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- PDB-6isr: structure of lipase mutant with Cys-His-Asp catalytic triad -

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Basic information

Entry
Database: PDB / ID: 6isr
Titlestructure of lipase mutant with 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
NICKEL (II) ION / DI(HYDROXYETHYL)ETHER / Lipase B
Similarity search - Component
Biological speciesPseudozyma antarctica (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 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 ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _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_asym_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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)67,7089
Polymers66,8832
Non-polymers8247
Water1,02757
1
A: Lipase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,8014
Polymers33,4421
Non-polymers3593
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area140 Å2
ΔGint-11 kcal/mol
Surface area12120 Å2
MethodPISA
2
B: Lipase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,9075
Polymers33,4421
Non-polymers4654
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area130 Å2
ΔGint-11 kcal/mol
Surface area12160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.288, 44.559, 132.947
Angle α, β, γ (deg.)90.00, 89.41, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Lipase B / CALB


Mass: 33441.734 Da / Num. of mol.: 2 / Mutation: T57A, A89T, W104V, S105C, 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
#2: Chemical ChemComp-NI / NICKEL (II) ION / Nickel


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL / Polyethylene glycol


Mass: 194.226 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 57 / 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: 46.88 %
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: BL18U1 / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 24, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 19262 / % possible obs: 99.6 % / Redundancy: 4.5 % / Rmerge(I) obs: 0.2 / Net I/σ(I): 8.818
Reflection shellResolution: 2.6→2.64 Å / Rmerge(I) obs: 0.824 / Mean I/σ(I) obs: 1.857 / Num. unique obs: 964

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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: 2.6→42.249 Å / SU ML: 0.31 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.42
RfactorNum. reflection% reflection
Rfree0.2279 876 4.88 %
Rwork0.1787 --
obs0.1812 17958 91.38 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.6→42.249 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4614 0 49 57 4720
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0084790
X-RAY DIFFRACTIONf_angle_d0.896561
X-RAY DIFFRACTIONf_dihedral_angle_d13.8282891
X-RAY DIFFRACTIONf_chiral_restr0.055757
X-RAY DIFFRACTIONf_plane_restr0.008858
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5824-2.74420.329770.23891888X-RAY DIFFRACTION61
2.7442-2.9560.30171570.23512752X-RAY DIFFRACTION90
2.956-3.25340.27081500.22173079X-RAY DIFFRACTION99
3.2534-3.72390.24971510.17663095X-RAY DIFFRACTION100
3.7239-4.69070.20591870.14923078X-RAY DIFFRACTION100
4.6907-42.25450.18381540.16143190X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: -12.6607 Å / Origin y: 12.1282 Å / Origin z: -33.353 Å
111213212223313233
T0.202 Å2-0.0045 Å20.0157 Å2-0.244 Å20.0112 Å2--0.2656 Å2
L-0.0705 °20.0099 °20.0531 °2-0.1138 °20.3759 °2--0.7371 °2
S-0.0095 Å °-0.0073 Å °0.004 Å °0.0046 Å °0.0117 Å °0.0017 Å °0.003 Å °-0.0214 Å °0.0028 Å °
Refinement TLS groupSelection details: all

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