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- PDB-8z2k: Substrate analog a013 bound form of PET-degrading cutinase mutant... -

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Basic information

Entry
Database: PDB / ID: 8z2k
TitleSubstrate analog a013 bound form of PET-degrading cutinase mutant Cut190**SS_S176A
ComponentsAlpha/beta hydrolase family protein
KeywordsHYDROLASE / PROTEIN ENGINEERING / POLYESTERASE / METAL BINDING / Aromatic ligand
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: 2.2 Å
AuthorsNumoto, N. / Kondo, F. / Bekker, G.J. / Liao, Z. / Yamashita, M. / Iida, A. / Ito, N. / Kamiya, N. / Oda, M.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP21H02120 Japan
CitationJournal: Int.J.Biol.Macromol. / Year: 2024
Title: Structural dynamics of the Ca 2+ -regulated cutinase towards structure-based improvement of PET degradation activity.
Authors: Numoto, N. / Kondo, F. / Bekker, G.J. / Liao, Z. / Yamashita, M. / Iida, A. / Ito, N. / Kamiya, N. / Oda, M.
History
DepositionApr 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release

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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)57,7795
Polymers57,0662
Non-polymers7133
Water1,44180
1
A: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9093
Polymers28,5331
Non-polymers3762
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8692
Polymers28,5331
Non-polymers3361
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)84.390, 84.390, 64.780
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: 28533.098 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, MINT15_00360 / Plasmid: pQE80L / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta-gami B(DE3) / References: UniProt: W0TJ64, cutinase
#2: Chemical ChemComp-A1L0O / 4-[oxidanyl(2-phenylmethoxyethoxy)phosphoryl]benzoic acid


Mass: 336.276 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H17O6P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 80 / 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: 2.33 Å3/Da / Density % sol: 47.3 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 6.8
Details: 50 mM Ammonium sulfate, 0.1 M PIPES pH 6.8, 12-18% (w/v) PEG 3,350, 0.5 mM CaCl2, and 2 mM ligands

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL45XU / Wavelength: 0.9 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 12, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.2→49 Å / Num. obs: 26260 / % possible obs: 100 % / Redundancy: 7.9 % / CC1/2: 0.983 / Rsym value: 0.259 / Net I/σ(I): 8.3
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 7.7 % / Mean I/σ(I) obs: 1.9 / Num. unique obs: 2722 / CC1/2: 0.455 / Rsym value: 1.15 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7CTS

7cts


Resolution: 2.2→48.48 Å / Cross valid method: FREE R-VALUE / σ(F): 2.78 / Phase error: 21.5169
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1952 1345 5.12 %
Rwork0.1724 24915 -
obs0.2 26260 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 19.1 Å2
Refinement stepCycle: LAST / Resolution: 2.2→48.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3968 0 47 80 4095
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00224132
X-RAY DIFFRACTIONf_angle_d0.53275630
X-RAY DIFFRACTIONf_chiral_restr0.0426601
X-RAY DIFFRACTIONf_plane_restr0.0059738
X-RAY DIFFRACTIONf_dihedral_angle_d12.7441502
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2-2.280.28471420.24372554X-RAY DIFFRACTION94.7
2.28-2.370.27971280.24152460X-RAY DIFFRACTION95.05
2.37-2.480.26931400.22832504X-RAY DIFFRACTION94.7
2.48-2.610.25141300.22442476X-RAY DIFFRACTION95.01
2.61-2.770.20621320.22132468X-RAY DIFFRACTION94.92
2.77-2.990.26081300.19882491X-RAY DIFFRACTION95.04
2.99-3.290.26071360.20082494X-RAY DIFFRACTION94.83
3.29-3.760.1911300.20232488X-RAY DIFFRACTION95.03
3.76-4.740.16651340.16152493X-RAY DIFFRACTION94.9
4.74-48.480.19511430.17492487X-RAY DIFFRACTION94.49

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