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

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Basic information

Entry
Database: PDB / ID: 5kqw
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 1 / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / Alpha-Beta Complex / 3-Layer(aba) Sandwich / Alpha Beta / DI(HYDROXYETHYL)ETHER
Function and homology information
Biological speciesPseudomonas (RNA similarity group I)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.23 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 6, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 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 1.2Oct 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 / pdbx_struct_conn_angle / struct_conn / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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.ptnr2_symmetry / _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
C: 4-aminobutyrate transaminase
D: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,20810
Polymers208,7414
Non-polymers4676
Water10,160564
1
A: 4-aminobutyrate transaminase
B: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,6696
Polymers104,3712
Non-polymers2994
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11650 Å2
ΔGint-76 kcal/mol
Surface area28830 Å2
MethodPISA
2
C: 4-aminobutyrate transaminase
D: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,5394
Polymers104,3712
Non-polymers1682
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11250 Å2
ΔGint-78 kcal/mol
Surface area28720 Å2
MethodPISA
Unit cell
Length a, b, c (Å)66.051, 125.179, 110.496
Angle α, β, γ (deg.)90.00, 101.97, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: 0 / Beg auth comp-ID: ASP / Beg label comp-ID: ASP / Refine code: 0

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11VALVALAA3 - 45823 - 478
21VALVALBB3 - 45823 - 478
12LEULEUAA3 - 45923 - 479
22LEULEUCC3 - 45923 - 479
13LEULEUAA3 - 45923 - 479
23LEULEUDD3 - 45923 - 479
14VALVALBB3 - 45823 - 478
24VALVALCC3 - 45823 - 478
15VALVALBB3 - 45823 - 478
25VALVALDD3 - 45823 - 478
16LEULEUCC3 - 45923 - 479
26LEULEUDD3 - 45923 - 479

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
4-aminobutyrate transaminase /


Mass: 52185.277 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas (RNA similarity group I) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: beta-alanine-pyruvate transaminase
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 564 / 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: 2.14 Å3/Da / Density % sol: 42.54 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 150 plus 150 nL drops with protein at 10 mg/mL and reservoir conditions of 15% PEG 8000, 17% glycerol, 150 mM MgCl2, 100 mM Tris pH 7.2. Seeding used to nucleate crystal formation.

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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 315 / 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: 2.23→47.2 Å / Num. obs: 85391 / % possible obs: 99.8 % / Redundancy: 3.8 % / CC1/2: 0.988 / Rmerge(I) obs: 0.13 / Net I/σ(I): 9.2
Reflection shellResolution: 2.23→2.27 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.739 / Mean I/σ(I) obs: 2.3 / CC1/2: 0.654 / % possible all: 99.1

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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: 2.23→47.2 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.939 / SU B: 5.813 / SU ML: 0.138 / Cross valid method: THROUGHOUT / ESU R: 0.3 / ESU R Free: 0.185 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.19198 4237 5 %RANDOM
Rwork0.16374 ---
obs0.16514 81119 99.74 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 22.983 Å2
Baniso -1Baniso -2Baniso -3
1-0.16 Å2-0 Å2-0.88 Å2
2---0.35 Å20 Å2
3---0.51 Å2
Refinement stepCycle: 1 / Resolution: 2.23→47.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14005 0 30 564 14599
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.01914447
X-RAY DIFFRACTIONr_bond_other_d0.0020.0213702
X-RAY DIFFRACTIONr_angle_refined_deg1.561.95619590
X-RAY DIFFRACTIONr_angle_other_deg0.995331564
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.92651841
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.68923.77626
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.386152329
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.2871581
X-RAY DIFFRACTIONr_chiral_restr0.0920.22105
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.02116514
X-RAY DIFFRACTIONr_gen_planes_other0.0020.023263
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_it1.6672.1217352
X-RAY DIFFRACTIONr_mcbond_other1.6652.1217351
X-RAY DIFFRACTIONr_mcangle_it2.5313.1759197
X-RAY DIFFRACTIONr_mcangle_other2.5313.1759198
X-RAY DIFFRACTIONr_scbond_it2.6262.4377095
X-RAY DIFFRACTIONr_scbond_other2.6262.4377095
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.2043.53210394
X-RAY DIFFRACTIONr_long_range_B_refined5.72740.76761634
X-RAY DIFFRACTIONr_long_range_B_other5.72740.76761635
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A300420.05
12B300420.05
21A300520.05
22C300520.05
31A302480.04
32D302480.04
41B302460.05
42C302460.05
51B300760.05
52D300760.05
61C299200.05
62D299200.05
LS refinement shellResolution: 2.23→2.288 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.289 350 -
Rwork0.281 5929 -
obs--99.07 %

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