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- PDB-8ibl: MES bound form of PET-degrading cutinase Cut190 with thermostabil... -

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Basic information

Entry
Database: PDB / ID: 8ibl
TitleMES bound form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202H and S176A inactivation
ComponentsAlpha/beta hydrolase family protein
KeywordsHYDROLASE / PROTEIN ENGINEERING / POLYESTERASE / DISULFIDE BOND / METAL BINDING / Ligand complex
Function / homologyPlatelet-activating factor acetylhydrolase, isoform II / : / carboxylic ester hydrolase activity / Alpha/Beta hydrolase fold / metal ion binding / Cutinase
Function and homology information
Biological speciesSaccharomonospora viridis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsEmori, M. / Numoto, N. / Kamiya, N. / Oda, M.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Biorxiv / Year: 2023
Title: Improvement of thermostability and activity of PET-degrading enzyme Cut190 towards a detailed understanding and application of the enzymatic reaction mechanism.
Authors: Numoto, N. / Kamiya, N. / Oda, M.
History
DepositionFeb 10, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature
Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha/beta hydrolase family protein
B: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,3218
Polymers57,7792
Non-polymers5426
Water1,09961
1
A: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1604
Polymers28,8901
Non-polymers2713
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-10 kcal/mol
Surface area10640 Å2
MethodPISA
2
B: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1604
Polymers28,8901
Non-polymers2713
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-10 kcal/mol
Surface area10650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.869, 83.869, 64.723
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number143
Space group name H-MP3
Space group name HallP3
Symmetry operation#1: x,y,z
#2: -y,x-y,z
#3: -x+y,-x,z

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Components

#1: Protein Alpha/beta hydrolase family protein / Cutinase


Mass: 28889.537 Da / Num. of mol.: 2
Mutation: Q123H,Q138A, S176A, N202H, S226P, R228S, D250C, E296C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomonospora viridis (bacteria) / Gene: Cut190, SAMN02982918_2340 / Production host: Escherichia coli (E. coli) / References: UniProt: W0TJ64, cutinase
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H13NO4S / Feature type: SUBJECT OF INVESTIGATION / Comment: pH buffer*YM
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 61 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: 2.27 Å3/Da / Density % sol: 45.92 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.1 M MES monohydrate pH 6.5, 0.2 M Ammonium sulfate, and 30% (w/v) PEG MME 5,000

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Data collection

DiffractionMean temperature: 95 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-17A / Wavelength: 0.9 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 9, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.6→33 Å / Num. obs: 15681 / % possible obs: 99.9 % / Redundancy: 5.4 % / Biso Wilson estimate: 36.34 Å2 / CC1/2: 0.98 / Rsym value: 0.276 / Net I/σ(I): 5.8
Reflection shellResolution: 2.61→2.76 Å / Num. unique obs: 2506 / CC1/2: 0.642 / Rsym value: 1.22

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→32.36 Å / SU ML: 0.3911 / Cross valid method: FREE R-VALUE / σ(F): 1.98 / Phase error: 29.2956
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2632 775 4.95 %
Rwork0.2106 14889 -
obs0.2132 15664 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 36.35 Å2
Refinement stepCycle: LAST / Resolution: 2.6→32.36 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4062 0 28 61 4151
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00254200
X-RAY DIFFRACTIONf_angle_d0.55635722
X-RAY DIFFRACTIONf_chiral_restr0.0431614
X-RAY DIFFRACTIONf_plane_restr0.0055748
X-RAY DIFFRACTIONf_dihedral_angle_d14.51311548
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.760.29791220.28912472X-RAY DIFFRACTION99.77
2.76-2.970.33981220.25142489X-RAY DIFFRACTION99.92
2.98-3.270.30811260.21812478X-RAY DIFFRACTION100
3.27-3.750.2821380.21992478X-RAY DIFFRACTION100
3.75-4.720.23231220.17762508X-RAY DIFFRACTION99.96
4.72-32.360.22111450.19162464X-RAY DIFFRACTION99.69

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