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

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Basic information

Entry
Database: PDB / ID: 5kqu
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 / PYRIDOXAL-5'-PHOSPHATE
Function and homology information
Biological speciesPseudomonas (RNA similarity group I)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.62 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 2.0May 30, 2018Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Other / Refinement description
Category: atom_site / atom_sites ...atom_site / atom_sites / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_oper_list / pdbx_validate_close_contact / pdbx_validate_symm_contact / refine / refine_hist / refine_ls_shell / reflns / reflns_shell
Item: _atom_site.Cartn_x / _atom_sites.fract_transf_matrix[2][1] ..._atom_site.Cartn_x / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[3][2] / _pdbx_nonpoly_scheme.auth_seq_num / _refine.aniso_B[1][2] / _refine.aniso_B[2][3] / _refine.details / _refine.ls_R_factor_R_free / _refine.ls_R_factor_R_work / _refine.ls_R_factor_obs / _refine.ls_percent_reflns_obs / _refine.pdbx_starting_model / _refine_hist.pdbx_number_atoms_ligand / _refine_hist.pdbx_number_atoms_protein / _refine_ls_shell.d_res_low / _reflns.pdbx_CC_half / _reflns_shell.pdbx_CC_half
Revision 2.1Jan 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 2.2Oct 4, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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
E: 4-aminobutyrate transaminase
F: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)313,53712
Polymers312,0556
Non-polymers1,4836
Water2,630146
1
A: 4-aminobutyrate transaminase
D: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,5124
Polymers104,0182
Non-polymers4942
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11890 Å2
ΔGint-81 kcal/mol
Surface area28880 Å2
MethodPISA
2
B: 4-aminobutyrate transaminase
C: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,5124
Polymers104,0182
Non-polymers4942
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12030 Å2
ΔGint-78 kcal/mol
Surface area29060 Å2
MethodPISA
3
E: 4-aminobutyrate transaminase
F: 4-aminobutyrate transaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,5124
Polymers104,0182
Non-polymers4942
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11870 Å2
ΔGint-80 kcal/mol
Surface area29160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.104, 146.886, 131.776
Angle α, β, γ (deg.)90.00, 97.68, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16B
26C
17B
27D
18B
28E
19B
29F
110C
210D
111C
211E
112C
212F
113D
213E
114D
214F
115E
215F

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASPASPVALVALAA3 - 45823 - 478
21ASPASPVALVALBB3 - 45823 - 478
12PHEPHEVALVALAA4 - 45824 - 478
22PHEPHEVALVALCC4 - 45824 - 478
13ASPASPLEULEUAA3 - 45923 - 479
23ASPASPLEULEUDD3 - 45923 - 479
14ASPASPLEULEUAA3 - 45923 - 479
24ASPASPLEULEUEE3 - 45923 - 479
15ASPASPVALVALAA3 - 45823 - 478
25ASPASPVALVALFF3 - 45823 - 478
16THRTHRVALVALBB2 - 45822 - 478
26THRTHRVALVALCC2 - 45822 - 478
17ASPASPVALVALBB3 - 45823 - 478
27ASPASPVALVALDD3 - 45823 - 478
18ASPASPVALVALBB3 - 45823 - 478
28ASPASPVALVALEE3 - 45823 - 478
19THRTHRVALVALBB2 - 45822 - 478
29THRTHRVALVALFF2 - 45822 - 478
110ASPASPVALVALCC3 - 45823 - 478
210ASPASPVALVALDD3 - 45823 - 478
111ASPASPVALVALCC3 - 45823 - 478
211ASPASPVALVALEE3 - 45823 - 478
112METMETLEULEUCC1 - 45921 - 479
212METMETLEULEUFF1 - 45921 - 479
113ASPASPLEULEUDD3 - 45923 - 479
213ASPASPLEULEUEE3 - 45923 - 479
114ASPASPVALVALDD3 - 45823 - 478
214ASPASPVALVALFF3 - 45823 - 478
115ASPASPVALVALEE3 - 45823 - 478
215ASPASPVALVALFF3 - 45823 - 478

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

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Components

#1: Protein
4-aminobutyrate transaminase


Mass: 52009.090 Da / Num. of mol.: 6
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
#2: Chemical
ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H10NO6P
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 146 / 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: 2.28 Å3/Da / Density % sol: 46.02 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 150 plus 150 nL drops with protein at 10 mg/mL and a reservoir of 15% PEG 8000, 13% glycerol, 155 mM MgCl2, 100 mM Tris pH 8.5

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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: 2.62→48.9 Å / Num. obs: 83580 / % possible obs: 99.7 % / Redundancy: 7.5 % / Rmerge(I) obs: 0.138 / Net I/σ(I): 11.3
Reflection shellResolution: 2.62→2.67 Å / Redundancy: 6.9 % / Rmerge(I) obs: 0.742 / Mean I/σ(I) obs: 2.5 / % possible all: 95.1

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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: 2.62→48.9 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.917 / SU B: 11.579 / SU ML: 0.232 / Cross valid method: THROUGHOUT / ESU R Free: 0.304 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22 4128 4.9 %RANDOM
Rwork0.188 ---
obs0.19 79422 99.6 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 41.19 Å2
Baniso -1Baniso -2Baniso -3
1-1.51 Å20 Å20.5 Å2
2---0.51 Å20 Å2
3----1.09 Å2
Refinement stepCycle: LAST / Resolution: 2.62→48.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20959 0 90 146 21195
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.01921589
X-RAY DIFFRACTIONr_bond_other_d0.0020.0220505
X-RAY DIFFRACTIONr_angle_refined_deg1.5041.96229275
X-RAY DIFFRACTIONr_angle_other_deg0.986347151
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.64352753
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.13223.412929
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.49153473
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.14415144
X-RAY DIFFRACTIONr_chiral_restr0.0860.23176
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.02124637
X-RAY DIFFRACTIONr_gen_planes_other0.0020.024883
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_it2.373.95311012
X-RAY DIFFRACTIONr_mcbond_other2.3683.95311011
X-RAY DIFFRACTIONr_mcangle_it3.685.92513765
X-RAY DIFFRACTIONr_mcangle_other3.6815.92513766
X-RAY DIFFRACTIONr_scbond_it2.8034.29210577
X-RAY DIFFRACTIONr_scbond_other2.8034.29210578
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.5076.30715511
X-RAY DIFFRACTIONr_long_range_B_refined7.15775.83990351
X-RAY DIFFRACTIONr_long_range_B_other7.15675.84790324
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)
11A295520.04
12B295520.04
21A294320.04
22C294320.04
31A294680.06
32D294680.06
41A294800.05
42E294800.05
51A294720.05
52F294720.05
61B296420.05
62C296420.05
71B295200.05
72D295200.05
81B295000.05
82E295000.05
91B296140.05
92F296140.05
101C294060.06
102D294060.06
111C294300.05
112E294300.05
121C296220.05
122F296220.05
131D294660.06
132E294660.06
141D294820.06
142F294820.06
151E295720.04
152F295720.04
LS refinement shellResolution: 2.62→2.69 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.321 298 -
Rwork0.297 5627 -
obs--96.09 %

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