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- PDB-8e9n: Crystal structure of E. coli aspartate aminotransferase mutant VF... -

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Basic information

Entry
Database: PDB / ID: 8e9n
TitleCrystal structure of E. coli aspartate aminotransferase mutant VFIY in the ligand-free form at 278 K
ComponentsAspartate aminotransferase
KeywordsTRANSFERASE / Enzyme / Designed Protein / Mutant Protein
Function / homology
Function and homology information


L-phenylalanine biosynthetic process / L-phenylalanine biosynthetic process from chorismate via phenylpyruvate / L-tyrosine:2-oxoglutarate aminotransferase activity / aspartate catabolic process / aspartate transaminase / L-aspartate:2-oxoglutarate aminotransferase activity / pyridoxal phosphate binding / protein homodimerization activity / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Aspartate/other aminotransferase / Aminotransferases, class-I, pyridoxal-phosphate-binding site / Aminotransferases class-I pyridoxal-phosphate attachment site. / Aminotransferase, class I/classII / Aminotransferase class I and II / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
PYRIDOXAL-5'-PHOSPHATE / Aspartate aminotransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.88 Å
AuthorsChica, R.A. / St-Jacques, A.D. / Rodriguez, J.M. / Thompson, M.C.
Funding support Canada, United States, 5items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2016-04831 Canada
Ontario Early Researcher AwardsER14-10-139 Canada
Canada Foundation for Innovation26503 Canada
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124149 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124169 United States
CitationJournal: Nat Commun / Year: 2023
Title: Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.
Authors: St-Jacques, A.D. / Rodriguez, J.M. / Eason, M.G. / Foster, S.M. / Khan, S.T. / Damry, A.M. / Goto, N.K. / Thompson, M.C. / Chica, R.A.
History
DepositionAug 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Oct 11, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Oct 18, 2023Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aspartate aminotransferase
B: Aspartate aminotransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,3986
Polymers89,7112
Non-polymers6864
Water5,927329
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8550 Å2
ΔGint-70 kcal/mol
Surface area28560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)143.830, 143.830, 81.570
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number173
Space group name H-MP63
Space group name HallP6c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: -x,-y,z+1/2

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Components

#1: Protein Aspartate aminotransferase / AspAT / Transaminase A


Mass: 44855.570 Da / Num. of mol.: 2 / Mutation: K37F, T43I, N64Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: aspC, b0928, JW0911 / Production host: Escherichia coli (E. coli) / References: UniProt: P00509, aspartate transaminase
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H10NO6P
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 329 / 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: 2.71 Å3/Da / Density % sol: 54.7 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: HEPES, ammonium sulfate, maleate, PEG 400

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Data collection

DiffractionMean temperature: 278 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.1158 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jun 1, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1158 Å / Relative weight: 1
ReflectionResolution: 1.88→124.43 Å / Num. obs: 76900 / % possible obs: 98.7 % / Redundancy: 10.3 % / Biso Wilson estimate: 32.7 Å2 / CC1/2: 0.997 / Net I/σ(I): 7.3
Reflection shellResolution: 1.88→1.91 Å / Num. unique obs: 3787 / CC1/2: 0.301

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
xia2data reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1X28

1x28


Resolution: 1.88→124.43 Å / SU ML: 0.2216 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 18.3685
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1783 7760 10.09 %
Rwork0.1505 69124 -
obs0.1533 76884 98.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 39.34 Å2
Refinement stepCycle: LAST / Resolution: 1.88→124.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6152 0 10 329 6491
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00726612
X-RAY DIFFRACTIONf_angle_d0.83899025
X-RAY DIFFRACTIONf_chiral_restr0.0455986
X-RAY DIFFRACTIONf_plane_restr0.0051207
X-RAY DIFFRACTIONf_dihedral_angle_d20.9142367
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.88-1.90.33812460.32692205X-RAY DIFFRACTION95.59
1.9-1.920.31762590.32272290X-RAY DIFFRACTION97.63
1.92-1.950.32192560.30272268X-RAY DIFFRACTION97.79
1.95-1.970.31422550.28712260X-RAY DIFFRACTION97.37
1.97-20.30062550.27412239X-RAY DIFFRACTION96.37
2-2.030.26772550.25732267X-RAY DIFFRACTION97.45
2.03-2.060.27362580.23862292X-RAY DIFFRACTION98.49
2.06-2.090.26212570.24292256X-RAY DIFFRACTION97.48
2.09-2.120.26722550.23852309X-RAY DIFFRACTION98.77
2.12-2.150.25952590.21942275X-RAY DIFFRACTION97.95
2.15-2.190.2292510.19992278X-RAY DIFFRACTION98.52
2.19-2.230.20812760.18822284X-RAY DIFFRACTION98.99
2.23-2.270.20222810.17582273X-RAY DIFFRACTION97.78
2.27-2.320.22532580.17892308X-RAY DIFFRACTION98.65
2.32-2.370.19342280.16492310X-RAY DIFFRACTION99.33
2.37-2.430.1992330.15792349X-RAY DIFFRACTION98.44
2.43-2.490.17142530.15252284X-RAY DIFFRACTION98.64
2.49-2.550.20612620.15322304X-RAY DIFFRACTION98.81
2.55-2.630.18212580.14982336X-RAY DIFFRACTION99.12
2.63-2.710.19342620.14712321X-RAY DIFFRACTION99.31
2.71-2.810.19552620.14472296X-RAY DIFFRACTION99.11
2.81-2.920.17872540.13982310X-RAY DIFFRACTION99.3
2.92-3.060.15942860.13862308X-RAY DIFFRACTION99.5
3.06-3.220.20112860.15242304X-RAY DIFFRACTION99.58
3.22-3.420.18852590.13762347X-RAY DIFFRACTION99.39
3.42-3.680.15082500.11952355X-RAY DIFFRACTION99.81
3.68-4.050.12532620.10372344X-RAY DIFFRACTION99.73
4.05-4.640.12062590.09712368X-RAY DIFFRACTION99.77
4.64-5.840.13032560.11432368X-RAY DIFFRACTION99.89
5.85-124.430.15092690.14862416X-RAY DIFFRACTION99.7

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