PDB-8e9v: Crystal structure of E. coli aspartate aminotransferase mutant VF...

ID or keywords:

Entry

Database: PDB / ID: 8e9v

Title

Crystal structure of E. coli aspartate aminotransferase mutant VFIT in the ligand-free form at 303 K

Components

Aspartate aminotransferase

Aspartate transaminase

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 aminotransferase activity / L-phenylalanine biosynthetic process /

aspartate transaminase /

L-aspartate:2-oxoglutarate aminotransferase activity /

pyridoxal phosphate binding / protein homodimerization activity / identical protein binding /

cytosol /

cytoplasm
Similarity search - Function

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.01 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	Oct 5, 2022	Provider: repository / Type: Initial release
Revision 1.1	Nov 2, 2022	Group: Database references / Category: citation Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2	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 1.3	Oct 18, 2023	Group: Refinement description / Category: pdbx_initial_refinement_model

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8e9v.cif.gz	547.9 KB	Display	PDBx/mmCIF format
PDB format	pdb8e9v.ent.gz	380.7 KB	Display	PDB format
PDBx/mmJSON format	8e9v.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/e9/8e9v ftp://data.pdbj.org/pub/pdb/validation_reports/e9/8e9v	HTTPS FTP

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Related structure data

Related structure data	8e9cC 8e9dC 8e9jC 8e9kC 8e9lC 8e9mC 8e9nC 8e9oC 8e9pC 8e9qC 8e9rC 8e9sC 8e9tC 8e9uC 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

90.3 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)	143.830, 143.830, 81.570
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 / Aspartate transaminase / AspAT / Transaminase A Mass: 44793.500 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source 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 / Pyridoxal phosphate Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₈H₁₀NO₆P
#3: Chemical	ChemComp-SO4 / SULFATE ION / Sulfate Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO₄
#4: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 222 / 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: 2.72 Å³/Da / Density % sol: 54.76 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / Details: HEPES, ammonium sulfate, PEG 400, maleate

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Data collection

Diffraction	Mean temperature: 303 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: 2.01→124.96 Å / Num. obs: 64155 / % possible obs: 100 % / Redundancy: 5 % / B_iso Wilson estimate: 34.87 Å² / CC_1/2: 0.993 / Net I/σ(I): 5.5
Reflection shell	Resolution: 2.01→2.04 Å / Num. unique obs: 3220 / CC_1/2: 0.357

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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: 2.01→71.92 Å / SU ML: 0.2595 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 20.7249
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.1961	6464	10.09 %
R_work	0.1684	57614	-
obs	0.1712	64078	99.75 %

Solvent computation

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

Displacement parameters

B_iso mean: 41.94 Å²

Refinement step

Cycle: LAST / Resolution: 2.01→71.92 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6118	0	0	222	6340

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0022	6377
X-RAY DIFFRACTION	f_angle_d	0.5169	8688
X-RAY DIFFRACTION	f_chiral_restr	0.0385	957
X-RAY DIFFRACTION	f_plane_restr	0.0028	1156
X-RAY DIFFRACTION	f_dihedral_angle_d	20.3589	2279

