PDB-4f5j: Rational Design and Directed Evolution for Conversion of Substrat...

ID or keywords:

Entry

Database: PDB / ID: 4f5j

Title

Rational Design and Directed Evolution for Conversion of Substrate Specificity from E.coli Aspartate Aminotransferase to Tyrosine Aminotransferase: Mutant P5.

Components

Aspartate aminotransferase

Keywords

TRANSFERASE / Aminotransferase

Function / homology

Function and homology information

L-phenylalanine biosynthetic process / L-phenylalanine biosynthetic process from chorismate via phenylpyruvate / L-tyrosine-2-oxoglutarate transaminase 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

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

MOLECULAR REPLACEMENT / Resolution: 1.954 Å

Authors

Addington, T.A. / Fisher, A.J. / Toney, M.D.

Citation

Journal: J.Mol.Biol. / Year: 2013
Title: Janus: prediction and ranking of mutations required for functional interconversion of enzymes.
Authors: Addington, T.A. / Mertz, R.W. / Siegel, J.B. / Thompson, J.M. / Fisher, A.J. / Filkov, V. / Fleischman, N.M. / Suen, A.A. / Zhang, C. / Toney, M.D.

History

Deposition	May 13, 2012	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Feb 13, 2013	Provider: repository / Type: Initial release
Revision 1.1	Feb 27, 2013	Group: Database references
Revision 1.2	Apr 24, 2013	Group: Database references

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	4f5j.cif.gz	329.9 KB	Display	PDBx/mmCIF format
PDB format	pdb4f5j.ent.gz	279.2 KB	Display	PDB format
PDBx/mmJSON format	4f5j.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	4f5j_validation.pdf.gz	436.4 KB	Display	wwPDB validaton report
Full document	4f5j_full_validation.pdf.gz	441.5 KB	Display
Data in XML	4f5j_validation.xml.gz	39.1 KB	Display
Data in CIF	4f5j_validation.cif.gz	60.4 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/f5/4f5j ftp://data.pdbj.org/pub/pdb/validation_reports/f5/4f5j	HTTPS FTP

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Related structure data

Related structure data	4f5fC 4f5gC 4f5hC 4f5iC 4f5kC 4f5mC 4f5l C: citing same article (ref.)
Similar structure data	4f5h-d 1g7x-1 1ars-1 4f5l-d 5ax8-1 5vwq-2 1g7w-1 3pd6-1 1g4v-1 4f5k-d 1g4x-1 Search similar-shape structures by Omokage search (details) Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Aspartate aminotransferase

B: Aspartate aminotransferase

89.5 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

Idetical with deposited unit
defined by author&software

Unit cell

Length a, b, c (Å)	59.706, 102.985, 139.344
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein	Aspartate aminotransferase / AspAT / Transaminase A Mass: 44764.332 Da / Num. of mol.: 2 Mutation: I39V, N40D, I43V, N74T, I78L, I81L, T114S, S145A, V146I, I197A, F220I, F222I, A228G, Y254C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: AAT, aspC, b0928, JW0911 / Plasmid: pET45b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00509, aspartate transaminase
#2: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 942 / Source method: isolated from a natural source / Formula: H₂O

#1: Protein

Aspartate aminotransferase / AspAT / Transaminase A

Mass: 44764.332 Da / Num. of mol.: 2
Mutation: I39V, N40D, I43V, N74T, I78L, I81L, T114S, S145A, V146I, I197A, F220I, F222I, A228G, Y254C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Escherichia coli (E. coli) / Strain: K12 / Gene: AAT, aspC, b0928, JW0911 / Plasmid: pET45b / Production host:

Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00509, aspartate transaminase

#2: Water

ChemComp-HOH / water

Mass: 18.015 Da / Num. of mol.: 942 / Source method: isolated from a natural source / Formula: H₂O

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.39 Å³/Da / Density % sol: 48.59 %
Crystal grow	Temperature: 277 K / Method: vapor diffusion, hanging drop / pH: 5 Details: 8% PEG 4000, 0.1 M Sodium Acetate, pH 5.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

Diffraction	Mean temperature: 85 K
Diffraction source	Source: ROTATING ANODE / Type: RIGAKU RU300 / Wavelength: 1.54 Å
Detector	Type: BRUKER SMART 6000 / Detector: CCD / Date: Aug 7, 2010 / Details: Mirrors
Radiation	Monochromator: Osmic Mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.54 Å / Relative weight: 1
Reflection	Resolution: 1.95→100 Å / Num. all: 63173 / Num. obs: 63086 / % possible obs: 99.9 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 5.76 % / B_iso Wilson estimate: 17.9 Å² / R_merge(I) obs: 0.0844 / R_sym value: 0.0844 / Net I/σ(I): 13.88
Reflection shell	Resolution: 1.95→2.05 Å / Redundancy: 3.78 % / R_merge(I) obs: 0.4078 / Mean I/σ(I) obs: 2.32 / Num. unique all: 8303 / R_sym value: 0.4078 / % possible all: 99

