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- PDB-3hht: A mutant of the nitrile hydratase from Geobacillus pallidus havin... -

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Basic information

Entry
Database: PDB / ID: 3hht
TitleA mutant of the nitrile hydratase from Geobacillus pallidus having enhanced thermostability
Components
  • Nitrile hydratase alpha subunit
  • Nitrile hydratase beta subunit
KeywordsLYASE / Alpha and beta proteins (a+b)
Function / homology
Function and homology information


nitrile hydratase / indole-3-acetonitrile nitrile hydratase activity / : / transition metal ion binding
Similarity search - Function
Nitrile hydratase, alpha subunit / Nitrile hydratase, beta subunit / Nitrile hydratase, beta subunit / Nitrile Hydratase; Chain A / Nitrile hydratase alpha /Thiocyanate hydrolase gamma / Nitrile hydratase beta subunit domain / Nitrile hydratase beta subunit, N-terminal / : / Nitrile hydratase beta subunit, C-terminal / Nitrile hydratase beta subunit, N-terminal ...Nitrile hydratase, alpha subunit / Nitrile hydratase, beta subunit / Nitrile hydratase, beta subunit / Nitrile Hydratase; Chain A / Nitrile hydratase alpha /Thiocyanate hydrolase gamma / Nitrile hydratase beta subunit domain / Nitrile hydratase beta subunit, N-terminal / : / Nitrile hydratase beta subunit, C-terminal / Nitrile hydratase beta subunit, N-terminal / Nitrile hydratase alpha subunit /Thiocyanate hydrolase gamma subunit / Nitrile hydratase alpha /Thiocyanate hydrolase gamma / Nitrile hydratase, alpha chain / Nitrile hydratase alpha /Thiocyanate hydrolase gamma superfamily / SH3 type barrels. - #50 / Electron transport accessory-like domain superfamily / Cyclin A; domain 1 / SH3 type barrels. / Roll / Alpha-Beta Complex / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
COBALT (III) ION / nitrile hydratase / Nitrile hydratase subunit beta
Similarity search - Component
Biological speciesGeobacillus pallidus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.16 Å
AuthorsVan Wyk, J.C. / Sewell, B.T. / Cowan, D.A. / Sayed, M.F. / Tsekoa, T.L. / Tastan Bishop, A.O.
CitationJournal: To be published
Title: The high-resolution structure of a Cobalt containing nitrile hydratase
Authors: Van Wyk, J.C. / Tastan Bishop, A.O. / Cowan, D.A. / Sayed, M.F. / Tsekoa, T.L. / Sewell, B.T.
History
DepositionMay 17, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / 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
Revision 1.3Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nitrile hydratase alpha subunit
B: Nitrile hydratase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,2595
Polymers51,1292
Non-polymers1303
Water9,350519
1
A: Nitrile hydratase alpha subunit
B: Nitrile hydratase beta subunit
hetero molecules

A: Nitrile hydratase alpha subunit
B: Nitrile hydratase beta subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,51810
Polymers102,2584
Non-polymers2606
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_777-y+2,-x+2,-z+5/21
Buried area22080 Å2
ΔGint-132 kcal/mol
Surface area33190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)105.832, 105.832, 83.723
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein Nitrile hydratase alpha subunit


Mass: 24674.207 Da / Num. of mol.: 1 / Mutation: D4G
Source method: isolated from a genetically manipulated source
Details: The details of pNH14k are described in Cameron, DA et al (2005) Biochim. Biophys Acta 1725:35-46
Source: (gene. exp.) Geobacillus pallidus (bacteria) / Strain: RAPc8 / Plasmid: pNH14k / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q84FS5, nitrile hydratase
#2: Protein Nitrile hydratase beta subunit


Mass: 26454.971 Da / Num. of mol.: 1 / Mutation: F36L, L103S, Y127N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus pallidus (bacteria) / Strain: RAPc8 / Plasmid: pNH14k / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q84FS6, nitrile hydratase
#3: Chemical ChemComp-3CO / COBALT (III) ION / Cobalt


Mass: 58.933 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Co
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 519 / 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: 2.29 Å3/Da / Density % sol: 46.29 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 30% PEG400, 100mM magnesium chloride, 100mM MES (2[N-Morpholino]ethanesulfonic acid), 10-40mg/ml protein, pH 6.5, vapor diffusion, hanging drop, temperature 295K

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.979 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jul 18, 2007 / Details: mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.16→26.99 Å / Num. obs: 162627 / % possible obs: 99.6 % / Redundancy: 5.58 % / Rmerge(I) obs: 0.047 / Χ2: 0.94 / Scaling rejects: 6861
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allΧ2% possible all
1.16-1.25.410.34.188510161391.33100
1.2-1.255.50.2584.489572161831.2100
1.25-1.315.540.2115.390078161581.11100
1.31-1.385.590.1676.490970161771.01100
1.38-1.465.630.129891746161820.95100
1.46-1.575.660.09210.792876162740.83100
1.57-1.735.670.07513.293072162830.79100
1.73-1.985.680.07313.493674163850.76100
1.98-2.55.710.04521.594650164830.71100
2.5-26.995.430.0254289532163630.7596.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
d*TREK9.7Ldata scaling
PHASERphasing
REFMACrefinement
PDB_EXTRACT3.005data extraction
MxCuBEdata collection
d*TREKdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2dpp

2dpp


Resolution: 1.16→26.99 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.978 / WRfactor Rfree: 0.145 / WRfactor Rwork: 0.128 / Occupancy max: 1 / Occupancy min: 0.07 / FOM work R set: 0.923 / SU B: 0.727 / SU ML: 0.016 / SU R Cruickshank DPI: 0.028 / SU Rfree: 0.028 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.028 / ESU R Free: 0.028 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES: REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.144 8144 5 %RANDOM
Rwork0.127 ---
obs0.128 162458 99.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 60.41 Å2 / Biso mean: 15.944 Å2 / Biso min: 7.04 Å2
Baniso -1Baniso -2Baniso -3
1-0.01 Å20 Å20 Å2
2--0.01 Å20 Å2
3----0.01 Å2
Refinement stepCycle: LAST / Resolution: 1.16→26.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3506 0 3 522 4031
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0223608
X-RAY DIFFRACTIONr_bond_other_d0.0010.022525
X-RAY DIFFRACTIONr_angle_refined_deg1.7261.9684895
X-RAY DIFFRACTIONr_angle_other_deg0.98636126
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1485435
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.79723.371175
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.9415625
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.361529
X-RAY DIFFRACTIONr_chiral_restr0.1130.2515
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.0213995
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02745
X-RAY DIFFRACTIONr_mcbond_it2.5361.52159
X-RAY DIFFRACTIONr_mcbond_other1.3241.5864
X-RAY DIFFRACTIONr_mcangle_it3.46723503
X-RAY DIFFRACTIONr_scbond_it5.03231449
X-RAY DIFFRACTIONr_scangle_it6.8064.51390
X-RAY DIFFRACTIONr_rigid_bond_restr2.68636133
X-RAY DIFFRACTIONr_sphericity_free15.3063559
X-RAY DIFFRACTIONr_sphericity_bonded5.94636036
LS refinement shellResolution: 1.16→1.19 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.219 631 -
Rwork0.197 11308 -
all-11939 -
obs--99.99 %

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