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- PDB-1t2n: Structure of a thermostable triple mutant of Bacillus subtilis li... -

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Basic information

Entry
Database: PDB / ID: 1t2n
TitleStructure of a thermostable triple mutant of Bacillus subtilis lipase obtained through directed evolution
ComponentsLipase
KeywordsHYDROLASE / ALPHA/BETA HYDROLASE
Function / homology
Function and homology information


lipase activity / triacylglycerol lipase / triglyceride lipase activity / lipid catabolic process / extracellular region
Similarity search - Function
Lipase EstA/Esterase EstB / Lipase (class 2) / Alpha/Beta hydrolase fold, catalytic domain / Alpha/Beta hydrolase fold / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsRajakumara, E. / Sankaranarayanan, R.
Citation
Journal: J.Mol.Biol. / Year: 2004
Title: Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.
Authors: Acharya, P. / Rajakumara, E. / Sankaranarayanan, R. / Rao, N.M.
#1: Journal: Acta Crystallogr.,Sect.D / Year: 2004
Title: Crystallization and preliminary X-ray crystallographic investigations on several thermostable forms of a Bacillus subtilis lipase.
Authors: Rajakumara, E. / Acharya, P. / Ahmad, S. / Shanmugam, V.M. / Rao, N.M. / Sankaranarayanan, R.
History
DepositionApr 22, 2004Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Sep 10, 2014Group: Database references
Revision 1.4Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / 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 / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Aug 23, 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

Downloads & links

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Assembly

Deposited unit
A: Lipase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,4992
Polymers19,4601
Non-polymers391
Water3,459192
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)75.863, 75.863, 102.68
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3
DetailsThe monomer found in the asu is the biologically functional unit.

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Components

#1: Protein Lipase / Triacylglycerol lipase


Mass: 19459.875 Da / Num. of mol.: 1 / Mutation: L114P, A132D, N166Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: LIPA, LIP, BSU02700 / Plasmid: pET-21b / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P37957, triacylglycerol lipase
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 192 / 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: 3 Å3/Da / Density % sol: 58.6 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 9.5
Details: PEG 3350, ethanolamine, n-octyl-beta-D-glucoside, sodium sulfate, pH 9.5, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU300 / Wavelength: 1.5418 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Feb 1, 2003 / Details: Osmic
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.8→25 Å / Num. all: 20416 / Num. obs: 20289 / % possible obs: 99.4 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 2.2 % / Biso Wilson estimate: 20.3 Å2 / Rsym value: 0.026 / Net I/σ(I): 32
Reflection shellResolution: 1.8→1.86 Å / Redundancy: 2 % / Mean I/σ(I) obs: 4 / Num. unique all: 1918 / Rsym value: 0.161 / % possible all: 94.5

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Processing

Software
NameVersionClassification
CNS1.1refinement
DENZOdata reduction
SCALEPACKdata scaling
CCP4(MOLREP)phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1I6W

1i6w


Resolution: 1.8→24.14 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 1521273.67 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
Details: The ion present in the coordinates was named potassium based on the B-factor during refinement.
RfactorNum. reflection% reflectionSelection details
Rfree0.259 1037 5.1 %RANDOM
Rwork0.226 ---
all0.228 20289 --
obs0.228 20289 99.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 111.926 Å2 / ksol: 0.520093 e/Å3
Displacement parametersBiso mean: 23.3 Å2
Baniso -1Baniso -2Baniso -3
1-1.72 Å21.69 Å20 Å2
2--1.72 Å20 Å2
3----3.45 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.27 Å0.23 Å
Luzzati d res low-5 Å
Luzzati sigma a0.13 Å0.1 Å
Refinement stepCycle: LAST / Resolution: 1.8→24.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1357 0 1 192 1550
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.005
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d24
X-RAY DIFFRACTIONc_improper_angle_d0.73
X-RAY DIFFRACTIONc_mcbond_it1.231.5
X-RAY DIFFRACTIONc_mcangle_it1.852
X-RAY DIFFRACTIONc_scbond_it1.922
X-RAY DIFFRACTIONc_scangle_it2.752.5
LS refinement shellResolution: 1.8→1.86 Å / Rfactor Rfree error: 0.036 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.369 104 5 %
Rwork0.294 1815 -
obs--94.8 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3ION.PARAMCNS_ION.TOP

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