PDB-8e9c: Crystal structure of E. coli aspartate aminotransferase mutant AI...

ID or keywords:

Entry

Database: PDB / ID: 8e9c

Title

Crystal structure of E. coli aspartate aminotransferase mutant AIFS in the ligand-free form at 100 K

Components

Aspartate aminotransferase

Keywords

TRANSFERASE / Enzyme / Designed Protein / Mutant Protein

Function / homology

Function and homology information

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

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.18 Å

Authors

Chica, R.A. / St-Jacques, A.D. / Rodriguez, J.M. / Thompson, M.C.

Funding support

Canada,

United States, 5items

Organization	Grant number	Country
Natural Sciences and Engineering Research Council (NSERC, Canada)	RGPIN-2016-04831	Canada
Ontario Early Researcher Awards	ER14-10-139	Canada
Canada Foundation for Innovation	26503	Canada
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	GM124149	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	GM124169	United States

Citation

Journal: Nat Commun / Year: 2023
Title: Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.
Authors: St-Jacques, A.D. / Rodriguez, J.M. / Eason, M.G. / Foster, S.M. / Khan, S.T. / Damry, A.M. / Goto, N.K. / Thompson, M.C. / Chica, R.A.

History

Deposition	Aug 26, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Nov 2, 2022	Provider: repository / Type: Initial release
Revision 1.1	Oct 11, 2023	Group: Data collection / Database references Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2	Oct 18, 2023	Group: Refinement description / Category: pdbx_initial_refinement_model

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	8e9c.cif.gz	533.3 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	8e9c_validation.pdf.gz	461.6 KB	Display	wwPDB validaton report
Full document	8e9c_full_validation.pdf.gz	462.3 KB	Display
Data in XML	8e9c_validation.xml.gz	29 KB	Display
Data in CIF	8e9c_validation.cif.gz	40.6 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/e9/8e9c ftp://data.pdbj.org/pub/pdb/validation_reports/e9/8e9c	HTTPS FTP

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Related structure data

Related structure data	8e9dC 8e9jC 8e9kC 8e9lC 8e9mC 8e9nC 8e9oC 8e9pC 8e9qC 8e9rC 8e9sC 8e9tC 8e9uC 8e9vC 1x28S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	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
Related items in Molecule of the Month	#259 - Jul 2021 Designed Proteins and Citizen Science similarity (1) #70 - Oct 2005 Designer Proteins similarity (1)

Deposited unit

A: Aspartate aminotransferase

B: Aspartate aminotransferase

hetero molecules

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

1

Idetical with deposited unit
defined by author&software
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	141.630, 141.630, 80.920
Angle α, β, γ (deg.)	90.000, 90.000, 120.000
Int Tables number	173
Space group name H-M	P6₃
Space group name Hall	P6c
Symmetry operation	#1: x,y,z #2: x-y,x,z+1/2 #3: y,-x+y,z+1/2 #4: -y,x-y,z #5: -x+y,-x,z #6: -x,-y,z+1/2

#1: Protein	Aspartate aminotransferase / AspAT / Transaminase A Mass: 44751.422 Da / Num. of mol.: 2 / Mutation: V35A K37I T43F N64S Source method: isolated from a genetically manipulated source Details: PLP cofactor forms covalent imine bond with catalytic lysine (K248) epsilon amino group Source: (gene. exp.) Escherichia coli (E. coli) / Gene: aspC, b0928, JW0911 / Production host: Escherichia coli (E. coli) / References: UniProt: P00509, aspartate transaminase
#2: Chemical	ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₈H₁₀NO₆P
#3: Chemical	ChemComp-SO4 / SULFATE ION Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO₄
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 172 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	N

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.62 Å³/Da / Density % sol: 53.01 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / Details: Ammonium sulfate, HEPES, PEG-400, maleate

