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- PDB-8ibm: Sulfate bound form of PET-degrading cutinase Cut190 with thermost... -

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Basic information

Entry
Database: PDB / ID: 8ibm
TitleSulfate 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 / homologyCutinase / PET hydrolase-like / : / 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: 2.2 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

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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)58,0516
Polymers57,7792
Non-polymers2724
Water1,09961
1
A: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0263
Polymers28,8901
Non-polymers1362
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area10570 Å2
MethodPISA
2
B: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0263
Polymers28,8901
Non-polymers1362
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area220 Å2
ΔGint-19 kcal/mol
Surface area10540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.755, 83.755, 64.752
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 / Plasmid: PQE80L / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta-gami B(DE3) / 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-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4 / Feature type: SUBJECT OF INVESTIGATION
#4: 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: 45.8 %
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: 2.2→37 Å / Num. obs: 25685 / % possible obs: 99.6 % / Redundancy: 5.5 % / Biso Wilson estimate: 38.44 Å2 / CC1/2: 0.997 / Rsym value: 0.125 / Net I/σ(I): 10.4
Reflection shellResolution: 2.2→2.33 Å / Redundancy: 5.5 % / Mean I/σ(I) obs: 1.4 / Num. unique obs: 4113 / CC1/2: 0.743 / Rsym value: 1.46 / % possible all: 98.5

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.2→36.27 Å / SU ML: 0.3375 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 38.2895
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2672 1270 4.96 %
Rwork0.2338 24322 -
obs0.2354 25592 99.21 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 48.01 Å2
Refinement stepCycle: LAST / Resolution: 2.2→36.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4062 0 12 61 4135
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00214184
X-RAY DIFFRACTIONf_angle_d0.47765702
X-RAY DIFFRACTIONf_chiral_restr0.0418614
X-RAY DIFFRACTIONf_plane_restr0.0051748
X-RAY DIFFRACTIONf_dihedral_angle_d11.75951530
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2-2.290.36421270.36892677X-RAY DIFFRACTION98.21
2.29-2.390.35641360.31952706X-RAY DIFFRACTION98.58
2.39-2.520.32511490.27352692X-RAY DIFFRACTION98.96
2.52-2.680.30831420.2932667X-RAY DIFFRACTION99.5
2.68-2.880.33771420.27312753X-RAY DIFFRACTION99.59
2.88-3.170.2661340.25592701X-RAY DIFFRACTION99.75
3.17-3.630.28871460.23492724X-RAY DIFFRACTION99.79
3.63-4.570.23711360.19242701X-RAY DIFFRACTION98.95
4.58-36.270.21331580.19192701X-RAY DIFFRACTION99.51

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