PDB-4f5g: Rational Design and Directed Evolution of E. coli Apartate Aminot...

ID or keywords:

Entry

Database: PDB / ID: 4f5g

Title

Rational Design and Directed Evolution of E. coli Apartate Aminotransferase to Tyrosine Aminotransferase: Mutant P2.

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: 1.67 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	4f5g.cif.gz	338.9 KB	Display	PDBx/mmCIF format
PDB format	pdb4f5g.ent.gz	284.4 KB	Display	PDB format
PDBx/mmJSON format	4f5g.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	4f5g_validation.pdf.gz	443.5 KB	Display	wwPDB validaton report
Full document	4f5g_full_validation.pdf.gz	453.5 KB	Display
Data in XML	4f5g_validation.xml.gz	42.1 KB	Display
Data in CIF	4f5g_validation.cif.gz	65.1 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/f5/4f5g ftp://data.pdbj.org/pub/pdb/validation_reports/f5/4f5g	HTTPS FTP

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Related structure data

Related structure data	4f5fC 4f5hC 4f5iC 4f5jC 4f5kC 4f5mC 4f5l C: citing same article (ref.)
Similar structure data	5eaa-1 3aat-1 1g4x-1 3zzk-1 1aic-d 1asf-1 1bqa-d 6czy-1 1x29-d 1b4x-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

89.4 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)	58.302, 102.575, 139.538
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: 44720.277 Da / Num. of mol.: 2 Mutation: I39V, N40D, I43V, N74T, I78L, I81L, T114S, S145A, V146I, I197A, F220I, F222I, A228G, Y254C, S283G, I351V 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.: 1060 / Source method: isolated from a natural source / Formula: H₂O

#1: Protein

Aspartate aminotransferase / AspAT / Transaminase A

Mass: 44720.277 Da / Num. of mol.: 2
Mutation: I39V, N40D, I43V, N74T, I78L, I81L, T114S, S145A, V146I, I197A, F220I, F222I, A228G, Y254C, S283G, I351V
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.: 1060 / 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: 2.33 Å³/Da / Density % sol: 47.26 %
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 13, 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: 1.67→57.689 Å / Num. all: 94375 / Num. obs: 93933 / % possible obs: 99.5 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 5.78 % / B_iso Wilson estimate: 17.5 Å² / R_merge(I) obs: 0.0532 / R_sym value: 0.0532 / Net I/σ(I): 18.71
Reflection shell	Resolution: 1.67→1.68 Å / Redundancy: 3.25 % / R_merge(I) obs: 0.375 / Mean I/σ(I) obs: 2.5 / Num. unique all: 13251 / R_sym value: 0.375 / % possible all: 99.4

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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: 1.67→57.689 Å / SU ML: 0.38 / σ(F): 0 / σ(I): 0 / Phase error: 17.51 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1905	4671	4.98 %
R_work	0.1633	-	-
obs	0.1646	93836	95.18 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / B_sol: 38.768 Å² / k_sol: 0.332 e/Å³

Displacement parameters

	B_aniso -1	B_aniso -2	B_aniso -3
1-	2.1499 Å²	-0 Å²	-0 Å²
2-	-	-0.2124 Å²	-0 Å²
3-	-	-	-1.9374 Å²

Refine analyze

Luzzati sigma a obs: 0.38 Å

Refinement step

Cycle: LAST / Resolution: 1.67→57.689 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6162	0	0	1060	7222

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	6440
X-RAY DIFFRACTION	f_angle_d	1.152	8760
X-RAY DIFFRACTION	f_dihedral_angle_d	12.712	2359
X-RAY DIFFRACTION	f_chiral_restr	0.073	966
X-RAY DIFFRACTION	f_plane_restr	0.005	1165