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.1158 Å
Detector	Type: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jun 1, 2021
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.1158 Å / Relative weight: 1
Reflection	Resolution: 2.18→67.54 Å / Num. obs: 48326 / % possible obs: 100 % / Redundancy: 20 % / CC_1/2: 0.997 / R_merge(I) obs: 0.126 / Net I/σ(I): 8.3
Reflection shell	Resolution: 2.18→2.22 Å / Redundancy: 38.6 % / R_merge(I) obs: 3.553 / Num. unique obs: 2387 / CC_1/2: 0.47 / R_pim(I) all: 0.575 / R_rim(I) all: 3.6 / Net I/σ(I) obs: 1.2 / % possible all: 100

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Processing

Software

Name	Version	Classification
PHENIX	1.17.1_3660	refinement
xia2		data reduction
DIALS		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 1X28

1x28

Resolution: 2.18→67.54 Å / SU ML: 0.3022 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.183
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2236	4875	10.1 %
R_work	0.1891	43406	-
obs	0.1925	48281	99.92 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL

Displacement parameters

B_iso mean: 47.94 Å²

Refinement step

Cycle: LAST / Resolution: 2.18→67.54 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5983	0	5	172	6160

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0024	6168
X-RAY DIFFRACTION	f_angle_d	0.535	8388
X-RAY DIFFRACTION	f_chiral_restr	0.0389	924
X-RAY DIFFRACTION	f_plane_restr	0.0031	1111
X-RAY DIFFRACTION	f_dihedral_angle_d	20.4399	2198

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 (%)
2.18-2.2	0.3546	178	0.3184	1408	X-RAY DIFFRACTION	99.62
2.2-2.23	0.3273	182	0.2768	1430	X-RAY DIFFRACTION	100
2.23-2.26	0.348	170	0.2715	1404	X-RAY DIFFRACTION	99.81
2.26-2.29	0.2947	164	0.258	1453	X-RAY DIFFRACTION	99.75
2.29-2.32	0.2498	131	0.2395	1460	X-RAY DIFFRACTION	99.69
2.32-2.35	0.2869	145	0.2478	1438	X-RAY DIFFRACTION	100
2.35-2.38	0.3335	156	0.2402	1449	X-RAY DIFFRACTION	99.94
2.38-2.42	0.2747	150	0.234	1468	X-RAY DIFFRACTION	100
2.42-2.46	0.2976	169	0.2417	1419	X-RAY DIFFRACTION	99.94
2.46-2.5	0.2649	160	0.2163	1440	X-RAY DIFFRACTION	99.94
2.5-2.54	0.2685	162	0.2139	1459	X-RAY DIFFRACTION	99.88
2.54-2.58	0.2601	152	0.2091	1461	X-RAY DIFFRACTION	100
2.58-2.63	0.2474	168	0.2105	1421	X-RAY DIFFRACTION	100
2.63-2.69	0.2411	159	0.2073	1433	X-RAY DIFFRACTION	99.94
2.69-2.75	0.2598	162	0.2096	1424	X-RAY DIFFRACTION	99.87
2.75-2.81	0.2828	170	0.2171	1460	X-RAY DIFFRACTION	99.88
2.81-2.88	0.2799	155	0.2122	1448	X-RAY DIFFRACTION	99.88
2.88-2.96	0.24	190	0.1998	1420	X-RAY DIFFRACTION	99.94
2.96-3.05	0.2704	169	0.2021	1425	X-RAY DIFFRACTION	99.94
3.05-3.14	0.2516	173	0.1993	1442	X-RAY DIFFRACTION	100
3.14-3.26	0.2062	163	0.1881	1450	X-RAY DIFFRACTION	100
3.26-3.39	0.2229	159	0.1825	1453	X-RAY DIFFRACTION	99.94
3.39-3.54	0.2073	158	0.1813	1467	X-RAY DIFFRACTION	100
3.54-3.73	0.1978	145	0.1714	1464	X-RAY DIFFRACTION	100
3.73-3.96	0.2065	183	0.1605	1422	X-RAY DIFFRACTION	100
3.96-4.27	0.1759	146	0.1541	1474	X-RAY DIFFRACTION	100
4.27-4.7	0.1773	167	0.1479	1452	X-RAY DIFFRACTION	100
4.7-5.37	0.1759	159	0.1585	1477	X-RAY DIFFRACTION	100
5.38-6.77	0.2222	176	0.1826	1465	X-RAY DIFFRACTION	100
6.77-10	0.1714	154	0.1714	1520	X-RAY DIFFRACTION	99.88