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 (%)
2.01-2.03	0.3709	214	0.329	1860	X-RAY DIFFRACTION	96.69
2.03-2.05	0.3372	212	0.3173	1912	X-RAY DIFFRACTION	99.95
2.05-2.08	0.3189	212	0.3093	1905	X-RAY DIFFRACTION	99.67
2.08-2.11	0.3033	216	0.3027	1904	X-RAY DIFFRACTION	99.72
2.11-2.13	0.3165	216	0.2975	1924	X-RAY DIFFRACTION	99.95
2.13-2.16	0.3031	216	0.2834	1904	X-RAY DIFFRACTION	99.95
2.16-2.19	0.2918	212	0.2684	1891	X-RAY DIFFRACTION	99.9
2.19-2.23	0.2575	233	0.2588	1914	X-RAY DIFFRACTION	99.91
2.23-2.26	0.3007	234	0.2418	1908	X-RAY DIFFRACTION	99.86
2.26-2.3	0.2743	234	0.2347	1887	X-RAY DIFFRACTION	100
2.3-2.34	0.2584	188	0.2268	1938	X-RAY DIFFRACTION	100
2.34-2.38	0.2459	187	0.212	1925	X-RAY DIFFRACTION	99.95
2.38-2.43	0.2504	204	0.2049	1950	X-RAY DIFFRACTION	99.81
2.43-2.48	0.2301	218	0.1996	1927	X-RAY DIFFRACTION	99.81
2.48-2.53	0.2509	211	0.206	1900	X-RAY DIFFRACTION	99.91
2.53-2.59	0.2304	208	0.1975	1943	X-RAY DIFFRACTION	99.77
2.59-2.65	0.2496	219	0.1966	1901	X-RAY DIFFRACTION	99.72
2.65-2.72	0.2306	212	0.1874	1898	X-RAY DIFFRACTION	99.01
2.72-2.8	0.2121	225	0.1784	1915	X-RAY DIFFRACTION	99.91
2.8-2.9	0.2348	203	0.1654	1927	X-RAY DIFFRACTION	99.95
2.9-3	0.1925	248	0.1621	1908	X-RAY DIFFRACTION	100
3-3.12	0.2165	233	0.1659	1915	X-RAY DIFFRACTION	99.91
3.12-3.26	0.1975	221	0.1599	1894	X-RAY DIFFRACTION	100
3.26-3.43	0.1787	208	0.1504	1954	X-RAY DIFFRACTION	99.95
3.43-3.65	0.1542	205	0.1304	1920	X-RAY DIFFRACTION	99.95
3.65-3.93	0.151	214	0.1222	1955	X-RAY DIFFRACTION	100
3.93-4.32	0.1428	210	0.1123	1933	X-RAY DIFFRACTION	100
4.33-4.95	0.1312	233	0.1102	1930	X-RAY DIFFRACTION	100
4.95-6.24	0.1625	210	0.1404	1969	X-RAY DIFFRACTION	100
6.24-71.92	0.1395	208	0.1482	2003	X-RAY DIFFRACTION	99.64

