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- PDB-8ekg: MHETase variant Thr159Val, Met192Tyr, Tyr252Phe, Tyr503Trp -

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Basic information

Entry
Database: PDB / ID: 8ekg
TitleMHETase variant Thr159Val, Met192Tyr, Tyr252Phe, Tyr503Trp
ComponentsMono(2-hydroxyethyl) terephthalate hydrolase
KeywordsHYDROLASE / Protein Engineering / MHETase / Plastic degradation
Function / homology
Function and homology information


mono(ethylene terephthalate) hydrolase / carboxylic ester hydrolase activity / xenobiotic catabolic process / cell outer membrane / metal ion binding
Similarity search - Function
Tannase/feruloyl esterase / Tannase and feruloyl esterase / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / Mono(2-hydroxyethyl) terephthalate hydrolase
Similarity search - Component
Biological speciesIdeonella sakaiensis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.65 Å
AuthorsSaunders, J.W. / Frkic, R.L. / Jackson, C.J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Biochemistry / Year: 2024
Title: Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.
Authors: Saunders, J.W. / Damry, A.M. / Vongsouthi, V. / Spence, M.A. / Frkic, R.L. / Gomez, C. / Yates, P.A. / Matthews, D.S. / Tokuriki, N. / McLeod, M.D. / Jackson, C.J.
History
DepositionSep 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 6, 2024Group: Structure summary / Category: 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: Mono(2-hydroxyethyl) terephthalate hydrolase
B: Mono(2-hydroxyethyl) terephthalate hydrolase
C: Mono(2-hydroxyethyl) terephthalate hydrolase
D: Mono(2-hydroxyethyl) terephthalate hydrolase
E: Mono(2-hydroxyethyl) terephthalate hydrolase
F: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)375,64828
Polymers374,2346
Non-polymers1,41422
Water7,260403
1
A: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,5814
Polymers62,3721
Non-polymers2083
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,97710
Polymers62,3721
Non-polymers6059
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4753
Polymers62,3721
Non-polymers1022
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,5374
Polymers62,3721
Non-polymers1643
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,6675
Polymers62,3721
Non-polymers2944
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: Mono(2-hydroxyethyl) terephthalate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4122
Polymers62,3721
Non-polymers401
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)63.967, 120.514, 126.290
Angle α, β, γ (deg.)90.440, 95.150, 90.200
Int Tables number1
Space group name H-MP1

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
Mono(2-hydroxyethyl) terephthalate hydrolase / MHET hydrolase / MHETase


Mass: 62372.414 Da / Num. of mol.: 6 / Mutation: T159V, Y503W, M192Y, Y252F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ideonella sakaiensis (bacteria) / Strain: NBRC 110686 / TISTR 2288 / 201-F6 / Gene: ISF6_0224 / Plasmid: pET28a(+) / Production host: Escherichia coli (E. coli) / Strain (production host): SMG96 / Variant (production host): Shuffle
References: UniProt: A0A0K8P8E7, mono(ethylene terephthalate) hydrolase

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Non-polymers , 5 types, 425 molecules

#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 403 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: 2.59 Å3/Da / Density % sol: 52.59 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.3 / Details: 20% PEG 6000, 0.2 M MgCl2, 0.1 M HEPES pH 7.3

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 15, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.65→43.87 Å / Num. obs: 104762 / % possible obs: 96 % / Redundancy: 1.8 % / Biso Wilson estimate: 45.98 Å2 / CC1/2: 0.986 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.112 / Rrim(I) all: 0.158 / Net I/σ(I): 4.5 / Num. measured all: 191573
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.65-2.71.90.836967351970.3540.8351.182196.6
14.51-43.871.80.01811216110.9980.0180.0261390.4

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimless0.7.7data scaling
PDB_EXTRACT3.27data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6qz4