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	2.67801173343	-0.449326111352	1.7393011269	2.80454365116	0.649602410634	1.42274037074	0.142483293718	-0.405576834568	-0.455889788357	0.027095510234	-0.210536722314	0.421334445042	0.423937585433	-0.295031891349	-0.0274616159472	0.609835436654	-0.141353788586	0.225824440494	0.51066962645	0.0536853511775	0.370492486786	50.2757078452	-6.39649497278	5.38285052223
2	1.45924619823	-0.65889780583	-0.255757829638	1.65156424583	0.546379353156	2.3872103944	-0.0327879113025	0.0117223436555	0.169664736995	0.0517126656527	0.0400040341515	-0.154126873588	-0.115307330674	0.149292188206	-0.0067930117157	0.300445620447	-0.0114538074192	-0.0263241779578	0.23061573854	-0.0165464881356	0.337394886365	58.9324300045	19.2208227202	-3.22799201883
3	4.37736320073	-4.73125176754	-2.24504797867	9.72933061658	4.82586942517	4.81478909998	-0.341576521112	-0.276649585665	0.533430317072	0.260767002605	0.336941178832	-0.254765207072	0.126793868949	0.530233228181	-0.0204098871883	0.380683533813	-0.0117298861717	-0.0821215676166	0.438450628739	0.00257672774671	0.374152894212	77.1119288694	9.82609545503	8.86449217785
4	4.15129170968	0.0466473603184	1.75618014253	4.40813802715	-1.45278413444	7.45371279828	0.276848316233	0.0599986394316	-0.0921215489136	-0.485910952514	0.19559286893	0.448563835499	1.01601791422	-0.837001624241	-0.373374441477	0.462837481356	-0.01459414656	-0.0872013675977	0.496147566569	0.0292326790931	0.327418731152	73.5314159916	-6.47887825931	0.48609733018
5	5.16139677929	-2.55058996562	-1.79036279331	6.54175498629	5.16447151893	4.08269948573	0.217759418506	0.549457236575	-0.39769928555	-0.153358494844	-0.337191450182	0.598078345073	-0.285380211245	-0.463682942113	0.0719304231502	0.482491220551	0.111911072864	-0.00628945343173	0.286496512992	0.0766861357126	0.403713495516	41.9345590454	31.4520533841	-3.31867996556
6	3.37934545751	-3.26691289702	0.289091803733	5.15220182613	0.432265938662	0.279633881427	-0.380343226968	-0.0555923182819	-0.150659296041	0.299464689395	0.716351222566	-0.0261757834267	0.204867163575	0.444931973086	-0.318751978452	0.676648320997	0.0623965066374	0.173655007451	0.523638439442	-0.0230190317346	0.449066280223	37.2348145249	33.6798203286	20.9983177971
7	1.18777327086	-0.868454582109	-0.63036684277	1.49889538791	0.667201307365	1.41170874128	-0.325574519209	0.0381176074307	-0.406858985928	0.371580689065	-0.0292777789319	0.440425335628	0.424492729416	-0.169849655084	0.187484201519	0.498066066608	-0.0593843798686	0.151466779173	0.247048974962	-0.0570246858612	0.501011003812	32.7285653031	7.82773866264	11.1250404013
8	2.50044052769	-2.58250049336	-0.142505367501	4.58610394157	0.548688334816	1.10732412512	-0.256675207367	0.0753405524318	-0.170043121103	0.0188806991146	-0.00874574003431	0.262607449005	0.186047164714	-0.0405416480889	0.198765498977	0.407421553786	-0.0197972067288	0.14307956877	0.322786690435	-0.060119249905	0.355591790938	25.2658002457	30.2841189134	21.4561055526

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid -2 through 46 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 47 through 300 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 301 through 331 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 332 through 396 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid -2 through 22 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 23 through 46 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 47 through 300 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 301 through 396 )

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