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- PDB-5kr5: Directed Evolution of Transaminases By Ancestral Reconstruction. ... -

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Basic information

Entry
Database: PDB / ID: 5kr5
TitleDirected Evolution of Transaminases By Ancestral Reconstruction. Using Old Proteins for New Chemistries
Components4-aminobutyrate transaminase
KeywordsTRANSFERASE / phylogenetics / directed evolution / transaminase / protein engineering
Function / homologyAspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / 3-Layer(aba) Sandwich / Alpha Beta / DI(HYDROXYETHYL)ETHER / PYRIDOXAL-5'-PHOSPHATE
Function and homology information
Biological speciesPseudomonas (RNA similarity group I)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 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 7, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.db_id
Revision 1.2Jan 30, 2019Group: Data collection / Database references / Category: citation / citation_author
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 1.3Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn / struct_conn_type / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 4-aminobutyrate transaminase
B: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,00110
Polymers104,0682
Non-polymers9338
Water6,449358
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12860 Å2
ΔGint-104 kcal/mol
Surface area28650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)98.451, 102.538, 199.781
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: _ / Ens-ID: 1 / Beg auth comp-ID: SER / Beg label comp-ID: SER / End auth comp-ID: ALA / End label comp-ID: ALA / Refine code: _ / Auth seq-ID: 3 - 455 / Label seq-ID: 23 - 475

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

#1: Protein 4-aminobutyrate transaminase


Mass: 52033.895 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas (RNA similarity group I) / Plasmid: pET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: beta-alanine-pyruvate transaminase
#2: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H10NO6P / References: beta-alanine-pyruvate transaminase
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 358 / 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: 2.42 Å3/Da / Density % sol: 49.22 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 150 plus 150 nL drops with protein at 4 mg/mL and reservoir conditions of 15% PEG 8000, 11 mM calcium acetate, 100 mM Tris pH 7.1. Microcrystals were used as nucleating agents to obtain full size crystals.

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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: Mar 1, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.1→48.6 Å / Num. obs: 59261 / % possible obs: 99.6 % / Redundancy: 7.3 % / CC1/2: 0.994 / Rmerge(I) obs: 0.17 / Net I/σ(I): 9.5
Reflection shellResolution: 2.1→2.15 Å / Redundancy: 7.3 % / Rmerge(I) obs: 0.899 / Mean I/σ(I) obs: 2.2 / CC1/2: 0.67 / % possible all: 97.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5kqt

5kqt


Resolution: 2.1→48.6 Å / Cor.coef. Fo:Fc: 0.938 / Cor.coef. Fo:Fc free: 0.908 / SU B: 6.161 / SU ML: 0.155 / Cross valid method: THROUGHOUT / ESU R: 0.23 / ESU R Free: 0.186 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23987 2892 4.9 %RANDOM
Rwork0.20425 ---
obs0.20597 56252 99.36 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 29.309 Å2
Baniso -1Baniso -2Baniso -3
1--1 Å20 Å2-0 Å2
2--1.09 Å20 Å2
3----0.09 Å2
Refinement stepCycle: 1 / Resolution: 2.1→48.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6957 0 54 358 7369
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0197218
X-RAY DIFFRACTIONr_bond_other_d0.0020.026815
X-RAY DIFFRACTIONr_angle_refined_deg1.4241.9569806
X-RAY DIFFRACTIONr_angle_other_deg0.953315684
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.2455917
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.70923.758314
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.351151128
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.2331540
X-RAY DIFFRACTIONr_chiral_restr0.0820.21065
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0218239
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021633
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_it1.3432.8473659
X-RAY DIFFRACTIONr_mcbond_other1.342.8463658
X-RAY DIFFRACTIONr_mcangle_it2.1764.2624579
X-RAY DIFFRACTIONr_mcangle_other2.1764.2634580
X-RAY DIFFRACTIONr_scbond_it1.523.0423559
X-RAY DIFFRACTIONr_scbond_other1.523.0423559
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.4894.4935228
X-RAY DIFFRACTIONr_long_range_B_refined5.24153.75431124
X-RAY DIFFRACTIONr_long_range_B_other5.24153.75431124
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 29746 / 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: 2.097→2.151 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.299 206 -
Rwork0.276 4038 -
obs--97.59 %

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