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- PDB-5cln: Crystal structure of a 4-oxalocrotonate tautomerase mutant at 2.7... -

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Basic information

Entry
Database: PDB / ID: 5cln
TitleCrystal structure of a 4-oxalocrotonate tautomerase mutant at 2.7 Angstrom
Components2-hydroxymuconate tautomerase
KeywordsISOMERASE / 4-Oxalocrotonate tautomerase / beta-alpha-beta structural motif / tautomerase superfamily
Function / homology
Function and homology information


xylene catabolic process / 2-hydroxymuconate tautomerase / toluene catabolic process / isomerase activity
Similarity search - Function
4-oxalocrotonate tautomerase, Pseudomonas-type / 4-oxalocrotonate tautomerase / Tautomerase enzyme / Macrophage Migration Inhibitory Factor / Macrophage Migration Inhibitory Factor / Tautomerase/MIF superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
2-hydroxymuconate tautomerase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.71 Å
AuthorsThunnissen, A.M.W.H. / Poddar, H.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
European Research Council242293 Netherlands
CitationJournal: Nat Commun / Year: 2016
Title: Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases.
Authors: van der Meer, J.Y. / Poddar, H. / Baas, B.J. / Miao, Y. / Rahimi, M. / Kunzendorf, A. / van Merkerk, R. / Tepper, P.G. / Geertsema, E.M. / Thunnissen, A.M. / Quax, W.J. / Poelarends, G.J.
History
DepositionJul 16, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 9, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2016Group: Database references
Revision 1.2Jan 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase
D: 2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase
G: 2-hydroxymuconate tautomerase
H: 2-hydroxymuconate tautomerase
I: 2-hydroxymuconate tautomerase
J: 2-hydroxymuconate tautomerase
K: 2-hydroxymuconate tautomerase
L: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)73,77512
Polymers73,77512
Non-polymers00
Water90150
1
A: 2-hydroxymuconate tautomerase
B: 2-hydroxymuconate tautomerase
C: 2-hydroxymuconate tautomerase
D: 2-hydroxymuconate tautomerase
E: 2-hydroxymuconate tautomerase
F: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)36,8886
Polymers36,8886
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12960 Å2
ΔGint-56 kcal/mol
Surface area13810 Å2
MethodPISA
2
G: 2-hydroxymuconate tautomerase
H: 2-hydroxymuconate tautomerase
I: 2-hydroxymuconate tautomerase

G: 2-hydroxymuconate tautomerase
H: 2-hydroxymuconate tautomerase
I: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)36,8886
Polymers36,8886
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_558-x,y,-z+31
Buried area12410 Å2
ΔGint-60 kcal/mol
Surface area14210 Å2
MethodPISA
3
K: 2-hydroxymuconate tautomerase

K: 2-hydroxymuconate tautomerase

J: 2-hydroxymuconate tautomerase

L: 2-hydroxymuconate tautomerase

J: 2-hydroxymuconate tautomerase

L: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)36,8886
Polymers36,8886
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_457-x-1,y,-z+21
crystal symmetry operation3_445x-1/2,y-1/2,z1
crystal symmetry operation3_455x-1/2,y+1/2,z1
crystal symmetry operation4_447-x-1/2,y-1/2,-z+21
crystal symmetry operation4_457-x-1/2,y+1/2,-z+21
MethodPISA
4
L: 2-hydroxymuconate tautomerase

J: 2-hydroxymuconate tautomerase

J: 2-hydroxymuconate tautomerase

L: 2-hydroxymuconate tautomerase

K: 2-hydroxymuconate tautomerase

K: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)36,8886
Polymers36,8886
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation1_545x,y-1,z1
crystal symmetry operation2_547-x,y-1,-z+21
crystal symmetry operation2_557-x,y,-z+21
crystal symmetry operation3_545x+1/2,y-1/2,z1
crystal symmetry operation4_447-x-1/2,y-1/2,-z+21
MethodPISA
5
J: 2-hydroxymuconate tautomerase

