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- PDB-4izt: The E41Q mutant of the amidase from Nesterenkonia sp. AN1 showing... -

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Basic information

Entry
Database: PDB / ID: 4izt
TitleThe E41Q mutant of the amidase from Nesterenkonia sp. AN1 showing covalent addition of the acetamide moiety of fluoroacetamide at the active site cysteine
ComponentsAmidase
Keywordshydrolase/substrate / fluoroacetamide / acetamide / hydrolase-substrate complex
Function / homology
Function and homology information


amidase / N-carbamoylputrescine amidase 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 domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / 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: 1.92 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
Revision 1.1Nov 20, 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: Amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,3134
Polymers30,1001
Non-polymers2133
Water3,477193
1
A: Amidase
hetero molecules

A: Amidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,6268
Polymers60,2002
Non-polymers4266
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555x,-y,-z1
Buried area4110 Å2
ΔGint1 kcal/mol
Surface area19540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.580, 115.450, 64.930
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-486-

HOH

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Components

#1: Protein Amidase


Mass: 30099.768 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


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


Mass: 77.058 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H4FNO
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 193 / 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: 2.38 Å3/Da / Density % sol: 48.41 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.1 M Tris-HCL, 2.0 M ammonium sulfate, pH 8.5, VAPOR DIFFUSION, SITTING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.8856 Å
DetectorDetector: CCD / Date: Nov 15, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8856 Å / Relative weight: 1
ReflectionResolution: 1.92→32.47 Å / Num. all: 22375 / Num. obs: 22375 / % possible obs: 100 % / Redundancy: 7.3 % / Rsym value: 0.108 / Net I/σ(I): 13
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
1.92-2.027.40.4131.82376132190.413100
2.02-2.157.40.3012.52250630350.301100
2.15-2.297.40.2343.22126728760.234100
2.29-2.487.40.1764.31985226840.176100
2.48-2.727.40.1345.51840824920.134100
2.72-3.047.40.1056.91649522350.105100
3.04-3.517.30.097.31473320080.09100
3.51-4.297.30.0659.71242917030.065100
4.29-6.077.20.04312.3956913350.043100
6.07-32.4656.70.0361552607880.03699.4

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 30.19 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å45.54 Å
Translation2.5 Å45.54 Å

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.3.15data scaling
PHASER2.1.4phasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.92→32.47 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.954 / WRfactor Rfree: 0.1733 / WRfactor Rwork: 0.1369 / Occupancy max: 1 / Occupancy min: 0.3 / FOM work R set: 0.9024 / SU B: 2.494 / SU ML: 0.073 / SU R Cruickshank DPI: 0.1207 / SU Rfree: 0.1152 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.121 / 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
Rfree0.1818 1131 5.1 %RANDOM
Rwork0.145 ---
obs0.1469 22358 100 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 76.05 Å2 / Biso mean: 18.7967 Å2 / Biso min: 2.6 Å2
Baniso -1Baniso -2Baniso -3
1-1.08 Å20 Å20 Å2
2---0.12 Å20 Å2
3----0.96 Å2
Refinement stepCycle: LAST / Resolution: 1.92→32.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1968 0 14 193 2175
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0330.0222092
X-RAY DIFFRACTIONr_angle_refined_deg2.2631.9912867
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1655283
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.8323.8394
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.82515320
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.631519
X-RAY DIFFRACTIONr_chiral_restr0.2250.2317
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.0221664
X-RAY DIFFRACTIONr_mcbond_it1.5181.51350
X-RAY DIFFRACTIONr_mcangle_it2.46622167
X-RAY DIFFRACTIONr_scbond_it3.9833742
X-RAY DIFFRACTIONr_scangle_it6.3464.5690
LS refinement shellResolution: 1.92→1.97 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.242 84 -
Rwork0.194 1544 -
all-1628 -
obs--100 %

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