[English] 日本語
Yorodumi
- PDB-7puo: Structure of a fused 4-OT variant engineered for asymmetric Micha... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7puo
TitleStructure of a fused 4-OT variant engineered for asymmetric Michael addition reactions
Components2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
KeywordsLYASE / Protein engineering / 4-oxalocrotonate tautomerase / Michael addition / carbon-carbon lyase
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.35 Å
AuthorsRozeboom, H.J. / Thunnissen, A.M.W.H. / Poelarends, G.J.
Funding support Netherlands, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)724.016.002 Netherlands
Netherlands Organisation for Scientific Research (NWO)713.015.003 Netherlands
CitationJournal: Angew.Chem.Int.Ed.Engl. / Year: 2022
Title: Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme.
Authors: Xu, G. / Kunzendorf, A. / Crotti, M. / Rozeboom, H.J. / Thunnissen, A.W.H. / Poelarends, G.J.
History
DepositionSep 30, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Structure summary / Category: citation / citation_author / entity
Item: _citation.journal_abbrev / _citation.page_last ..._citation.journal_abbrev / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.name / _entity.pdbx_ec
Revision 1.2Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
D: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)82,36810
Polymers82,1136
Non-polymers2554
Water1,49583
1
A: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,1494
Polymers41,0573
Non-polymers921
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
D: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,2206
Polymers41,0573
Non-polymers1633
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)51.860, 100.915, 153.117
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))
d_2ens_1(chain "B" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))
d_3ens_1(chain "C" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))
d_4ens_1(chain "D" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))
d_5ens_1(chain "E" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))
d_6ens_1(chain "F" and (resid 1 through 3 or resid 5 through 62 or resid 64 through 126))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1PROALAA1 - 3
d_12ens_1ILEARGA5 - 62
d_13ens_1GLYLYSA64 - 126
d_21ens_1PROALAB1 - 3
d_22ens_1ILEARGB5 - 62
d_23ens_1GLYLYSB64 - 126
d_31ens_1PROALAD1 - 3
d_32ens_1ILEARGD5 - 62
d_33ens_1GLYLYSD64 - 126
d_41ens_1PROALAE1 - 3
d_42ens_1ILEARGE5 - 62
d_43ens_1GLYLYSE64 - 126
d_51ens_1PROALAG1 - 3
d_52ens_1ILEARGG5 - 62
d_53ens_1GLYLYSG64 - 126
d_61ens_1PROALAH1 - 3
d_62ens_1ILEARGH5 - 62
d_63ens_1GLYLYSH64 - 126

NCS oper:
IDCodeMatrixVector
1given(0.0623423021672, 0.879403255864, -0.471978125485), (0.542821090399, 0.366963048059, 0.755435890845), (0.837531313551, -0.303295293295, -0.454481313022)-24.0532514447, 16.008557518, 13.8656109577
2given(-0.100621719144, -0.515482534419, -0.850971812897), (-0.524120426104, -0.699546282778, 0.485729119153), (-0.845679045843, 0.494886608185, -0.199785375995)-18.724938737, 3.11344096306, -21.9712132035
3given(0.999980956185, 0.0051429649702, -0.00341133091312), (0.00330119213984, 0.021273917312, 0.999768234429), (0.00521434537976, -0.999760456486, 0.02125653426)12.9490651794, -25.1997599448, -25.1459699798
4given(0.0531905578807, 0.88962865558, -0.453576476157), (0.856843797036, -0.273926069164, -0.436787380899), (-0.512824991622, -0.365411225611, -0.776843075637)-11.3085056434, -11.0188032178, -40.8558212051
5given(-0.107567986134, -0.52917107895, -0.841669232871), (-0.860382818891, 0.473748260175, -0.18789356279), (0.498167174065, 0.703946414995, -0.506249850864)-5.80818493592, -47.1509138172, -28.9775956668

-
Components

#1: Protein
2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase


Mass: 13685.563 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68- ...Details: The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.,The parent protein for protein engineering was 4-OT from Pseudomonas putida (uniprot Q01468). Residues 63-67 form an engineered linker sequence fusing two monomers of 4-OT (residues 1-62, 68-129). The following mutations were introduced by protein engineering and directed evolution: R11H, E22D, L31D, P34T, T36S, M45L, R61P, D80G, V90A, T103K, V107I, I108F, M112I, F117A, I119V, A124L, R128L.
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: xylH / Production host: Escherichia coli BL21(DE3) (bacteria) / References: 2-hydroxymuconate tautomerase
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 83 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

-
Experimental details

-
Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

-
Sample preparation

CrystalDensity Matthews: 2.44 Å3/Da / Density % sol: 49.58 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Purified enzyme (16 mg/ml in 5 mM potassium phosphate buffer, pH 7.3) was crystallised in drops prepared by 1:1 mixing with a crystallization reservoir solution containing 20% PEG 2000, 10 ...Details: Purified enzyme (16 mg/ml in 5 mM potassium phosphate buffer, pH 7.3) was crystallised in drops prepared by 1:1 mixing with a crystallization reservoir solution containing 20% PEG 2000, 10 mM urea in 0.1 M Bis-Tris, pH 7.0.