J: 2-hydroxymuconate tautomerase

L: 2-hydroxymuconate tautomerase

L: 2-hydroxymuconate tautomerase

K: 2-hydroxymuconate tautomerase

K: 2-hydroxymuconate tautomerase


Theoretical massNumber of molelcules
Total (without water)36,8886
Polymers36,8886
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_557-x,y,-z+21
crystal symmetry operation1_565x,y+1,z1
crystal symmetry operation2_567-x,y+1,-z+21
crystal symmetry operation3_555x+1/2,y+1/2,z1
crystal symmetry operation4_457-x-1/2,y+1/2,-z+21
Buried area13050 Å2
ΔGint-48 kcal/mol
Surface area13990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.163, 87.258, 97.284
Angle α, β, γ (deg.)90.000, 113.730, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C
41chain D
51chain E
61chain F
71chain G
81chain H
91chain I
101chain J
111chain K
121chain L

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: ALA / End label comp-ID: ALA / Auth seq-ID: 1 - 57 / Label seq-ID: 1 - 57

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain AAA
2chain BBB
3chain CCC
4chain DDD
5chain EEE
6chain FFF
7chain GGG
8chain HHH
9chain III
10chain JJJ
11chain KKK
12chain LLL

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Components

#1: Protein
2-hydroxymuconate tautomerase / / 4-oxalocrotonate tautomerase / 4-OT


Mass: 6147.944 Da / Num. of mol.: 12 / Fragment: UNP residues 2-58 / Mutation: M45Y, F50A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: xylH / Plasmid: pJexpress 414 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q01468, 2-hydroxymuconate tautomerase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 50 / Source method: isolated from a natural source / Formula: H2O

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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: 2.3 Å3/Da / Density % sol: 46.41 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.2 M sodium formate, 0.1 M Bis-Tris Propane, 20% PEG 3350

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: ROTATING ANODE / Type: BRUKER AXS MICROSTAR / Wavelength: 1.5418 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jan 17, 2015 / Details: HELIOS OPTICS
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.71→57.6 Å / Num. obs: 18204 / % possible obs: 99.4 % / Redundancy: 4.2 % / Rmerge(I) obs: 0.148 / Net I/av σ(I): 1.8 / Net I/σ(I): 6.9 / Num. measured all: 76379
Reflection shellResolution: 2.71→2.84 Å / Rmerge(I) obs: 0.714 / Mean I/σ(I) obs: 1.8 / % possible all: 95.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
Aimlessdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4X19

4x19


Resolution: 2.71→57.6 Å / SU ML: 0.35 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 27.83 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2634 886 4.88 %
Rwork0.2325 17284 -
obs0.234 18170 99.25 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 105.66 Å2 / Biso mean: 40.722 Å2 / Biso min: 20.55 Å2
Refinement stepCycle: final / Resolution: 2.71→57.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5172 0 0 50 5222
Biso mean---30.56 -
Num. residues----684
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0055220
X-RAY DIFFRACTIONf_angle_d0.9887032
X-RAY DIFFRACTIONf_chiral_restr0.041852
X-RAY DIFFRACTIONf_plane_restr0.003900
X-RAY DIFFRACTIONf_dihedral_angle_d14.3041980
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A3919X-RAY DIFFRACTION9.174TORSIONAL
12B3919X-RAY DIFFRACTION9.174TORSIONAL
13C3919X-RAY DIFFRACTION9.174TORSIONAL
14D3919X-RAY DIFFRACTION9.174TORSIONAL
15E3919X-RAY DIFFRACTION9.174TORSIONAL
16F3919X-RAY DIFFRACTION9.174TORSIONAL
17G3919X-RAY DIFFRACTION9.174TORSIONAL
18H3919X-RAY DIFFRACTION9.174TORSIONAL
19I3919X-RAY DIFFRACTION9.174TORSIONAL
110J3919X-RAY DIFFRACTION9.174TORSIONAL
111K3919X-RAY DIFFRACTION9.174TORSIONAL
112L3919X-RAY DIFFRACTION9.174TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7063-2.87590.32461470.29612767291497
2.8759-3.09790.31841270.291529123039100
3.0979-3.40970.2811620.256328733035100
3.4097-3.90290.25631330.229429123045100
3.9029-4.91690.20621400.188929173057100
4.9169-57.63080.27041770.2182903308099

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