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- PDB-5kqt: Directed Evolution of Transaminases By Ancestral Reconstruction. ... -

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Basic information

Entry
Database: PDB / ID: 5kqt
TitleDirected Evolution of Transaminases By Ancestral Reconstruction. Using Old Proteins for New Chemistries
Components4-aminoburyrate transaminase
KeywordsTRANSFERASE / phylogenetics / directed evolution / transaminase / protein engineering
Function / homology
Function and homology information


transaminase activity / pyridoxal phosphate binding
Similarity search - Function
Aminotransferases class-III pyridoxal-phosphate attachment site. / Aminotransferase class-III / Aminotransferase class-III / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase ...Aminotransferases class-III pyridoxal-phosphate attachment site. / Aminotransferase class-III / Aminotransferase class-III / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase / Alpha-Beta Complex / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PYRIDOXAL-5'-PHOSPHATE / Uncharacterized protein
Similarity search - Component
Biological speciesPseudomonas sp. AAC (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.99 Å
AuthorsWilding, M. / Newman, J. / Peat, T.S. / Scott, C.
CitationJournal: Green Chemistry / Year: 2017
Title: Reverse engineering: transaminase biocatalyst development using ancestral sequence reconstruction
Authors: Wilding, M. / Peat, T.S. / Kalyaanamoorthy, S. / Newman, J. / Scott, C. / Jermiin, L.S.
History
DepositionJul 6, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Database references / Derived calculations
Category: citation / citation_author / pdbx_struct_special_symmetry
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Oct 4, 2023Group: 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 4-aminoburyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,0285
Polymers51,5581
Non-polymers4714
Water3,279182
1
A: 4-aminoburyrate transaminase
hetero molecules

A: 4-aminoburyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,05710
Polymers103,1152
Non-polymers9428
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/61
Buried area12330 Å2
ΔGint-91 kcal/mol
Surface area28170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)66.346, 66.346, 343.314
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522
Components on special symmetry positions
IDModelComponents
11A-501-

SO4

21A-607-

HOH

31A-767-

HOH

41A-782-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein 4-aminoburyrate transaminase


Mass: 51557.598 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas sp. AAC (bacteria) / Gene: FG99_14885 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: A0A081YAN7, beta-alanine-pyruvate transaminase

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Non-polymers , 5 types, 186 molecules

#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H10NO6P
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 182 / 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: 2.12 Å3/Da / Density % sol: 41.85 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.8
Details: 150 nL plus 150 nL drops of protein at 5 mg/mL and reservoir which consisted of 1.6 M ammonium sulfate, 1% dioxane, 50 mM MES pH 6.8

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: May 9, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 1.99→47.8 Å / Num. obs: 32214 / % possible obs: 99.9 % / Redundancy: 20.7 % / CC1/2: 0.998 / Rmerge(I) obs: 0.136 / Net I/σ(I): 17.6
Reflection shellResolution: 1.99→2.1 Å / Redundancy: 20.7 % / Rmerge(I) obs: 0.72 / Mean I/σ(I) obs: 4.9 / CC1/2: 0.91 / % possible all: 99.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3gju

3gju


Resolution: 1.99→47.8 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.917 / SU B: 4.621 / SU ML: 0.125 / Cross valid method: THROUGHOUT / ESU R: 0.192 / ESU R Free: 0.167 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23695 1625 5.1 %RANDOM
Rwork0.19569 ---
obs0.19778 30450 99.89 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 23.889 Å2
Baniso -1Baniso -2Baniso -3
1--0.28 Å2-0.14 Å2-0 Å2
2---0.28 Å2-0 Å2
3---0.89 Å2
Refinement stepCycle: 1 / Resolution: 1.99→47.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3447 0 27 182 3656
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0193578
X-RAY DIFFRACTIONr_bond_other_d0.0020.023412
X-RAY DIFFRACTIONr_angle_refined_deg1.3891.9564855
X-RAY DIFFRACTIONr_angle_other_deg0.95537823
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7025462
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.57823.203153
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.5315562
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.2721525
X-RAY DIFFRACTIONr_chiral_restr0.0790.2532
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0214116
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02827
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.0572.3171842
X-RAY DIFFRACTIONr_mcbond_other1.0572.3161841
X-RAY DIFFRACTIONr_mcangle_it1.6413.4692306
X-RAY DIFFRACTIONr_mcangle_other1.6413.472307
X-RAY DIFFRACTIONr_scbond_it1.2432.4571735
X-RAY DIFFRACTIONr_scbond_other1.2432.4571735
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.0163.6292549
X-RAY DIFFRACTIONr_long_range_B_refined3.66143.97715314
X-RAY DIFFRACTIONr_long_range_B_other3.66143.97715314
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.991→2.042 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.355 125 -
Rwork0.292 2134 -
obs--98.65 %

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