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 (%)
1.67-1.684	0.3601	22	0.2718	402	X-RAY DIFFRACTION	13
1.684-1.7038	0.3191	93	0.251	1678	X-RAY DIFFRACTION	55
1.7038-1.7246	0.2909	163	0.2393	3009	X-RAY DIFFRACTION	98
1.7246-1.7464	0.2456	175	0.2221	3058	X-RAY DIFFRACTION	99
1.7464-1.7694	0.2598	157	0.211	3088	X-RAY DIFFRACTION	99
1.7694-1.7936	0.2284	142	0.2015	3059	X-RAY DIFFRACTION	100
1.7936-1.8193	0.2796	144	0.1937	3093	X-RAY DIFFRACTION	99
1.8193-1.8464	0.2313	173	0.1849	3045	X-RAY DIFFRACTION	99
1.8464-1.8753	0.2136	148	0.1782	3083	X-RAY DIFFRACTION	99
1.8753-1.906	0.2003	167	0.1706	3026	X-RAY DIFFRACTION	99
1.906-1.9389	0.2122	188	0.163	3062	X-RAY DIFFRACTION	99
1.9389-1.9742	0.2027	187	0.1646	2996	X-RAY DIFFRACTION	99
1.9742-2.0121	0.188	172	0.1688	3100	X-RAY DIFFRACTION	99
2.0121-2.0532	0.1995	166	0.1616	3038	X-RAY DIFFRACTION	99
2.0532-2.0978	0.2052	148	0.1652	3095	X-RAY DIFFRACTION	99
2.0978-2.1467	0.2022	180	0.1583	3076	X-RAY DIFFRACTION	99
2.1467-2.2003	0.193	134	0.154	3105	X-RAY DIFFRACTION	100
2.2003-2.2598	0.1825	175	0.1471	3094	X-RAY DIFFRACTION	100
2.2598-2.3263	0.1826	182	0.1452	3108	X-RAY DIFFRACTION	100
2.3263-2.4014	0.1912	169	0.1478	3106	X-RAY DIFFRACTION	100
2.4014-2.4872	0.1711	148	0.1515	3127	X-RAY DIFFRACTION	100
2.4872-2.5868	0.1829	166	0.1548	3121	X-RAY DIFFRACTION	100
2.5868-2.7046	0.1785	175	0.1518	3128	X-RAY DIFFRACTION	100
2.7046-2.8471	0.1742	157	0.156	3134	X-RAY DIFFRACTION	100
2.8471-3.0255	0.1753	157	0.1524	3174	X-RAY DIFFRACTION	100
3.0255-3.2591	0.1556	162	0.1597	3163	X-RAY DIFFRACTION	100
3.2591-3.587	0.179	148	0.1544	3182	X-RAY DIFFRACTION	100
3.587-4.106	0.1555	173	0.1455	3191	X-RAY DIFFRACTION	100
4.106-5.1726	0.1669	148	0.1525	3236	X-RAY DIFFRACTION	100
5.1726-57.7238	0.2199	152	0.1901	3388	X-RAY DIFFRACTION	99

