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- PDB-4izw: The E41L mutant of the amidase from Nesterenkonia sp. AN1 showing... -

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Basic information

Entry
Database: PDB / ID: 4izw
TitleThe E41L mutant of the amidase from Nesterenkonia sp. AN1 showing covalent addition of the acetamide moiety of fluoroacetamide at the active site cysteine
ComponentsAmidase
KeywordsHYDROLASE / active site / fluoroacetamide / acetamide
Function / homology
Function and homology information


amidase / indoleacetamide hydrolase activity / N-carbamoylputrescine amidase activity / beta-alanine biosynthetic process via 3-ureidopropionate / beta-ureidopropionase activity / putrescine biosynthetic process from arginine / amidase activity
Similarity search - Function
(R)-stereoselective amidase RamA-like / Nitrilase/N-carbamoyl-D-aminoacid amidohydrolase / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETAMIDE / 2-fluoroacetamide / Amidase
Similarity search - Component
Biological speciesNesterenkonia sp. 10004 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.6 Å
AuthorsKimani, S.W. / Sewell, B.T.
CitationJournal: To be Published
Title: Covalent modifications of the active site cysteine occur as a result of mutating the glutamate of the catalytic triad in the amidase from Nesterenkonia sp.
Authors: Kimani, S.W. / Hunter, R. / Vlok, M. / Watermeyer, J. / Sewell, B.T.
History
DepositionJan 30, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2014Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,3755
Polymers30,0851
Non-polymers2904
Water2,918162
1
A: Amidase
hetero molecules

A: Amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,75010
Polymers60,1702
Non-polymers5808
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555x,-y,-z1
Buried area4170 Å2
ΔGint-1 kcal/mol
Surface area19540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.626, 114.382, 64.767
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-522-

HOH

21A-547-

HOH

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Components

#1: Protein Amidase /


Mass: 30084.795 Da / Num. of mol.: 1 / Mutation: E41Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nesterenkonia sp. 10004 (bacteria) / Strain: AN1 / Gene: Nit2 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: D0VWZ1, amidase
#2: Chemical ChemComp-ACM / ACETAMIDE / Acetamide


Mass: 59.067 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H5NO
#3: Chemical ChemComp-FTM / 2-fluoroacetamide / Fluoroacetamide


Mass: 77.058 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H4FNO
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 162 / 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: 2.33 Å3/Da / Density % sol: 47.16 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M HEPES sodium, 2% PEG 400, 2.0 M ammonium sulfate, pH 7.5, VAPOR DIFFUSION, SITTING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.9184 Å
DetectorDetector: CCD / Date: Jul 10, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.6→42.87 Å / Num. obs: 35169 / % possible obs: 94 % / Redundancy: 2.61 % / Rmerge(I) obs: 0.076 / Χ2: 0.98 / Net I/σ(I): 7.9 / Scaling rejects: 695
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allΧ2% possible all
1.6-1.662.590.3353929735870.9996.9
1.66-1.722.60.2223.3928535730.9596.6
1.72-1.82.60.2413.5930935750.9896.5
1.8-1.92.610.1954927535430.9895.9
1.9-2.022.610.1515.2930435461.0495.2
2.02-2.172.630.1186.5912334680.9494.1
2.17-2.392.620.1067.2924835080.9393.6
2.39-2.742.640.0898.9930635010.9393.1
2.74-3.452.560.05913.3890034350.9290.7
3.45-42.872.670.03824.2955434331.1487.6

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 31.3 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å42.9 Å
Translation2.5 Å42.9 Å

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Processing

Software
NameVersionClassificationNB
d*TREKdata scaling
d*TREK9.7Ldata reduction
PHASER2.1.1phasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→42.87 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.953 / WRfactor Rfree: 0.2186 / WRfactor Rwork: 0.1914 / Occupancy max: 1 / Occupancy min: 0.5 / FOM work R set: 0.8639 / SU B: 1.582 / SU ML: 0.054 / SU R Cruickshank DPI: 0.0902 / SU Rfree: 0.0903 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.09 / ESU R Free: 0.09 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.213 1765 5 %RANDOM
Rwork0.183 ---
obs0.1845 35166 93.97 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 72.62 Å2 / Biso mean: 19.9973 Å2 / Biso min: 9.85 Å2
Baniso -1Baniso -2Baniso -3
1-0.15 Å20 Å20 Å2
2--0.01 Å20 Å2
3----0.17 Å2
Refinement stepCycle: LAST / Resolution: 1.6→42.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1961 0 19 162 2142
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0360.0222052
X-RAY DIFFRACTIONr_angle_refined_deg2.5691.9942798
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2035266
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.21823.84691
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.09515315
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.81518
X-RAY DIFFRACTIONr_chiral_restr0.2350.2311
X-RAY DIFFRACTIONr_gen_planes_refined0.0160.0221611
X-RAY DIFFRACTIONr_mcbond_it1.6291.51319
X-RAY DIFFRACTIONr_mcangle_it2.56622114
X-RAY DIFFRACTIONr_scbond_it4.0063733
X-RAY DIFFRACTIONr_scangle_it6.5554.5681
LS refinement shellResolution: 1.6→1.642 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.33 136 -
Rwork0.298 2506 -
all-2642 -
obs--96.95 %

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