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- PDB-6isq: structure of Lipase mutant with oxided Cys-His-Asp catalytic triad -

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Basic information

Entry
Database: PDB / ID: 6isq
Titlestructure of Lipase mutant with oxided Cys-His-Asp catalytic triad
ComponentsLipase B
KeywordsHYDROLASE / Calb
Function / homology
Function and homology information


triacylglycerol lipase / triglyceride lipase activity / lipid catabolic process
Similarity search - Function
Alpha/Beta hydrolase fold, catalytic domain / Alpha/Beta hydrolase fold / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / ISOPROPYL ALCOHOL / TRIETHYLENE GLYCOL / Lipase B
Similarity search - Component
Biological speciesPseudozyma antarctica (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.86 Å
AuthorsCen, Y.X. / Zhou, J.H. / Wu, Q.
CitationJournal: Nat Commun / Year: 2019
Title: Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.
Authors: Cen, Y. / Singh, W. / Arkin, M. / Moody, T.S. / Huang, M. / Zhou, J. / Wu, Q. / Reetz, M.T.
History
DepositionNov 18, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 22, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lipase B
B: Lipase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,67211
Polymers66,9472
Non-polymers7259
Water5,405300
1
A: Lipase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,0798
Polymers33,4741
Non-polymers6067
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area250 Å2
ΔGint-2 kcal/mol
Surface area12310 Å2
MethodPISA
2
B: Lipase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,5933
Polymers33,4741
Non-polymers1192
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area240 Å2
ΔGint-2 kcal/mol
Surface area12330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.691, 132.375, 52.422
Angle α, β, γ (deg.)90.00, 90.04, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Lipase B / CALB


Mass: 33473.734 Da / Num. of mol.: 2 / Mutation: T57A, A89T, W104V, S105CSD, V149G, A281Y, A282Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudozyma antarctica (fungus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P41365, triacylglycerol lipase

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Non-polymers , 5 types, 309 molecules

#2: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#3: Chemical ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL / Isopropyl alcohol


Mass: 60.095 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL / Polyethylene glycol


Mass: 150.173 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14O4
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 300 / 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: 2.32 Å3/Da / Density % sol: 46.9 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop
Details: 0.1M sodium acetate, pH 6.0 25% PEG4000 8% isopropanol 0.5% N,N-dimethyldodecylamine oxide(DDAO)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Nov 25, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 1.86→50 Å / Num. obs: 51204 / % possible obs: 99.7 % / Redundancy: 5.5 % / Rmerge(I) obs: 0.141 / Net I/σ(I): 14
Reflection shellResolution: 1.86→1.89 Å / Rmerge(I) obs: 0.675 / Mean I/σ(I) obs: 2 / Num. unique obs: 2499

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1TCA

1tca


Resolution: 1.86→37.038 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 18.75
RfactorNum. reflection% reflection
Rfree0.1893 2554 5.07 %
Rwork0.1605 --
obs0.162 50329 97.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.86→37.038 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4701 0 48 300 5049
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0184968
X-RAY DIFFRACTIONf_angle_d1.4566815
X-RAY DIFFRACTIONf_dihedral_angle_d16.0981794
X-RAY DIFFRACTIONf_chiral_restr0.096780
X-RAY DIFFRACTIONf_plane_restr0.01901
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8545-1.89020.2431170.21652180X-RAY DIFFRACTION81
1.8902-1.92880.25491450.2062491X-RAY DIFFRACTION92
1.9288-1.97070.24421160.19932619X-RAY DIFFRACTION95
1.9707-2.01650.25881560.19762655X-RAY DIFFRACTION98
2.0165-2.0670.22041510.17262549X-RAY DIFFRACTION98
2.067-2.12280.19681830.16462716X-RAY DIFFRACTION100
2.1228-2.18530.22981310.16092699X-RAY DIFFRACTION100
2.1853-2.25580.22491390.16072705X-RAY DIFFRACTION100
2.2558-2.33640.17161310.1582731X-RAY DIFFRACTION100
2.3364-2.430.18561460.16352662X-RAY DIFFRACTION100
2.43-2.54050.21821530.15982713X-RAY DIFFRACTION99
2.5405-2.67440.17121430.1652700X-RAY DIFFRACTION100
2.6744-2.84190.19561450.16492713X-RAY DIFFRACTION100
2.8419-3.06130.18011610.16682703X-RAY DIFFRACTION100
3.0613-3.36920.19221260.16332727X-RAY DIFFRACTION100
3.3692-3.85620.17261520.14562707X-RAY DIFFRACTION100
3.8562-4.85670.14231320.14262735X-RAY DIFFRACTION100
4.8567-37.04570.16931270.14422770X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 55.3872 Å / Origin y: 0.1516 Å / Origin z: 52.6137 Å
111213212223313233
T0.1252 Å20.0096 Å20.016 Å2-0.1202 Å20.0107 Å2--0.1245 Å2
L0.383 °20.0895 °20.1875 °2-0.1867 °20.0991 °2--0.342 °2
S-0.0012 Å °-0.0069 Å °0.0051 Å °0.0075 Å °-0.0025 Å °0.0086 Å °-0.0098 Å °-0.0028 Å °0.007 Å °
Refinement TLS groupSelection details: all

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