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	0.820464459406	-1.05997111253	0.922065242179	3.59665278373	-1.03987530144	1.01513263895	-0.266083264172	-0.304676907256	0.169978597373	0.113781670339	0.156575979706	0.294883546156	-0.0268261114008	-0.324774645995	0.09260811202	0.404381322035	-0.0129819588365	0.000161721937163	0.375915110723	-0.0151127618033	0.457990472489	21.0743259713	-50.72385848	7.19435771865
2	3.08335080085	2.64616820625	1.84592212687	7.05819334442	3.21960903786	3.93646541349	0.152701497742	-0.242489347472	-0.17746753654	0.588426096765	-0.172185026214	-0.351785608052	0.266214248163	-0.229610406858	-0.0151468233069	0.393282017346	-0.00314665115151	-0.023018388643	0.246544963651	0.0413542488248	0.24460988331	44.2079381719	-50.2026312639	21.2233479514
3	0.530575979607	0.0649123698848	-0.25070996851	1.66201558017	0.582140889116	1.97313603718	0.0114426715468	-0.0306233263902	0.0885996867335	0.0156571337054	0.0503511155744	-0.187362079968	-0.02419559517	0.219249433958	-0.0673856721707	0.241663724581	-0.0462951407357	-0.0127033849686	0.238591525232	-0.000660803075147	0.271292782133	47.7080579438	-36.2697358197	1.5236693082
4	0.670675484585	0.184241866858	0.178830772631	1.48226935911	0.695038792075	2.61556554067	-0.00375331713563	0.0905555014786	-0.0530578411453	-0.111835554792	0.0719733463576	-0.139865724678	0.137033555724	0.197021011648	-0.0633005737624	0.243960472955	-0.0267517551819	-0.00803955824677	0.24290319032	-0.00935824609852	0.265790327061	46.4208150817	-44.9473743675	-4.35826160813
5	4.41123175341	4.55212081434	2.48465253754	7.83241661276	3.33627716035	3.31055084457	0.150519445242	0.0109531143406	-0.218149471468	0.483545181028	-0.0252514124078	-0.638068384636	0.422412791081	0.014936799396	-0.128835809164	0.374183600974	0.0683462150789	-0.0323512956042	0.25232632763	0.0328246842081	0.271733149461	47.8342404452	-62.7142898454	11.0658206725
6	1.7070426992	0.316982791039	-0.333737506983	3.06346479284	-0.570442658366	2.24297550877	0.0244858425639	0.162573184593	-0.0138987452994	-0.192906264109	0.122856673729	0.151116852629	0.163065531216	-0.426121197428	-0.176707487859	0.426406762291	-0.0148340218329	-0.0238033231345	0.372188441523	0.0129013770867	0.301736857927	31.809816152	-67.3733197971	2.81012685813
7	0.319004204262	0.665245457073	0.970285054449	2.20400531763	2.27622914724	2.97220320438	-0.217246591485	0.0284738950718	0.0400580629118	-0.299675254974	0.0563015521746	0.0833432051736	0.00121863369894	0.289473750824	0.149044043239	0.441204084606	-0.0412135369767	0.0202290979777	0.393775796813	-0.0203288631279	0.411204208375	50.2838801424	-17.3769990984	6.95452853299
8	1.41798396501	0.794987638521	-0.390271183809	7.77598946749	-0.937913464986	1.16308992501	0.199280958049	-0.292132623458	0.0561625635473	0.843292445037	-0.348913160539	-0.204887271288	0.117563655379	-0.0769133721269	0.136440133302	0.412835647493	-0.0665322742327	-0.0391098766149	0.354273722701	-0.0448424872724	0.245737791737	41.3414174249	-29.5478641712	29.2439055994
9	0.891284452554	-0.207403514332	0.455847473449	2.25703892721	0.617325051799	1.25529501202	0.0541142047263	-0.0966268179852	-0.0693447937589	0.452498532354	-0.300782299244	0.568017025397	0.13310197658	-0.368845141914	0.205387177633	0.340450003124	-0.128908079395	0.0828353461573	0.392945979227	-0.0743398164703	0.40727024542	22.6093030649	-37.1251391234	18.5707876742
10	1.8100815996	-0.326931036923	-0.137009746139	3.51527733917	0.770506688504	1.85070549176	-0.131246177446	0.110263927441	0.444880067115	-0.487902490392	-0.133033990625	0.499208012643	-0.502992202613	-0.236371221271	0.201005659457	0.389969522207	0.0243322396441	-0.139601879844	0.312030739947	-0.0240235900253	0.481614002484	24.9330255159	-15.5355967338	4.5509031213
11	2.14053144196	0.272693916536	0.685639741233	2.47187695757	0.469078987707	2.14491927616	-0.0824985213022	-0.0646639880331	0.341485073166	-0.081578804646	-0.147267673216	0.720398665656	-0.257712147773	-0.517240593756	0.282972461147	0.31067823446	0.045326858694	-0.0518630414377	0.374785494658	-0.123716303602	0.580580860393	19.7728509247	-15.7598415989	14.276093944
12	0.727468384356	0.132846757159	0.315589781339	1.69634923403	0.585083746158	1.68534751949	0.041633678724	-0.177744916031	0.125014307839	0.187120406874	-0.241660090904	0.487404510289	0.111603478777	-0.417818231381	0.153182786062	0.257193313606	-0.0613433917661	0.0185639541263	0.339576885684	-0.0852043575035	0.385242965724	24.3321746872	-31.3998132253	15.0816967091
13	2.67905714963	2.92039570517	-0.519059627804	3.18554251731	-0.657847505355	0.917452008135	0.28139629754	-0.218857408784	0.398935170322	0.968982356777	-0.308838612818	0.762765989495	0.0806299985702	-0.168467885013	-0.0315769648045	0.430590597138	-0.0200519765863	0.0544449889137	0.397662625303	-0.122876277276	0.372079363047	35.0916355614	-13.6020818838	31.9426046776
14	3.02330405649	0.66591606104	0.122461240377	2.30596468343	-0.0656109358012	2.17183260042	-0.0664102211564	0.269163488673	0.280418485713	-0.337409032586	-0.0833928968294	0.317425765023	-0.208730118899	-0.129119595149	0.0629206812969	0.380528930825	0.0293504653325	-0.0573976004058	0.294897169887	-0.0578072702554	0.41996860537	40.9401042783	-1.85876490499	18.0095733375
15	2.20376720877	0.682667052852	-0.159055638592	2.54269416978	0.118838428723	1.81212540728	0.0661558658675	0.110650507188	0.0335338416087	-0.074162718318	0.0508985920061	0.118004597893	0.0377493639714	0.0666686938833	0.0332309506181	0.304093994035	-0.0111635931342	-0.0117722601323	0.305472400315	-0.0655504377113	0.321656472308	45.5785966708	-5.3022824815	21.6586305416

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 0 through 32 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 33 through 59 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 60 through 130 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 131 through 300 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 301 through 331 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 332 through 396 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 0 through 32 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 33 through 59 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 60 through 101 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 102 through 168 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 169 through 222 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 223 through 300 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 301 through 331 )
14	X-RAY DIFFRACTION	14	chain 'B' and (resid 332 through 365 )
15	X-RAY DIFFRACTION	15	chain 'B' and (resid 366 through 396 )

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