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	0.0561	0.2096	-0.4594	0.3676	-0.3946	1.2621	-0.0349	0.1432	-0.3001	-0.2345	-0.1643	-0.3241	0.7161	0.3728	0.0809	0.2221	0.0969	0.043	0.3196	0.0455	0.22	2.7536	-8.8721	-35.8441
2	0.7624	-0.0039	0.0761	0.2393	0.0399	0.8962	0.0019	-0.0103	0.0766	-0.0149	-0.0149	-0.0091	-0.1567	0.0338	0.0312	0.0978	-0.002	0.0022	0.0518	-0.0066	0.0956	-3.1082	19.1416	-24.0415
3	0.2652	0.0184	0.3528	0.5815	-0.2515	2.1994	-0.0446	0.0622	-0.0758	-0.0292	-0.0182	0.0667	0.0362	-0.2154	0.0455	0.0905	-0.0112	-0.0184	0.1254	-0.0159	0.122	-19.8346	7.4587	-39.8259
4	0.4167	0.0482	0.0115	0.2846	0.1218	0.8296	-0.0285	0.0175	0.021	-0.0665	-0.0208	0.0712	-0.1422	-0.0862	0.0309	0.0968	0.0094	-0.0103	0.0672	-0.0033	0.0938	-11.7127	18.3915	-30.1431
5	1.2118	0.5705	0.4601	2.5783	1.7823	3.7918	-0.0242	0.1798	0.0326	-0.2711	0.1394	-0.1565	-0.3564	0.1263	-0.0191	0.1362	-0.0284	0.0078	0.1095	0.0383	0.1088	4.675	21.2672	-45.751
6	1.8441	-0.3597	-0.1258	1.4268	0.5903	1.7043	-0.0367	0.3047	-0.3405	-0.2293	0.0778	-0.0055	-0.0059	0.1799	-0.0326	0.1353	-0.0131	0.0042	0.1881	-0.0512	0.1554	1.8296	4.7995	-54.476
7	0.8957	-0.1509	0.948	-0.0307	0.1509	1.1587	-0.0891	-0.0223	0.142	0.018	-0.031	0.0548	-0.1676	-0.2395	0.0826	0.1501	0.0013	-0.0398	0.1911	-0.0112	0.1593	-14.721	19.7595	-3.5427
8	0.3064	0.0775	-0.1424	0.2849	-0.288	1.1893	0.0138	-0.0009	0.0426	0.0152	0.0146	-0.0546	-0.065	0.2058	-0.0026	0.0539	-0.0099	0.0009	0.1127	-0.0023	0.1205	10.5251	11.3361	-14.8393
9	0.6891	0.085	-0.1449	0.761	0.4173	1.5475	-0.0459	0.0091	-0.0893	-0.0028	0.0318	-0.0352	0.2124	0.075	0.0227	0.1047	0.0202	0.0068	0.0534	-0.0048	0.0934	-0.2515	-7.4964	-14.3113
10	0.5689	0.1056	-0.2048	0.243	-0.0977	1.3408	-0.0209	-0.0447	-0.1025	0.0399	-0.0077	0.0142	0.27	0.0038	0.015	0.1328	-0.0003	0.0239	0.0606	0.007	0.1054	-4.5489	-9.7716	-0.0577
11	0.6828	0.2328	0.036	0.3229	0.2123	1.0389	-0.0254	0.027	-0.0845	-0.024	0.0211	0.0023	0.1472	0.0675	0.0108	0.0832	0.0142	0.0065	0.0567	-0.0073	0.0776	2.5306	-2.9321	-17.1629
12	2.4912	-0.2646	-2.1483	0.5919	0.1176	4.0628	-0.0084	-0.258	-0.0513	0.01	0.0495	-0.0343	0.09	0.5307	-0.0225	0.0781	0.0097	-0.0196	0.1405	-0.0053	0.1018	11.7211	6.5089	6.3776
13	1.0124	-0.2963	-0.1977	1.1321	0.1968	1.2239	-0.002	-0.1875	0.0192	0.0927	0.0249	0.1415	-0.0444	-0.0759	-0.0258	0.0912	0.0007	0.0209	0.1205	-0.01	0.1104	-4.2884	10.9343	15.0559

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 43:127)
3	X-RAY DIFFRACTION	3	CHAIN A AND (RESSEQ 128:188)
4	X-RAY DIFFRACTION	4	CHAIN A AND (RESSEQ 189:310)
5	X-RAY DIFFRACTION	5	CHAIN A AND (RESSEQ 311:341)
6	X-RAY DIFFRACTION	6	CHAIN A AND (RESSEQ 342:406)
7	X-RAY DIFFRACTION	7	CHAIN B AND (RESSEQ 8:42)
8	X-RAY DIFFRACTION	8	CHAIN B AND (RESSEQ 43:81)
9	X-RAY DIFFRACTION	9	CHAIN B AND (RESSEQ 82:140)
10	X-RAY DIFFRACTION	10	CHAIN B AND (RESSEQ 141:244)
11	X-RAY DIFFRACTION	11	CHAIN B AND (RESSEQ 245:310)
12	X-RAY DIFFRACTION	12	CHAIN B AND (RESSEQ 311:341)
13	X-RAY DIFFRACTION	13	CHAIN B AND (RESSEQ 342:406)

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