PDB-8e9l: Crystal structure of E. coli aspartate aminotransferase mutant VF...

ID or keywords:

Entry

Database: PDB / ID: 8e9l

Title

Crystal structure of E. coli aspartate aminotransferase mutant VFIT in the ligand-free form at 278 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 / cytosol / cytoplasm
Similarity search - Function

Biological species

Escherichia coli (E. coli)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.31 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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PDBx/mmCIF format	8e9l.cif.gz	538 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	8e9l_validation.pdf.gz	454.8 KB	Display	wwPDB validaton report
Full document	8e9l_full_validation.pdf.gz	455.6 KB	Display
Data in XML	8e9l_validation.xml.gz	28.9 KB	Display
Data in CIF	8e9l_validation.cif.gz	40.7 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/e9/8e9l ftp://data.pdbj.org/pub/pdb/validation_reports/e9/8e9l	HTTPS FTP

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Related structure data

Related structure data	8e9cC 8e9dC 8e9jC 8e9kC 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

90.3 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 / AspAT / Transaminase A Mass: 44793.500 Da / Num. of mol.: 2 / Mutation: K37F, T43I, N64T 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 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.: 157 / 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, maleate, PEG 400, ammonium sulfate

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Data collection

Diffraction	Mean temperature: 278 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: 2.31→124.38 Å / Num. obs: 42181 / % possible obs: 100 % / Redundancy: 10.3 % / B_iso Wilson estimate: 37.64 Å² / CC_1/2: 0.982 / R_merge(I) obs: 0.2 / Net I/σ(I): 3.3
Reflection shell	Resolution: 2.31→2.35 Å / Num. unique obs: 2069 / CC_1/2: 0.295

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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: 2.31→124.38 Å / SU ML: 0.3344 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 22.4923
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2218	4197	9.96 %
R_work	0.1829	37946	-
obs	0.1868	42143	99.6 %

Solvent computation

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

Displacement parameters

B_iso mean: 43.93 Å²

Refinement step

Cycle: LAST / Resolution: 2.31→124.38 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6105	0	0	157	6262

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0034	6293
X-RAY DIFFRACTION	f_angle_d	0.4998	8571
X-RAY DIFFRACTION	f_chiral_restr	0.0388	952
X-RAY DIFFRACTION	f_plane_restr	0.0027	1136
X-RAY DIFFRACTION	f_dihedral_angle_d	20.773	2227

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 (%)
2.31-2.34	0.3637	102	0.3229	1155	X-RAY DIFFRACTION	89.09
2.34-2.36	0.3574	125	0.3215	1295	X-RAY DIFFRACTION	99.86
2.36-2.39	0.3447	131	0.2994	1270	X-RAY DIFFRACTION	100
2.39-2.42	0.3212	129	0.3016	1255	X-RAY DIFFRACTION	100
2.42-2.45	0.3073	136	0.2823	1240	X-RAY DIFFRACTION	99.71
2.45-2.49	0.3231	152	0.2812	1260	X-RAY DIFFRACTION	100
2.49-2.52	0.3163	135	0.2813	1269	X-RAY DIFFRACTION	99.93
2.52-2.56	0.3158	142	0.277	1262	X-RAY DIFFRACTION	100
2.56-2.6	0.2781	138	0.265	1252	X-RAY DIFFRACTION	99.93
2.6-2.64	0.2998	138	0.2779	1284	X-RAY DIFFRACTION	100
2.64-2.69	0.3495	134	0.2548	1239	X-RAY DIFFRACTION	100
2.69-2.74	0.2889	159	0.2475	1259	X-RAY DIFFRACTION	100
2.74-2.79	0.2582	138	0.2471	1272	X-RAY DIFFRACTION	100
2.79-2.85	0.2844	144	0.2509	1263	X-RAY DIFFRACTION	100
2.85-2.91	0.3359	131	0.236	1276	X-RAY DIFFRACTION	100
2.91-2.98	0.2611	160	0.2143	1235	X-RAY DIFFRACTION	100
2.98-3.05	0.2529	157	0.2137	1267	X-RAY DIFFRACTION	100
3.05-3.13	0.2799	157	0.2058	1239	X-RAY DIFFRACTION	100
3.14-3.23	0.23	148	0.1967	1258	X-RAY DIFFRACTION	100
3.23-3.33	0.2411	143	0.1796	1254	X-RAY DIFFRACTION	100
3.33-3.45	0.2235	135	0.1753	1288	X-RAY DIFFRACTION	100
3.45-3.59	0.2229	130	0.1694	1286	X-RAY DIFFRACTION	100
3.59-3.75	0.1811	127	0.1516	1272	X-RAY DIFFRACTION	100
3.75-3.95	0.1766	159	0.1312	1261	X-RAY DIFFRACTION	100
3.95-4.2	0.1738	139	0.1198	1265	X-RAY DIFFRACTION	100
4.2-4.52	0.1668	143	0.1183	1288	X-RAY DIFFRACTION	100
4.52-4.98	0.1413	145	0.1123	1270	X-RAY DIFFRACTION	100
4.98-5.7	0.172	137	0.132	1291	X-RAY DIFFRACTION	100
5.7-7.18	0.153	153	0.1557	1276	X-RAY DIFFRACTION	100
7.18-124.38	0.1635	130	0.1394	1345	X-RAY DIFFRACTION	99.8

