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PDB | 1T4M | STRUCTURE OF A THERMOSTABLE DOUBLE MUTANT OF BACILLUS SUBTILIS LIPASE OBTAINED THROUGH DIRECTED EVOLUTION

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Internal ID
22458
PDB Entry ID
1T4M
Title
STRUCTURE OF A THERMOSTABLE DOUBLE MUTANT OF BACILLUS SUBTILIS LIPASE OBTAINED THROUGH DIRECTED EVOLUTION
Authors
Rajakumara, E.,Sankaranarayanan, R.
Primary Citation
AuthorsAcharya, P., Rajakumara, E., Sankaranarayanan, R., Rao, N.M.
TitleStructural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase
JournalJ.Mol.Biol., v.341, pp.1271 - 1281, 2004
AbstractView
Pubmed15321721
DOI10.1016/j.jmb.2004.06.059
 
History
Deposition: 2004-04-30Release: 2004-11-23Last Modified: 2009-02-24
Experimental Method
TypeX-RAY DIFFRACTION
Parameters
Resolution [Å]R-Value (Obs.)R-Value (Work)R-FreeSpace Group
2.000.2070.2060.257H 3
Unit Cell
Length [Å]a76.01b76.01c103.09
Angles [°]alpha90.00beta90.00gamma120.00
Molecular Description
PolymerMoleculeMutationFragmentChainsEC NumberOther details
1LIPASE AA132D, N166YA3.1.1.3
Functional Class
Source
PolymerScientific NameCommon NameExpression System
1Bacillus subtilisEscherichia coli BL21(DE3)
Related PDB Entries
Chemical Component
IdentifierNameFormulaLigand StructureLigand Interaction
KPOTASSIUM IONK1[View][View]
SCOP Classification
CATH Classification
Structural Details
KeywordHYDROLASE
TextALPHA/BETA HYDROLASE, HYDROLASE
 
Polymeric Molecules
Chain A
DescriptionLIPASE A
MutationA132D, N166Y
Polymer typepolypeptide
Formula Weight19476.1
Source Methodgenetically manipulated
Entity NameTriacylglycerol lipase
Entity Name SysE.C.3.1.1.3
 
Entity Poly
EntityChiralityLinkageMonomer# Mon.StrandTypeDetails
1n/anonon/aApolypeptide(L)n/a
Ligands And Prosthetic Groups
IDNameFormulaWeightLigand Structure
KPOTASSIUM IONK139.098View
EC and Associated Pathways
ChainsIUBMBKEGG EnzymeMetaCycReactome
A3.1.1.33.1.1.33.1.1.33.1.1.3
Natural/Genetically Modified Source
Scientific NameBacillus subtilis
Source GenusBacillus
GeneLip A
Host Common Namen/a
Host Scientific NameEscherichia coli BL21(DE3)
Host GenusEscherichia coli BL21(DE3)
Host Cell Linen/a
Host StrainBL21(DE3)
Host Vector TypePLASMID
Host PlasmidpET-21b
 
Crystallization Experiments
MethodVAPOR DIFFUSION, HANGING DROP
PH9.5
Temperature100.0
DetailsPEG 3350, ethanolamine, n-octyl-beta-D-glucoside, sodium sulfate., pH 9.5, VAPOR DIFFUSION, HANGING DROP, temperature 100K
 
Unit Cell
Length a (Å)Length b Å)Length c (Å)
76.0176.01103.09
Angle Alpha (°)Angle Beta (°)Angle Gamma (°)
90.0090.00120.00
Space Group
H 3
Diffraction Detector
DetectorIMAGE PLATE
TypeMARRESEARCH
Detailsosmic
Collection Date2003-04-03
 
Diffraction Radiation
Monochromatorn/a
Diffraction ProtocolSINGLE WAVELENGTH
Wavelengthn/a
Wavelength List1.5418
 
Diffraction Source
SourceROTATING ANODE
TypeRIGAKU RU300
Siten/a
Beamlinen/a
 
Reflection Details
Observed Criterion Sigma (F)0.0
Observed Criterion Sigma (I)0.0
Resolution (High)2.00
Resolution (Low)25.00
Number Reflections (All) 14102
Number Reflections (Observed)14102
Percent Possible (Observed)94.0
R Merge I (Observed)n/a
Net I Over Average Sigma (I)n/a
B (Isotropic) From Wilson Plot27.9
Redundancy2.1
 
High Resolution Shell Details
Resolution(High)2.00
Resolution(Low)2.07
Percent Possible(All) 63.20
R Merge I(Observed)n/a
Mean I Over Sigma(Observed)2.4
R Sym I(Observed)0.241
Redundancy1.9
Num Unique Reflections(All)793
 
Refinement Statistics
Structure Solution MethodMOLECULAR REPLACEMENT
Resolution (High)2.00
Resolution (Low)24.19
Cut-off Sigma (F)0.0
Cut-off Sigma (I)n/a
Number of Reflections (All)14102
Number of Reflections (Observed)14102
Number of Reflections (R-Free) 712
Percent Reflections (Observed)94.20
R-Factor (All)0.207
R-Factor (Observed)0.207
R-Work0.206
R-Free0.257
R-Free Selection DetailsRANDOM
 
Temperature Factor Modeling
Isotropic Thermal ModelRESTRAINED
Mean Isotropic B Value32.200
Anisotropic B[1][1]3.80
Anisotropic B[1][2]3.77
Anisotropic B[1][3]0.00
Anisotropic B[2][2]3.80
Anisotropic B[2][3]0.00
Anisotropic B[3][3]-7.60
 
Resolution Shells
Shell Resol (High)2.00
Shell Resolution (Low)n/a
Number of Reflections (Observed)924
Number of Reflections (R-Free)51
Number of Reflections (R-Work)873
R-Factor (R-Work)0.320
R-Factor (R-Free)0.352
R-Free Error0.050
Percent Reflections (Observed)61.50
 
RMS Deviations
Parameter TypeDeviation From Ideal
c_bond_d0.006
c_angle_deg1.2
c_dihedral_angle_d24.0
c_improper_angle_d0.70
Coordinate Error
Luzzati ESD (Observed)0.27
Luzzati Sigma A (Observed)0.24
Luzzati Resolution Cutoff (Low)5.0
Luzzati ESD (R-Free Set)0.33
Luzzati Sigma A (R-Free Set)0.28
 
Number of Non-Hydrogen Atoms Used in Refinement
Protein Atoms1358
Nucleic Acid Atoms0
Heterogen Atoms1
Solvent Atoms183
 
Programs
Data Collectionn/a
Data Reduction (intensity integration)DENZO
Data Reduction (data scaling)SCALEPACK
Structure SolutionMOLREP
Structure RefinementCNS 1.1
 
Software
Classificationdata collection
Software NameDENZO
 
Classificationdata reduction
Software NameSCALEPACK
 
Classificationmodel building
Software NameMOLREP
Software Version CCP4
 
Classificationrefinement
Software NameCNS
Software Version 1.1
 
Sequence Chain
A
Sequence
  1   AEHNPVVMVH  GIGGASFNFA  GIKSYLVSQG  WSRDKLYAVD  FWDKTGTNYN  NGPVLSRFVQ

 61   KVLDETGAKK  VDIVAHSMGG  ANTLYYIKNL  DGGNKVANVV  TLGGANRLTT  GKALPGTDPN

121   QKILYTSIYS  SDDMIVMNYL  SRLDGARNVQ  IHGVGHIGLL  YSSQVYSLIK  EGLNGGGQNT

181   N