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- PDB-7b4j: Thermostable omega transaminase PjTA-R6 variant W58M/F86L/R417L e... -

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Basic information

Entry
Database: PDB / ID: 7b4j
TitleThermostable omega transaminase PjTA-R6 variant W58M/F86L/R417L engineered for asymmetric synthesis of enantiopure bulky amines
ComponentsAspartate aminotransferase family protein
KeywordsTRANSFERASE / Aminotransferase / Transaminase / Amines 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
4'-DEOXY-4'-AMINOPYRIDOXAL-5'-PHOSPHATE / SUCCINIC ACID / Aspartate aminotransferase family protein
Similarity search - Component
Biological speciesPseudomonas sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.9 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 1.1Sep 22, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / diffrn_source / pdbx_database_proc
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._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
A: Aspartate aminotransferase family protein
B: Aspartate aminotransferase family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,8645
Polymers101,2502
Non-polymers6143
Water6,882382
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-66 kcal/mol
Surface area28210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)97.822, 97.822, 118.716
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A7 - 454
2010B7 - 454

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Components

#1: Protein Aspartate aminotransferase family protein


Mass: 50624.773 Da / Num. of mol.: 2 / Mutation: W58M, F86L, R417L
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-PMP / 4'-DEOXY-4'-AMINOPYRIDOXAL-5'-PHOSPHATE / PYRIDOXAMINE-5'-PHOSPHATE


Mass: 248.173 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H13N2O5P
#3: Chemical ChemComp-SIN / SUCCINIC ACID / Succinic acid


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


Mass: 18.015 Da / Num. of mol.: 382 / 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: 2.8 Å3/Da / Density % sol: 56.15 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.6
Details: Drop were prepared by mixing 1ul of protein solution (~10 mg/ml in 20 mM HEPES pH 7.5, 100 mM NaCl, and 20 uM PLP buffer ) with 1ul of reservoir solution. The reservoir contained 0.7-1M ...Details: Drop were prepared by mixing 1ul of protein solution (~10 mg/ml in 20 mM HEPES pH 7.5, 100 mM NaCl, and 20 uM PLP buffer ) with 1ul of reservoir solution. The reservoir contained 0.7-1M Succinic acid pH 7.6. Crystals formed after 48h.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: DIAMOND / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jul 18, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.9→50.8 Å / Num. obs: 87797 / % possible obs: 100 % / Redundancy: 4.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.054 / Rpim(I) all: 0.027 / Rrim(I) all: 0.06 / Net I/σ(I): 15.1
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
1.9-1.934.90.7372202844590.70.3680.8261.8100
10.23-50.754.70.017283359810.0090.01959.899.4

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Processing

Software
NameVersionClassification
Aimless0.7.4data scaling
REFMAC5.8.0258refinement
PDB_EXTRACT3.27data extraction
DIALS1.14.13data processing
Coot0.9.2model building
REFMAC5.8.0258phasing
xia2data processing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.9→50.8 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.961 / SU B: 3.402 / SU ML: 0.095 / Cross valid method: FREE R-VALUE / σ(F): 0 / ESU R: 0.127 / ESU R Free: 0.119 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2076 4254 4.8 %RANDOM
Rwork0.1778 ---
obs0.1792 83494 99.99 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 112.33 Å2 / Biso mean: 33.469 Å2 / Biso min: 16.14 Å2
Baniso -1Baniso -2Baniso -3
1-0.07 Å20 Å20 Å2
2--0.07 Å20 Å2
3----0.13 Å2
Refinement stepCycle: final / Resolution: 1.9→50.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6901 0 40 398 7339
Biso mean--42.58 39.45 -
Num. residues----901
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0137123
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176625
X-RAY DIFFRACTIONr_angle_refined_deg1.4581.6339658
X-RAY DIFFRACTIONr_angle_other_deg1.3991.56615372
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.8995905
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.79922.816348
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.902151166
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.9571536
X-RAY DIFFRACTIONr_chiral_restr0.0750.2904
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.028062
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021452
Refine LS restraints NCS

Ens-ID: 1 / Number: 14515 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.08 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 1.9→1.949 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.317 328 -
Rwork0.28 6146 -
all-6474 -
obs--99.98 %

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