(W/V) GLYCEROL 5 MM 2-MERCAPTOETHANOL, 50 MM HEPES, PH7.5 THEN SOAKED BY 50MM AMP AND 50 MM ASPARAGINE
Crystal grow
*PLUS
Temperature: 293 K / Method: vapor diffusion, sitting drop
Details: drop consists of equal volume of protein and reservoir solutions
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
130 mg/mlprotein1drop
220 mMHEPES1drop
310 %(w/v)glycerol1drop
45 mMbeta-mercaptoethanol1drop
545 %satammonium sulfate1reservoir
622 mMAsn1reservoir
788 mM1reservoirMgCl2
850 mMHEPES1reservoir
910 %(w/v)glycerol1reservoir
105 mMbeta-mercaptoethanol1reservoir

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Data collection

DiffractionMean temperature: 293 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.5418
DetectorType: RIGAKU / Detector: IMAGE PLATE / Date: Nov 1, 1995 / Details: MIRROR
RadiationMonochromator: MSC DOUBLE MIRROR / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionHighest resolution: 2.2 Å / Num. obs: 25168 / % possible obs: 72.1 % / Observed criterion σ(I): 1 / Redundancy: 2.8 % / Biso Wilson estimate: 10.7 Å2 / Rmerge(I) obs: 0.112 / Net I/σ(I): 5
Reflection shellResolution: 2.2→2.25 Å / Redundancy: 1.7 % / Rmerge(I) obs: 0.238 / Mean I/σ(I) obs: 2.2 / % possible all: 53.4
Reflection
*PLUS
Num. measured all: 71426
Reflection shell
*PLUS
% possible obs: 53.4 %

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Processing

Software
NameClassification
PROCESSdata collection
PROCESSdata reduction
X-PLORmodel building
X-PLORrefinement
PROCESSdata scaling
X-PLORphasing
RefinementStarting model: PDB ENTRY 11AS

11as


Resolution: 2.2→10 Å / Data cutoff high absF: 10000000 / Data cutoff low absF: 0.001 / Cross valid method: THROUGHOUT / σ(F): 2
RfactorNum. reflection% reflectionSelection details
Rfree0.287 2455 10 %RANDOM
Rwork0.164 ---
obs0.164 24815 71.1 %-
Displacement parametersBiso mean: 10.2 Å2
Refine analyzeLuzzati coordinate error obs: 0.25 Å / Luzzati d res low obs: 5 Å
Refinement stepCycle: LAST / Resolution: 2.2→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5118 0 46 203 5367
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_bond_d0.008
X-RAY DIFFRACTIONx_bond_d_na
X-RAY DIFFRACTIONx_bond_d_prot
X-RAY DIFFRACTIONx_angle_d
X-RAY DIFFRACTIONx_angle_d_na
X-RAY DIFFRACTIONx_angle_d_prot
X-RAY DIFFRACTIONx_angle_deg1.25
X-RAY DIFFRACTIONx_angle_deg_na
X-RAY DIFFRACTIONx_angle_deg_prot
X-RAY DIFFRACTIONx_dihedral_angle_d27.4
X-RAY DIFFRACTIONx_dihedral_angle_d_na
X-RAY DIFFRACTIONx_dihedral_angle_d_prot
X-RAY DIFFRACTIONx_improper_angle_d2.7
X-RAY DIFFRACTIONx_improper_angle_d_na
X-RAY DIFFRACTIONx_improper_angle_d_prot
X-RAY DIFFRACTIONx_mcbond_it
X-RAY DIFFRACTIONx_mcangle_it
X-RAY DIFFRACTIONx_scbond_it
X-RAY DIFFRACTIONx_scangle_it
LS refinement shellResolution: 2.2→2.3 Å / Total num. of bins used: 8
RfactorNum. reflection% reflection
Rfree0.345 250 10 %
Rwork0.244 2045 -
obs--54.9 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PARHCSDX.PROTOPHCSDX.PRO
X-RAY DIFFRACTION2PARAM19.SOLTOPH19.SOL
Software
*PLUS
Name: X-PLOR / Version: 3.1 / Classification: refinement
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_dihedral_angle_d
X-RAY DIFFRACTIONx_dihedral_angle_deg27.4
X-RAY DIFFRACTIONx_improper_angle_d
X-RAY DIFFRACTIONx_improper_angle_deg2.7

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