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- PDB-7b4i: Thermostable omega transaminase PjTA-R6 variant W58G engineered f... -

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Basic information

Entry
Database: PDB / ID: 7b4i
TitleThermostable omega transaminase PjTA-R6 variant W58G engineered for asymmetric synthesis of enantiopure bulky amines
ComponentsAspartate aminotransferase family protein
KeywordsTRANSFERASE / Aminotransferase / Transaminase / Amine synthesis / Enantioselective / Thermostable / Engineered
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 / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
PYRIDOXAL-5'-PHOSPHATE / SUCCINIC ACID / Aspartate aminotransferase family protein
Similarity search - Component
Biological speciesPseudomonas sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.7 Å
AuthorsCapra, N. / Rozeboom, H.J. / Thunnissen, A.M.W.H. / Janssen, D.B.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
European Union (EU)722610 Netherlands
CitationJournal: Acs Catalysis / Year: 2021
Title: Computational Redesign of an omega-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines.
Authors: Meng, Q. / Ramirez-Palacios, C. / Capra, N. / Hooghwinkel, M.E. / Thallmair, S. / Rozeboom, H.J. / Thunnissen, A.W.H. / Wijma, H.J. / Marrink, S.J. / Janssen, D.B.
History
DepositionDec 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 2.0Sep 22, 2021Group: Atomic model / Data collection / Database references
Category: atom_site / citation ...atom_site / citation / citation_author / diffrn_source / pdbx_database_proc
Item: _atom_site.calc_flag / _citation.journal_volume ..._atom_site.calc_flag / _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _diffrn_source.pdbx_synchrotron_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Aspartate aminotransferase family protein
BBB: Aspartate aminotransferase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,8705
Polymers101,2572
Non-polymers6123
Water7,710428
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12020 Å2
ΔGint-63 kcal/mol
Surface area28250 Å2
MethodPISA
Unit cell
Length a, b, c (Å)97.784, 97.784, 119.899
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Noncrystallographic symmetry (NCS)NCS domain: (Details: Chains AAA BBB)

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Components

#1: Protein Aspartate aminotransferase family protein


Mass: 50628.684 Da / Num. of mol.: 2 / Mutation: W58G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas sp. (bacteria) / Gene: CMK94_18730, DIU04_17820 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A2D8IND4
#2: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H10NO6P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-SIN / SUCCINIC ACID


Mass: 118.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6O4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 428 / 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: 2.83 Å3/Da / Density % sol: 56.54 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.6
Details: Drops were prepared by mixing 1 ul of protein solution (~10 mg/ml in 20 mM HEPES pH 7.5, 100 mM NaCl, 20 uM PLP buffer) with 1 ul of reservoir solution. Reservoir contained 0.7-1M succinic ...Details: Drops were prepared by mixing 1 ul of protein solution (~10 mg/ml in 20 mM HEPES pH 7.5, 100 mM NaCl, 20 uM PLP buffer) with 1 ul of reservoir solution. Reservoir contained 0.7-1M succinic acid pH 7.6. Crystals appeared after 48h.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: DIAMOND / Beamline: I24 / Wavelength: 0.9686 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 4, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 1.7→98 Å / Num. obs: 123358 / % possible obs: 99.9 % / Redundancy: 5.5 % / CC1/2: 0.999 / Rmerge(I) obs: 0.038 / Rpim(I) all: 0.027 / Rrim(I) all: 0.046 / Net I/σ(I): 17.4
Reflection shell

Diffraction-ID: 1 / Redundancy: 5.5 %

Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all
9.31-980.0261233580.9980.0180.032
1.7-1.730.45860580.8630.3250.563

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Processing

Software
NameVersionClassification
DIALS1.14.13data processing
Aimless0.7.4data scaling
Aimless0.7.4data reduction
Coot0.9.2model building
REFMAC5.8.0258refinement
REFMAC5.8.0258phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.7→98 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.968 / SU B: 2.005 / SU ML: 0.064 / Cross valid method: FREE R-VALUE / ESU R: 0.087 / ESU R Free: 0.083
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.191 6175 5 %
Rwork0.173 117147 -
all0.174 --
obs-123322 99.9 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 30.6 Å2
Baniso -1Baniso -2Baniso -3
1-0.117 Å2-0 Å20 Å2
2--0.117 Å20 Å2
3----0.233 Å2
Refinement stepCycle: LAST / Resolution: 1.7→98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6907 0 38 428 7373
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0137149
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176622
X-RAY DIFFRACTIONr_angle_refined_deg1.3971.6289691
X-RAY DIFFRACTIONr_angle_other_deg1.3861.56415363
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.735909
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.05122.563355
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.521151166
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.7111539
X-RAY DIFFRACTIONr_chiral_restr0.0730.2905
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.028116
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021481
X-RAY DIFFRACTIONr_nbd_refined0.2040.21436
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1650.26340
X-RAY DIFFRACTIONr_nbtor_refined0.1620.23561
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0760.23231
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1350.2415
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1510.29
X-RAY DIFFRACTIONr_nbd_other0.1740.229
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.180.214
X-RAY DIFFRACTIONr_mcbond_it1.9313.0833627
X-RAY DIFFRACTIONr_mcbond_other1.9253.0823625
X-RAY DIFFRACTIONr_mcangle_it2.6074.6194539
X-RAY DIFFRACTIONr_mcangle_other2.6084.6194539
X-RAY DIFFRACTIONr_scbond_it2.7913.4313522
X-RAY DIFFRACTIONr_scbond_other2.793.433523
X-RAY DIFFRACTIONr_scangle_it4.2625.0235152
X-RAY DIFFRACTIONr_scangle_other4.2625.0235153
X-RAY DIFFRACTIONr_lrange_it5.31537.4428059
X-RAY DIFFRACTIONr_lrange_other5.2637.2157976
X-RAY DIFFRACTIONr_ncsr_local_group_10.0660.0514460
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.740.2734710.2588593X-RAY DIFFRACTION99.4
1.74-1.790.2654290.2428435X-RAY DIFFRACTION99.8
1.79-1.840.2564640.2398199X-RAY DIFFRACTION99.96
1.84-1.90.254240.2247881X-RAY DIFFRACTION99.97
1.901-1.9630.2274240.2127745X-RAY DIFFRACTION100
1.963-2.0320.2373720.2077495X-RAY DIFFRACTION99.9746
2.03-2.110.2294120.2087137X-RAY DIFFRACTION99.72
2.11-2.190.2263390.2016925X-RAY DIFFRACTION99.53
2.19-2.290.2172930.1946719X-RAY DIFFRACTION99.99
2.29-2.40.222960.1866421X-RAY DIFFRACTION100
2.4-2.530.193330.1776065X-RAY DIFFRACTION100
2.53-2.690.2053290.1825669X-RAY DIFFRACTION100
2.69-2.870.2112640.1855446X-RAY DIFFRACTION100
2.87-3.10.2062650.195006X-RAY DIFFRACTION99.8
3.1-3.40.1882580.1774575X-RAY DIFFRACTION100
3.4-3.80.1652210.1554210X-RAY DIFFRACTION100
3.8-4.390.1452020.1293704X-RAY DIFFRACTION100
4.39-5.370.1531520.1293137X-RAY DIFFRACTION99.88
5.37-7.590.1531430.1512433X-RAY DIFFRACTION99.96
7.59-97.970.149840.141352X-RAY DIFFRACTION100

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