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Processing

Software

Name	Version	Classification
PROTEUM PLUS	PLUS	data collection
PHASER		phasing
PHENIX	(phenix.refine: 1.7.1_743)	refinement
PROTEUM PLUS	PLUS	data reduction
PROTEUM PLUS	PLUS	data scaling

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.954→51.653 Å / SU ML: 0.47 / σ(F): 1.34 / Phase error: 18.01 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.2036	3112	4.94 %
R_work	0.1614	-	-
obs	0.1634	62990	99.9 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / B_sol: 42.996 Å² / k_sol: 0.33 e/Å³

Displacement parameters

	B_aniso -1	B_aniso -2	B_aniso -3
1-	3.8311 Å²	0 Å²	-0 Å²
2-	-	-0.3769 Å²	-0 Å²
3-	-	-	-3.4542 Å²

Refine analyze

Luzzati sigma a obs: 0.47 Å

Refinement step

Cycle: LAST / Resolution: 1.954→51.653 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6168	0	0	942	7110

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	6408
X-RAY DIFFRACTION	f_angle_d	1.065	8712
X-RAY DIFFRACTION	f_dihedral_angle_d	12.937	2346
X-RAY DIFFRACTION	f_chiral_restr	0.074	963
X-RAY DIFFRACTION	f_plane_restr	0.005	1156

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
1.954-1.9846	0.2738	153	0.2289	2642	X-RAY DIFFRACTION	98
1.9846-2.0171	0.2464	150	0.2092	2666	X-RAY DIFFRACTION	100
2.0171-2.0519	0.233	163	0.1885	2666	X-RAY DIFFRACTION	100
2.0519-2.0892	0.2615	132	0.1739	2679	X-RAY DIFFRACTION	100
2.0892-2.1294	0.2102	132	0.1745	2713	X-RAY DIFFRACTION	100
2.1294-2.1728	0.2162	154	0.1623	2643	X-RAY DIFFRACTION	100
2.1728-2.2201	0.2279	126	0.1568	2729	X-RAY DIFFRACTION	100
2.2201-2.2717	0.218	147	0.1521	2669	X-RAY DIFFRACTION	100
2.2717-2.3285	0.2359	160	0.1558	2690	X-RAY DIFFRACTION	100
2.3285-2.3915	0.1866	142	0.1586	2720	X-RAY DIFFRACTION	100
2.3915-2.4619	0.2321	138	0.154	2682	X-RAY DIFFRACTION	100
2.4619-2.5413	0.2086	144	0.1611	2705	X-RAY DIFFRACTION	100
2.5413-2.6321	0.2358	137	0.157	2716	X-RAY DIFFRACTION	100
2.6321-2.7375	0.1952	141	0.1562	2721	X-RAY DIFFRACTION	100
2.7375-2.8621	0.1974	148	0.1558	2724	X-RAY DIFFRACTION	100
2.8621-3.013	0.1994	112	0.1543	2725	X-RAY DIFFRACTION	100
3.013-3.2017	0.2012	162	0.1646	2722	X-RAY DIFFRACTION	100
3.2017-3.4489	0.1905	126	0.1584	2770	X-RAY DIFFRACTION	100
3.4489-3.7959	0.187	141	0.1548	2739	X-RAY DIFFRACTION	100
3.7959-4.3449	0.1566	138	0.1354	2782	X-RAY DIFFRACTION	100
4.3449-5.4732	0.1752	139	0.1492	2814	X-RAY DIFFRACTION	100
5.4732-51.6711	0.201	127	0.1873	2961	X-RAY DIFFRACTION	100