6qz4


Resolution: 2.65→43.87 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.96 / Phase error: 27.11 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2408 4885 4.67 %
Rwork0.184 99816 -
obs0.1867 104701 95.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 154.34 Å2 / Biso mean: 54.2421 Å2 / Biso min: 25.61 Å2
Refinement stepCycle: final / Resolution: 2.65→43.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms24748 0 77 403 25228
Biso mean--67.9 43.13 -
Num. residues----3344
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.65-2.680.38941840.33753379356396
2.68-2.710.38561630.33353268343197
2.71-2.740.3611740.31743371354596
2.74-2.780.37271500.30983373352395
2.78-2.820.31831790.28393294347398
2.82-2.850.34041840.27243315349996
2.85-2.90.33681790.27363318349795
2.9-2.940.36751960.27933304350096
2.94-2.980.36871400.27113301344196
2.98-3.030.32521740.25743356353095
3.03-3.090.32351920.24923300349296
3.09-3.140.32131950.24933298349397
3.14-3.20.34241630.24643308347195
3.2-3.270.26791620.23793361352397
3.27-3.340.27841740.22483367354197
3.34-3.420.25421850.19153314349996
3.42-3.50.23581280.1823362349096
3.5-3.60.22481370.17773339347695
3.6-3.70.25731660.16563317348397
3.7-3.820.21531470.16163340348796
3.82-3.960.20061450.15173326347195
3.96-4.120.18131540.1473336349096
4.12-4.30.19141640.13683363352796
4.3-4.530.17081410.12483313345495
4.53-4.810.16351100.1263327343796
4.81-5.180.20341700.1363310348095
5.18-5.710.18651670.14183340350796
5.71-6.530.18891600.15343302346295
6.53-8.220.17551250.14383330345596
8.22-43.870.17941770.14053284346195
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.7333-0.27341.03660.8356-0.4121.3890.0417-0.16160.1392-0.1477-0.0785-0.0214-0.09730.00040.04860.37830.0502-0.04150.3289-0.07080.4935-0.6969-4.944358.1698
21.2877-0.09780.15310.69060.56911.4390.056-0.1873-0.2162-0.0073-0.0142-0.12570.22170.0518-0.01830.40540.0338-0.07930.21220.01890.463-4.0427-33.150455.5166
31.83571.57191.94641.96750.52334.31180.0746-0.26060.06680.0383-0.32250.42020.202-0.32710.25670.26140.0352-0.02240.3609-0.0890.4468-25.6088-25.855953.5778
43.2385-0.5388-1.5161.05410.78312.4911-0.02050.01410.1333-0.0397-0.00950.03080.0765-0.16350.04070.32940.0381-0.08180.3885-0.11540.4934-17.222534.897469.7411
50.7136-0.189-0.28691.90110.31380.51150.033-0.26990.30980.03410.02050.0045-0.18350.0173-0.05320.470.0509-0.07180.5616-0.27060.7221-6.962456.816984.5108
64.21791.6195-0.52212.9102-1.27251.6181-0.37390.33080.0294-0.26190.1006-0.39360.3185-0.03160.2780.40450.02530.06210.5131-0.09410.696347.799112.0819-35.2319
70.5518-0.4588-0.39771.19070.18311.0506-0.1856-0.13240.31190.34750.1522-0.388-0.14480.13610.08040.44310.058-0.20220.4378-0.11860.639536.358828.0945-12.4375
81.57430.1513-0.01352.06420.06571.4888-0.15640.05050.2091-0.04130.1544-0.0362-0.4123-0.21990.03920.43620.0888-0.06070.4065-0.05470.52572.35859.6084-38.5266
90.20570.1038-0.05810.284-0.11690.7536-0.00140.05060.01130.0034-0.01840.00670.03460.00620.01340.26460.0521-0.02120.3467-0.03940.402110.2389-10.3263-26.998
100.70210.2636-0.29140.8605-0.37991.5819-0.05340.112-0.08610.0382-0.0367-0.00110.1697-0.06180.09160.29350.0554-0.05130.3383-0.08960.465210.2185-20.2342-22.3703
111.7138-0.24420.94760.36370.03684.56910.052-0.0262-0.2002-0.0165-0.12850.10010.02790.51350.07080.31850.0353-0.01320.3764-0.03580.505133.0715-11.6011-21.4404
123.8128-1.85340.99242.583-0.78531.3981-0.1003-0.2228-0.32050.24010.36750.01630.0246-0.0009-0.20580.37250.0787-0.02930.374-0.07210.48120.26930.266355.042
130.1097-0.0634-0.15160.35230.05230.8074-0.0727-0.079-0.00910.00760.02830.0260.01880.00260.04260.30810.056-0.05720.3742-0.05950.472826.83180.941432.8978
143.3692.05181.32041.25420.92354.33080.03270.4708-0.2029-0.13390.1069-0.18620.18120.155-0.12060.23460.0369-0.00090.33110.01490.251130.6826-5.66786.5541
152.03370.21980.531.17510.39980.8637-0.01510.04550.0621-0.0444-0.0058-0.00580.01150.05780.01550.30220.0365-0.01120.34180.00120.342531.72112.037525.6738
162.81510.3904-1.87421.3078-0.24262.57850.0352-0.27920.15560.05080.1211-0.0617-0.05120.4498-0.12450.30980.0284-0.08430.4677-0.16580.53824.50432.774957.4275
170.6921-0.3644-0.32830.8410.1940.71330.114-0.05810.1373-0.22690.00790.0459-0.27210.0736-0.12650.4555-0.006-0.04060.2911-0.10630.499513.774747.397135.5895
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'D' and (resid 47 through 212 )D47 - 212
2X-RAY DIFFRACTION2chain 'D' and (resid 213 through 533 )D213 - 533
3X-RAY DIFFRACTION3chain 'D' and (resid 534 through 603 )D534 - 603
4X-RAY DIFFRACTION4chain 'E' and (resid 46 through 212 )E46 - 212
5X-RAY DIFFRACTION5chain 'E' and (resid 213 through 603 )E213 - 603
6X-RAY DIFFRACTION6chain 'F' and (resid 47 through 110 )F47 - 110
7X-RAY DIFFRACTION7chain 'F' and (resid 111 through 603 )F111 - 603
8X-RAY DIFFRACTION8chain 'A' and (resid 46 through 110 )A46 - 110
9X-RAY DIFFRACTION9chain 'A' and (resid 111 through 333 )A111 - 333
10X-RAY DIFFRACTION10chain 'A' and (resid 334 through 548 )A334 - 548
11X-RAY DIFFRACTION11chain 'A' and (resid 549 through 603 )A549 - 603
12X-RAY DIFFRACTION12chain 'B' and (resid 47 through 110 )B47 - 110
13X-RAY DIFFRACTION13chain 'B' and (resid 111 through 326 )B111 - 326
14X-RAY DIFFRACTION14chain 'B' and (resid 327 through 382 )B327 - 382
15X-RAY DIFFRACTION15chain 'B' and (resid 383 through 603 )B383 - 603
16X-RAY DIFFRACTION16chain 'C' and (resid 47 through 212 )C47 - 212
17X-RAY DIFFRACTION17chain 'C' and (resid 213 through 603 )C213 - 603

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