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- PDB-7qvh: The crystal structure of HotPETase, an evolved thermostable varia... -

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Basic information

Entry
Database: PDB / ID: 7qvh
TitleThe crystal structure of HotPETase, an evolved thermostable variant of IsPETase
ComponentsPoly(ethylene terephthalate) hydrolase
KeywordsHYDROLASE / PET-depolymerase / PETase
Function / homology
Function and homology information


acetylesterase activity / poly(ethylene terephthalate) hydrolase / carboxylic ester hydrolase activity / xenobiotic catabolic process / extracellular region
Similarity search - Function
Dienelactone hydrolase / Dienelactone hydrolase family / : / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
TRIETHYLENE GLYCOL / Poly(ethylene terephthalate) hydrolase
Similarity search - Component
Biological speciesIdeonella sakaiensis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.24 Å
AuthorsHardy, F.J. / Levy, C. / Green, A.P.
Funding support United Kingdom, 11items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M027023/1 United Kingdom
European Research Council (ERC)757991 United Kingdom
Engineering and Physical Sciences Research CouncilR031711 United Kingdom
Engineering and Physical Sciences Research CouncilR026815/1 United Kingdom
Engineering and Physical Sciences Research CouncilK014706/2 United Kingdom
Engineering and Physical Sciences Research CouncilK014668/1 United Kingdom
Engineering and Physical Sciences Research CouncilK014854/1 United Kingdom
Engineering and Physical Sciences Research CouncilK014714/1 United Kingdom
Engineering and Physical Sciences Research CouncilM013219/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)M017702/1 United Kingdom
Engineering and Physical Sciences Research CouncilS01778X/1 United Kingdom
CitationJournal: Nat Catal / Year: 2022
Title: Directed evolution of an efficient and thermostable PET depolymerase
Authors: Bell, E.L. / Smithson, R. / Kilbride, S. / Foster, J. / Hardy, F.J. / Ramachandran, S. / Tedstone, A.A. / Haigh, S.J. / Garforth, A.A. / Day, P.J.R. / Levy, C. / Shaver, M.P. / Green, A.P.
History
DepositionJan 21, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 3, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Poly(ethylene terephthalate) hydrolase
B: Poly(ethylene terephthalate) hydrolase
C: Poly(ethylene terephthalate) hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,7045
Polymers86,4043
Non-polymers3002
Water3,819212
1
A: Poly(ethylene terephthalate) hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9512
Polymers28,8011
Non-polymers1501
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Poly(ethylene terephthalate) hydrolase


Theoretical massNumber of molelcules
Total (without water)28,8011
Polymers28,8011
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Poly(ethylene terephthalate) hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9512
Polymers28,8011
Non-polymers1501
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)81.700, 74.855, 82.701
Angle α, β, γ (deg.)90.000, 118.574, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Poly(ethylene terephthalate) hydrolase / PET hydrolase / PETase / PET-digesting enzyme


Mass: 28801.248 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ideonella sakaiensis (bacteria) / Strain: NBRC 110686 / TISTR 2288 / 201-F6 / Gene: ISF6_4831 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0K8P6T7, poly(ethylene terephthalate) hydrolase
#2: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14O4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 212 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.57 Å3/Da / Density % sol: 52.15 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.85 M sodium citrate tribasic dehydrate, 0.1 M Tris pH 8.0 and 0.1 M sodium chloride (LMB screen HT96 H7 Molecular Dimensions).

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Oct 9, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.24→72.63 Å / Num. obs: 42290 / % possible obs: 99.81 % / Redundancy: 6.9 % / Biso Wilson estimate: 37.46 Å2 / CC1/2: 0.987 / Rmerge(I) obs: 0.2233 / Rpim(I) all: 0.09086 / Net I/σ(I): 5.13
Reflection shellResolution: 2.24→2.32 Å / Redundancy: 6.9 % / Mean I/σ(I) obs: 0.68 / Num. unique obs: 4173 / CC1/2: 0.66 / CC star: 0.892 / Rpim(I) all: 0.4946 / % possible all: 99.02

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
xia2data reduction
xia2data scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5xjh

5xjh


Resolution: 2.24→72.63 Å / SU ML: 0.3977 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 26.1399
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2514 1926 4.56 %
Rwork0.1978 40312 -
obs-42230 99.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 40.11 Å2
Refinement stepCycle: LAST / Resolution: 2.24→72.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5933 0 20 212 6165
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00266108
X-RAY DIFFRACTIONf_angle_d0.53948318
X-RAY DIFFRACTIONf_chiral_restr0.044917
X-RAY DIFFRACTIONf_plane_restr0.00381091
X-RAY DIFFRACTIONf_dihedral_angle_d8.5561876
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.24-2.30.3881480.34722804X-RAY DIFFRACTION98.24
2.3-2.360.35771840.31272810X-RAY DIFFRACTION99.87
2.36-2.430.33981660.28082846X-RAY DIFFRACTION99.9
2.43-2.510.33081330.26482861X-RAY DIFFRACTION99.77
2.51-2.60.31751140.25332897X-RAY DIFFRACTION100
2.6-2.70.25991400.23342864X-RAY DIFFRACTION99.97
2.7-2.820.27521590.22332853X-RAY DIFFRACTION99.9
2.82-2.970.25271470.21722847X-RAY DIFFRACTION99.93
2.97-3.160.26361360.1942891X-RAY DIFFRACTION100
3.16-3.40.26631340.19372900X-RAY DIFFRACTION99.97
3.4-3.740.22191330.17462888X-RAY DIFFRACTION99.97
3.74-4.280.19981080.15092925X-RAY DIFFRACTION100
4.28-5.40.18341190.15542926X-RAY DIFFRACTION99.93
5.4-72.630.2221050.1813000X-RAY DIFFRACTION99.49

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