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°²)	L12 (°²)	L13 (°²)	L22 (°²)	L23 (°²)	L33 (°²)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å²)	T12 (Å²)	T13 (Å²)	T22 (Å²)	T23 (Å²)	T33 (Å²)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	0.0114	0.0202	0.015	0.0562	-0.0486	0.1199	0.0505	0.0981	0.0519	0.0851	-0.0151	0.0353	-0.142	-0.0627	0.0331	0.1153	0.0643	-0.0663	0.171	-0.0108	0.1084	27.0847	111.9047	33.858
2	0.0047	-0.0144	-0.0141	0.018	0.0198	0.0168	0.0263	-0.0238	-0.0466	0.0302	0.0158	0.0063	0.0779	-0.0443	0.0073	0.0628	-0.0551	-0.0123	0.0948	0.0125	0.0721	22.153	85.3659	45.202
3	0.0019	0.0015	-0.0005	0.0138	-0.0075	0.0072	0.0077	-0.0148	-0.0565	0.0147	0.0247	0.0148	0.0804	-0.017	0.002	0.1346	0.0169	-0.0086	0.0619	0.0202	0.1228	41.46	74.7913	45.1508
4	0.0289	-0.0117	0.0197	0.0226	-0.0478	0.0485	-0.0163	0.0111	0.03	-0.0391	-0.0158	-0.0165	0.0707	0.0702	-0	0.0858	0.0041	0.0118	0.0868	0.0057	0.0886	47.4085	89.7242	32.5776
5	0.0067	0.0021	-0.0105	0.0015	-0.0125	0.0254	-0.0293	-0.0568	0.0183	-0.017	-0.0135	0.0023	0.0623	0.0147	0	0.0699	-0.0037	-0.0079	0.0704	0.0042	0.0806	39.0796	85.8126	47.2255
6	0.0176	0.0115	0.0039	0.0311	0.0061	0.001	-0.0003	0.0944	-0.0413	-0.0042	-0.0067	0.0328	0.0431	-0.0383	-0.0209	0.0555	-0.1155	-0.098	-0.065	-0.1314	-0.0561	25.0955	81.7881	24.107
7	0.0151	-0.02	0.001	0.0211	0.004	0.0228	-0.0141	0.0825	0.1043	-0.0877	0.0586	-0.01	0.0142	-0.0605	0.003	0.099	-0.0102	-0.0129	0.1312	0.036	0.1039	28.0349	97.9627	15.48
8	0.003	-0.0212	0.0006	0.0181	-0.003	0.0067	0.0195	0.0602	0.0067	0.0492	-0.0033	-0.0111	0.0614	0.0863	0.013	0.0879	0.0232	0.0729	0.1069	0.0201	0.092	44.8769	83.1855	66.1164
9	0.0034	0.0015	0.0103	0.0266	0.0082	0.0251	-0.0157	0.0162	-0.0451	0.0276	-0.001	-0.0277	-0.0051	-0.1161	-0.0042	0.034	-0.023	-0.0123	0.1076	0.0009	0.0753	18.9145	91.1322	55.7081
10	0.014	0.0107	-0.0119	0.0221	-0.0092	0.0256	-0.0318	0.0167	0.0038	-0.0346	0.0146	-0.0196	-0.0995	-0.0259	-0	0.0861	0.0131	-0.0108	0.0771	0.0075	0.0751	30.4642	110.8602	55.6543
11	0.0089	-0.0032	0.0097	0.0005	0.0053	0.013	-0.014	-0.0186	-0.0046	0.0171	-0.0161	-0.0386	-0.0698	0.0446	-0.0055	0.1053	-0.024	-0.0249	0.0734	0.0008	0.0762	41.6272	111.7656	70.4646
12	0.0066	-0.002	0.0131	0.0182	-0.0115	0.021	-0.0165	-0.0534	0.0389	0.0296	-0.0097	-0.0299	-0.1503	-0.1111	-0.0029	0.1165	0.0396	-0.0138	0.0606	-0.0069	0.0787	28.5273	113.7526	69.102
13	0.0065	0.0023	0.0076	-0.001	-0.0026	0.0228	-0.0272	0.0045	0.0073	-0.0234	0.0277	-0.0117	-0.0763	-0.0846	-0	0.0963	0.0104	-0.0102	0.072	0.0113	0.0809	27.247	105.5948	52.472
14	0.0178	0.0005	0.0014	0.0619	0.0099	0.0113	0.007	0.0061	0.0067	0.0632	0.0167	0.0335	-0.0035	-0.0738	0.0057	0.0578	0.0045	0.0331	0.1527	0.0108	0.0792	18.0374	96.4938	76.1066
15	0.0075	-0.0041	0.0059	0.0111	0.0025	0.0175	-0.0037	-0.0825	-0.0132	0.0285	0.0214	-0.0347	0.0058	-0.0014	0.0187	0.0499	0.0005	-0.012	0.0755	0.0217	0.0757	34.4149	91.6857	84.5091

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	CHAIN A AND (RESSEQ 8:42)
2	X-RAY DIFFRACTION	2	CHAIN A AND (RESSEQ 43:81)
3	X-RAY DIFFRACTION	3	CHAIN A AND (RESSEQ 82:104)
4	X-RAY DIFFRACTION	4	CHAIN A AND (RESSEQ 105:244)
5	X-RAY DIFFRACTION	5	CHAIN A AND (RESSEQ 245:310)
6	X-RAY DIFFRACTION	6	CHAIN A AND (RESSEQ 311:341)
7	X-RAY DIFFRACTION	7	CHAIN A AND (RESSEQ 342:406)
8	X-RAY DIFFRACTION	8	CHAIN B AND (RESSEQ 8:42)
9	X-RAY DIFFRACTION	9	CHAIN B AND (RESSEQ 43:81)
10	X-RAY DIFFRACTION	10	CHAIN B AND (RESSEQ 82:140)
11	X-RAY DIFFRACTION	11	CHAIN B AND (RESSEQ 141:188)
12	X-RAY DIFFRACTION	12	CHAIN B AND (RESSEQ 189:244)
13	X-RAY DIFFRACTION	13	CHAIN B AND (RESSEQ 245:310)
14	X-RAY DIFFRACTION	14	CHAIN B AND (RESSEQ 311:341)
15	X-RAY DIFFRACTION	15	CHAIN B AND (RESSEQ 342:406)

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