-
Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.965459 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Jun 19, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.965459 Å / Relative weight: 1
ReflectionResolution: 2.35→47.92 Å / Num. obs: 34025 / % possible obs: 99.3 % / Redundancy: 5.4 % / CC1/2: 0.998 / Rmerge(I) obs: 0.071 / Rpim(I) all: 0.034 / Rrim(I) all: 0.079 / Net I/σ(I): 13.3 / Num. measured all: 182172 / Scaling rejects: 24
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
2.35-2.435.30.9631710832290.6220.4591.0721.798.8
9.1-47.924.90.0432326630.9980.0190.04437.997.5

-
Phasing

PhasingMethod: molecular replacement

-
Processing

Software
NameVersionClassification
PHENIX1.20rc1-4395refinement
XDS20210205data reduction
Aimless0.7.7data scaling
PHASER2.7.0phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4X19

4x19


Resolution: 2.35→46.13 Å / SU ML: 0.3127 / Cross valid method: FREE R-VALUE / σ(F): 1.06 / Phase error: 25.5449
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2359 1708 5.03 %
Rwork0.1999 32255 -
obs0.2017 33963 98.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 63.06 Å2
Refinement stepCycle: LAST / Resolution: 2.35→46.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5733 0 14 83 5830
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00615811
X-RAY DIFFRACTIONf_angle_d0.84897806
X-RAY DIFFRACTIONf_chiral_restr0.0613920
X-RAY DIFFRACTIONf_plane_restr0.00721014
X-RAY DIFFRACTIONf_dihedral_angle_d11.69532210
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AX-RAY DIFFRACTIONTorsion NCS1.40814577571
ens_1d_3AX-RAY DIFFRACTIONTorsion NCS1.31154141935
ens_1d_4AX-RAY DIFFRACTIONTorsion NCS0.979773601865
ens_1d_5AX-RAY DIFFRACTIONTorsion NCS1.46301409637
ens_1d_6AX-RAY DIFFRACTIONTorsion NCS1.34227457966
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.35-2.420.36241400.32022606X-RAY DIFFRACTION98.39
2.42-2.50.29831340.28482646X-RAY DIFFRACTION99.29
2.5-2.590.28671390.25212685X-RAY DIFFRACTION98.98
2.59-2.690.26011490.23832623X-RAY DIFFRACTION99.43
2.69-2.810.31631290.23842682X-RAY DIFFRACTION99.86
2.81-2.960.30111490.25052672X-RAY DIFFRACTION99.58
2.96-3.150.26381590.25552679X-RAY DIFFRACTION99.4
3.15-3.390.27971400.22092677X-RAY DIFFRACTION99.3
3.39-3.730.27971390.2112706X-RAY DIFFRACTION98.92
3.73-4.270.19131410.17322695X-RAY DIFFRACTION98.51
4.27-5.380.18431330.16662727X-RAY DIFFRACTION98.11
5.38-46.130.2061560.16572857X-RAY DIFFRACTION98.21
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)
12.09292960314-0.7604899733940.4110690060162.774583377850.1594876607345.45099595788-0.125176956902-0.196216216107-0.09113318716660.215963795526-0.0075340360629-0.1187207414950.5413762628760.401983563180.1292174369770.396596969885-0.004074840785830.08744315367480.3817389794130.02951945363820.343736551908-10.44554987-0.705985884941.83906010706
22.71746904096-0.613021089714-0.5424882982012.730565649250.0590252458164.49419363226-0.164330190405-0.2660380537230.1743834602150.173872681889-0.04400737183130.45154417596-0.489407999546-0.5533225917950.2102829466940.4498904723350.005607185752490.01723090690470.448640660306-0.1148789935860.405975534206-26.113809815711.46555517084.51856528897
34.05126062932-1.02599728115-0.458274163844.081379016150.774376322866.71840125390.00368322741050.5242316630890.227490253276-0.446477028367-0.3285442548950.117028042535-0.695295763-0.2211007864590.3191794932050.442520907439-0.00360390119098-0.007766631671690.3743391065740.02312878947980.389288341492-19.29207712029.98308455759-13.9209624517
44.014809225020.2478392326752.081399175233.775124995880.1402433854825.39027503429-0.02970732855040.1092171678480.01731815958610.5404969830540.0934841860662-0.372435814348-0.3569181974250.32697573914-0.03758106522940.3339230336670.0454630728103-0.003240353263260.319765581174-0.03106669444190.3671570009642.5293093516-23.4193550118-24.4498299687
52.761711202470.2292999042211.117325499853.207513361951.117234768324.22962428938-0.0653808023740.1454644610520.153676240205-0.07125792895060.09813514118350.201106251017-0.50679116153-0.364219036429-0.01500032230210.3317512039640.090118826360.03739339389980.3294128905150.0626990376650.338974643631-13.2658989799-20.5285450871-36.6756261112
63.272433186390.357115723941.33900399624.116903558110.9136849945095.766083342250.3499541227220.434437272978-0.9202046416640.1146868375320.282893334518-0.2042449159941.059822665830.467481230946-0.5171099440040.4545957584770.150535370251-0.1551397605370.450655357335-0.08697242604540.570992211905-6.59380802384-38.9413825199-35.6738598053
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11(chain 'A' and resid 1 through 129)AA1 - 1291 - 129
22(chain 'B' and resid 1 through 129)BB1 - 1291 - 129
33(chain 'C' and resid 1 through 128)CD1 - 1281 - 128
44(chain 'D' and resid 1 through 129)DE1 - 1291 - 129
55(chain 'E' and resid 1 through 129)EG1 - 1291 - 129
66(chain 'F' and resid 1 through 126)FH1 - 1261 - 126

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more