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- PDB-3qzu: Crystal structure of Bacillus subtilis Lipase A 7-fold mutant; th... -

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Basic information

Entry
Database: PDB / ID: 3qzu
TitleCrystal structure of Bacillus subtilis Lipase A 7-fold mutant; the outcome of directed evolution towards thermostability
ComponentsLipase estA
KeywordsHYDROLASE / Alpha/beta hydrolase
Function / homology
Function and homology information


lipase activity / triacylglycerol lipase / triacylglycerol 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 subsp. subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsPijning, T. / Augustyniak, W. / Reetz, M.T. / Dijkstra, B.W.
CitationJournal: Protein Sci. / Year: 2012
Title: Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention.
Authors: Augustyniak, W. / Brzezinska, A.A. / Pijning, T. / Wienk, H. / Boelens, R. / Dijkstra, B.W. / Reetz, M.T.
History
DepositionMar 7, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2012Provider: repository / Type: Initial release
Revision 1.1Apr 18, 2012Group: Database references
Revision 1.2Sep 13, 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_ncs_dom_lim / 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_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)39,25611
Polymers38,5332
Non-polymers7249
Water8,683482
1
A: Lipase estA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,6746
Polymers19,2661
Non-polymers4085
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Lipase estA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,5825
Polymers19,2661
Non-polymers3164
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)59.163, 45.511, 77.613
Angle α, β, γ (deg.)90.00, 101.10, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLU / Beg label comp-ID: GLU / End auth comp-ID: ASN / End label comp-ID: ASN / Refine code: 5 / Auth seq-ID: 2 - 181 / Label seq-ID: 2 - 181

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

#1: Protein Lipase estA / Lipase A / Triacylglycerol lipase


Mass: 19266.498 Da / Num. of mol.: 2 / Mutation: R33Q, D34N, K35D, K112D, M134D, Y139C, I157M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis (bacteria)
Strain: 168 / Gene: BSU02700, estA, lip, lipA / Plasmid: pET22b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) RP / References: UniProt: P37957, triacylglycerol lipase
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 482 / 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: 2.66 Å3/Da / Density % sol: 53.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 24% (w/v) PEG 3350, 0.2 M (NH4)2)SO4, 0.1 M BisTris-HCl, 0.03 M BES-NaOH, 0.03% (w/v) NaN3, pH 6.0, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.8726 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 10, 2010 / Details: mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8726 Å / Relative weight: 1
ReflectionResolution: 1.71→45.51 Å / Num. all: 43880 / Num. obs: 43134 / % possible obs: 98.3 % / Observed criterion σ(I): 3 / Redundancy: 4.1 % / Biso Wilson estimate: 16.9 Å2 / Rmerge(I) obs: 0.159 / Net I/σ(I): 6.8
Reflection shellResolution: 1.71→1.81 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.822 / Mean I/σ(I) obs: 1.5 / Num. unique all: 18453 / % possible all: 88.2

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Processing

SoftwareName: REFMAC / Version: 5.5.0102 / Classification: refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1I6W

1i6w


Resolution: 1.85→76.16 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.907 / SU B: 2.725 / SU ML: 0.083 / Isotropic thermal model: isotropic / Cross valid method: THROUGHOUT / ESU R Free: 0.134 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22275 1700 5 %RANDOM
Rwork0.17274 ---
obs0.17525 32229 97.03 %-
all-33215 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 13.792 Å2
Baniso -1Baniso -2Baniso -3
1--0.6 Å20 Å20.37 Å2
2--0.15 Å20 Å2
3---0.59 Å2
Refinement stepCycle: LAST / Resolution: 1.85→76.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2700 0 42 482 3224
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0212805
X-RAY DIFFRACTIONr_angle_refined_deg1.1071.9483804
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8515367
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.83525.645124
X-RAY DIFFRACTIONr_dihedral_angle_3_deg10.4215442
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10158
X-RAY DIFFRACTIONr_chiral_restr0.0780.2421
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022132
X-RAY DIFFRACTIONr_mcbond_it0.6211.51779
X-RAY DIFFRACTIONr_mcangle_it1.14522843
X-RAY DIFFRACTIONr_scbond_it1.96731026
X-RAY DIFFRACTIONr_scangle_it3.2384.5958
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

NumberTypeRms dev position (Å)Weight position
712medium positional0.070.5
627loose positional0.215
712medium thermal2.052
627loose thermal2.0910
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.361 121 -
Rwork0.252 2444 -
obs--99 %

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