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- PDB-4gyl: The E142L mutant of the amidase from Geobacillus pallidus showing... -

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Basic information

Entry
Database: PDB / ID: 4gyl
TitleThe E142L mutant of the amidase from Geobacillus pallidus showing the result of Michael addition of acrylamide at the active site cysteine
ComponentsAliphatic amidase
KeywordsHYDROLASE / amidase / catalytic tetrad / amidase mechanism / acrylamide / Michael Adduct
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
PROPIONAMIDE / Aliphatic amidase
Similarity search - Component
Biological speciesBacillus sp. (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.9 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
DepositionSep 5, 2012Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Aug 21, 2013Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2014Group: Database references
Revision 1.2Oct 30, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn / 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_conn.pdbx_leaving_atom_flag / _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

Downloads & links

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Assembly

Deposited unit
A: Aliphatic amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,7333
Polymers38,6251
Non-polymers1092
Water4,378243
1
A: Aliphatic amidase
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)232,40118
Polymers231,7496
Non-polymers65112
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 area45390 Å2
ΔGint-315 kcal/mol
Surface area51350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)130.490, 130.490, 130.490
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-577-

HOH

21A-633-

HOH

31A-727-

HOH

41A-730-

HOH

51A-731-

HOH

61A-732-

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-ROP / PROPIONAMIDE


Mass: 73.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7NO
#3: Chemical ChemComp-CL / CHLORIDE ION


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


Mass: 18.015 Da / Num. of mol.: 243 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.4 Å3/Da / Density % sol: 48.68 % / Mosaicity: 0.226 °
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: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.542 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Oct 22, 2011 / Details: VariMax HF Confocal Optical System
RadiationMonochromator: VariMax HF Confocal Optical System / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.542 Å / Relative weight: 1
ReflectionResolution: 1.9→24.23 Å / Num. obs: 30455 / % possible obs: 99.9 % / Redundancy: 15.98 % / Rmerge(I) obs: 0.246 / Χ2: 1.19 / Net I/σ(I): 7.8 / Scaling rejects: 3679
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allΧ2Rejects% possible all
1.9-1.976.560.6882.11942729541.46399.1
1.97-2.059.570.6222.72875329931.35111100
2.05-2.1414.80.5763.64431629781.35244100
2.14-2.2518.290.5264.35543830071.35432100
2.25-2.3918.370.4585.15571029981.34632100
2.39-2.5818.510.425.55639130221.29451100
2.58-2.8418.570.3526.45690530411.21444100
2.84-3.2518.610.2359.55735030581.08448100
3.25-4.0918.490.13814.65804531170.91420100
4.09-24.2317.520.1116.85801232870.8543499.7

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Processing

Software
NameVersionClassificationNB
d*TREK9.9.9.5Ldata reduction
REFMACrefinement
PDB_EXTRACT3.11data extraction
HKL-2000data collection
d*TREKdata scaling
REFMAC5.7.0029phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.9→24.23 Å / Cor.coef. Fo:Fc: 0.961 / Cor.coef. Fo:Fc free: 0.949 / WRfactor Rfree: 0.1708 / WRfactor Rwork: 0.1427 / Occupancy max: 1 / Occupancy min: 0.33 / FOM work R set: 0.9067 / SU B: 2.475 / SU ML: 0.072 / SU R Cruickshank DPI: 0.129 / SU Rfree: 0.1161 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.13 / 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
Rfree0.1866 1537 5 %RANDOM
Rwork0.1631 ---
obs0.1643 30454 99.79 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 68.73 Å2 / Biso mean: 17.561 Å2 / Biso min: 10.24 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: LAST / Resolution: 1.9→24.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2649 0 6 243 2898
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0230.0192720
X-RAY DIFFRACTIONr_bond_other_d0.0060.022545
X-RAY DIFFRACTIONr_angle_refined_deg2.0751.9473683
X-RAY DIFFRACTIONr_angle_other_deg1.07235871
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.3085339
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.54924.597124
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.82415458
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.5641513
X-RAY DIFFRACTIONr_chiral_restr0.1370.2395
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.0213104
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02619
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.29 87 -
Rwork0.279 2111 -
all-2198 -
obs--98.79 %

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