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- PDB-4kzf: The mechanism of the amidases: The effect of the mutation E142L i... -

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Basic information

Entry
Database: PDB / ID: 4kzf
TitleThe mechanism of the amidases: The effect of the mutation E142L in the amidase from Geobacillus pallidus
ComponentsAliphatic amidase
KeywordsHYDROLASE / active site / chloride ion / cysteine 166 oxidation / amidase
Function / homology
Function and homology information


amidase / amidase activity
Similarity search - Function
Aliphatic amidase / : / Nitrilase/N-carbamoyl-D-aminoacid amidohydrolase / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesBacillus sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.85 Å
AuthorsWeber, B.W. / Sewell, B.T. / Kimani, S.W. / Varsani, A. / Cowan, D.A. / Hunter, R.
CitationJournal: J.Biol.Chem. / Year: 2013
Title: The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning.
Authors: Weber, B.W. / Kimani, S.W. / Varsani, A. / Cowan, D.A. / Hunter, R. / Venter, G.A. / Gumbart, J.C. / Sewell, B.T.
History
DepositionMay 29, 2013Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Aug 21, 2013Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2014Group: Database references
Revision 1.2Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / 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 / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aliphatic amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,6602
Polymers38,6251
Non-polymers351
Water3,693205
1
A: Aliphatic amidase
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)231,96212
Polymers231,7496
Non-polymers2136
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555-z,x,-y1
crystal symmetry operation11_555y,-z,-x1
crystal symmetry operation14_445-y-1/2,-x-1/2,-z+1/21
crystal symmetry operation18_445-x-1/2,z-1/2,y+1/21
crystal symmetry operation22_445z-1/2,-y-1/2,x+1/21
Buried area44440 Å2
ΔGint-310 kcal/mol
Surface area51700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)130.979, 130.979, 130.979
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number208
Space group name H-MP4232
Components on special symmetry positions
IDModelComponents
11A-506-

HOH

21A-538-

HOH

31A-562-

HOH

41A-581-

HOH

51A-593-

HOH

61A-605-

HOH

71A-615-

HOH

81A-617-

HOH

91A-658-

HOH

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Components

#1: Protein Aliphatic amidase / Acylamide amidohydrolase


Mass: 38624.875 Da / Num. of mol.: 1 / Mutation: E142L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. (bacteria) / Strain: RAPC8 / Gene: amiE, ami / Plasmid: pET21a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) PLysS / References: UniProt: Q9L543, amidase
#2: Chemical ChemComp-CL / CHLORIDE ION


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


Mass: 18.015 Da / Num. of mol.: 205 / 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: 2.42 Å3/Da / Density % sol: 49.26 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 1.2M sodium citrate, 0.4M sodium chloride, 0.1M sodium acetate, pH 5.6, vapor diffusion, hanging drop, temperature 294K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.8856 Å
DetectorDetector: CCD / Date: Jun 28, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8856 Å / Relative weight: 1
ReflectionResolution: 1.84→92.62 Å / Num. all: 33703 / Num. obs: 33703 / % possible obs: 99.7 % / Observed criterion σ(F): 3 / Observed criterion σ(I): 3 / Redundancy: 6.58 % / Biso Wilson estimate: 29.5 Å2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 10.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
REFMACrefinement
PDB_EXTRACT3.11data extraction
MxCuBEdata collection
d*TREKdata reduction
d*TREKdata scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2PLQ

2plq


Resolution: 1.85→36.3 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.945 / WRfactor Rfree: 0.1968 / WRfactor Rwork: 0.1812 / Occupancy max: 1 / Occupancy min: 0.33 / FOM work R set: 0.8783 / SU B: 2.712 / SU ML: 0.08 / SU R Cruickshank DPI: 0.1322 / SU Rfree: 0.1154 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.132 / ESU R Free: 0.115 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES: REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2028 1690 5.1 %RANDOM
Rwork0.1852 ---
all0.1861 33255 --
obs0.1861 33155 99.34 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 74.04 Å2 / Biso mean: 23.3198 Å2 / Biso min: 10.23 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: LAST / Resolution: 1.85→36.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2640 0 1 205 2846
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0240.0192722
X-RAY DIFFRACTIONr_bond_other_d0.0010.022548
X-RAY DIFFRACTIONr_angle_refined_deg1.9411.9423688
X-RAY DIFFRACTIONr_angle_other_deg0.95935879
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.1055340
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.25524.516124
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.04215461
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.5561513
X-RAY DIFFRACTIONr_chiral_restr0.1410.2394
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0213106
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02623
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.353 117 -
Rwork0.293 2134 -
all-2251 -
obs--93.44 %

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