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- PDB-8z2h: Substrate analog a010 bound form of PET-degrading cutinase mutant... -

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Basic information

Entry
Database: PDB / ID: 8z2h
TitleSubstrate analog a010 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: 1.8 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)57,6396
Polymers57,0662
Non-polymers5724
Water3,585199
1
A: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8193
Polymers28,5331
Non-polymers2862
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Alpha/beta hydrolase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8193
Polymers28,5331
Non-polymers2862
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)82.110, 82.110, 64.700
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-A1L0N / 4-[2-hydroxyethyloxy(oxidanyl)phosphoryl]benzoic acid


Mass: 246.154 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H11O6P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 199 / 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: 2.21 Å3/Da / Density % sol: 44.26 %
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: 1.8→48 Å / Num. obs: 44206 / % possible obs: 97.7 % / Redundancy: 2.6 % / CC1/2: 0.99 / Rsym value: 0.108 / Net I/σ(I): 7.5
Reflection shellResolution: 1.8→1.86 Å / Redundancy: 2.6 % / Mean I/σ(I) obs: 1.3 / Num. unique obs: 4092 / CC1/2: 0.361 / Rsym value: 0.788 / % possible all: 96.5

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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: 1.8→47.86 Å / Cross valid method: FREE R-VALUE / σ(F): 2.52 / Phase error: 22.5509
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.196 2271 5.14 %
Rwork0.1643 41935 -
obs0.1919 44206 97.75 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 20 Å2
Refinement stepCycle: LAST / Resolution: 1.8→47.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3998 0 34 199 4231
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00374142
X-RAY DIFFRACTIONf_angle_d0.6595646
X-RAY DIFFRACTIONf_chiral_restr0.0455606
X-RAY DIFFRACTIONf_plane_restr0.0071742
X-RAY DIFFRACTIONf_dihedral_angle_d12.88731530
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.840.26881470.28222561X-RAY DIFFRACTION91.24
1.84-1.880.31891340.27472614X-RAY DIFFRACTION92.14
1.88-1.930.28841330.25642576X-RAY DIFFRACTION91.41
1.93-1.980.28561380.25952586X-RAY DIFFRACTION91.93
1.98-2.040.27991310.25342614X-RAY DIFFRACTION92.24
2.04-2.110.2441400.24192610X-RAY DIFFRACTION91.74
2.11-2.180.21971390.23132602X-RAY DIFFRACTION91.85
2.18-2.270.21661360.22112605X-RAY DIFFRACTION92.38
2.27-2.370.24241430.21472596X-RAY DIFFRACTION92.25
2.37-2.50.23861350.20052649X-RAY DIFFRACTION93.08
2.5-2.650.23251420.1962625X-RAY DIFFRACTION93.75
2.65-2.860.19861460.1782653X-RAY DIFFRACTION93.91
2.86-3.140.20981290.16592679X-RAY DIFFRACTION94.56
3.15-3.60.22081470.15912696X-RAY DIFFRACTION94.66
3.6-4.530.20621410.15192671X-RAY DIFFRACTION94.62
4.54-47.860.181410.15512647X-RAY DIFFRACTION93.67

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