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- PDB-8e9r: Crystal structure of E. coli aspartate aminotransferase mutant VF... -

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Basic information

Entry
Database: PDB / ID: 8e9r
TitleCrystal structure of E. coli aspartate aminotransferase mutant VFCS 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 from chorismate via phenylpyruvate / L-tyrosine-2-oxoglutarate transaminase activity / L-phenylalanine biosynthetic 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 / 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 ...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 / 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 / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PYRIDOXAL-5'-PHOSPHATE / Aspartate aminotransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)90,2256
Polymers89,5392
Non-polymers6864
Water6,738374
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8400 Å2
ΔGint-69 kcal/mol
Surface area28580 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: 44769.461 Da / Num. of mol.: 2 / Mutation: K37F, T43C, N64S
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.: 374 / 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: 2.72 Å3/Da / Density % sol: 54.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: HEPES, maleate, ammonium sulfate, 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: 0.8855 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: 0.8855 Å / Relative weight: 1
ReflectionResolution: 1.9→124.73 Å / Num. obs: 75209 / % possible obs: 99.8 % / Redundancy: 20 % / Biso Wilson estimate: 30.07 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.2 / Net I/σ(I): 6.1
Reflection shellResolution: 1.9→1.93 Å / Num. unique obs: 3696 / CC1/2: 0.303

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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: 1.9→124.56 Å / SU ML: 0.2363 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 19.0782
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.191 7563 10.06 %
Rwork0.1528 67588 -
obs0.1567 75151 99.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 39.32 Å2
Refinement stepCycle: LAST / Resolution: 1.9→124.56 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6133 0 10 374 6517
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00496410
X-RAY DIFFRACTIONf_angle_d0.79348731
X-RAY DIFFRACTIONf_chiral_restr0.0438959
X-RAY DIFFRACTIONf_plane_restr0.00451162
X-RAY DIFFRACTIONf_dihedral_angle_d21.94642283
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.9-1.920.33912430.32592178X-RAY DIFFRACTION97.46
1.92-1.950.33592520.31452239X-RAY DIFFRACTION99.12
1.95-1.970.32832530.29452248X-RAY DIFFRACTION100
1.97-20.32272450.29182230X-RAY DIFFRACTION99.36
2-2.020.28612480.27312222X-RAY DIFFRACTION99.96
2.02-2.050.30542550.2642267X-RAY DIFFRACTION99.41
2.05-2.080.29452480.25552230X-RAY DIFFRACTION100
2.08-2.110.28592530.25272230X-RAY DIFFRACTION99.6
2.11-2.140.27772540.24152269X-RAY DIFFRACTION99.88
2.14-2.180.25032450.22742231X-RAY DIFFRACTION99.72
2.18-2.220.2532520.20462237X-RAY DIFFRACTION99.76
2.22-2.260.24482800.19062218X-RAY DIFFRACTION100
2.26-2.30.21552780.17692237X-RAY DIFFRACTION99.76
2.3-2.350.20652120.16822272X-RAY DIFFRACTION99.88
2.35-2.40.21392340.16482288X-RAY DIFFRACTION99.96
2.4-2.450.21332360.1592236X-RAY DIFFRACTION99.88
2.45-2.510.21782670.15552239X-RAY DIFFRACTION99.92
2.51-2.580.2012400.15492280X-RAY DIFFRACTION99.92
2.58-2.660.2082540.14832247X-RAY DIFFRACTION99.96
2.66-2.740.19482580.14492251X-RAY DIFFRACTION99.96
2.74-2.840.1962540.14722252X-RAY DIFFRACTION100
2.84-2.960.19932530.14422266X-RAY DIFFRACTION99.96
2.96-3.090.19462800.1422220X-RAY DIFFRACTION100
3.09-3.250.19172710.14642256X-RAY DIFFRACTION100
3.25-3.460.1612430.13082275X-RAY DIFFRACTION100
3.46-3.720.1492360.11212286X-RAY DIFFRACTION100
3.73-4.10.12642670.09762277X-RAY DIFFRACTION100
4.1-4.690.13162420.09442266X-RAY DIFFRACTION100
4.69-5.910.14532470.11682313X-RAY DIFFRACTION100
5.92-124.560.16882630.1492328X-RAY DIFFRACTION99.62

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