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	5.5116467054	0.491922109443	2.30882362909	0.665540722787	0.592656333268	1.6057787311	0.0871512208858	-0.677816089933	-0.0998023933553	0.0429401344799	-0.161738564034	0.277371563784	0.346972024031	-0.285409663879	0.0486897598322	0.444747521711	-0.0610888559475	0.0201202958414	0.446763897675	-0.00971572553893	0.415765232665	53.7985273873	-7.14111894742	5.0170890999
2	5.15703997705	-2.8596565915	-1.37089814846	6.92137284444	2.53316810768	3.79216603969	-0.25778055407	-0.434200363906	0.172293514732	0.546918113881	0.21001703703	-0.31256925334	0.361834275404	0.0519134945017	0.0439423629503	0.343007275655	0.02898757685	-0.0470130025389	0.333015924615	0.0224617706087	0.217884723349	66.271158234	12.8673628686	18.9702102942
3	1.46648297025	-0.38533778396	-0.495578030568	1.25905102273	0.33871825727	2.61721890242	0.00310150603647	-0.00990744606108	0.129329466788	-0.0626411500314	0.0382765950883	-0.106557375768	-0.0358488203665	0.164776916814	-0.0431582464244	0.26366034917	0.0107132485283	-0.00978150479039	0.222433054564	-0.0204967409352	0.26795677667	60.1400268325	19.2613852633	-4.81175390705
4	1.68761808542	-1.18184532374	0.0201234087702	2.31631421825	-0.4995937426	1.23482500978	0.0345918069781	-0.0258428104792	0.0467958026481	-0.273008577171	0.0105469344649	0.0718717035558	0.281746040621	0.0617748442966	-0.0354814692797	0.298525374813	-0.00423550602052	-0.0204635965736	0.407893616339	-0.000591118056849	0.270007357133	75.8612720101	-0.57486789622	3.57969124407
5	1.7249972605	-1.24586694741	-2.23213534627	1.15807031177	1.58224188775	5.044362869	0.173662445528	0.163114602982	-0.0339384310022	-0.410634112984	-0.0959198287426	0.122780765411	-0.418389985498	0.253472592227	-0.0575209786589	0.495664548905	0.0185301300138	0.0505089906576	0.405067100952	0.0323807087141	0.412567235897	40.5317557164	34.8114149987	4.20060777295
6	2.10045160849	-0.367437923014	-0.116520247923	1.70336399346	0.69950124953	1.53619553937	-0.16009344008	-0.0261394563578	-0.280609400613	0.137680654326	-0.0588590884762	0.38440380834	0.20970847534	-0.225834493841	0.201838899964	0.305573693102	-0.0128658800445	0.064182578992	0.242371565064	-0.0291899287464	0.338740217752	34.0458626103	9.40562386565	12.2647362603
7	5.22335588271	-5.84714820475	-0.311124356842	8.35784986156	0.170510501854	0.799812480974	-0.727128121565	-0.785310126975	-0.575969790631	0.989656331291	0.694884921994	0.75296080482	0.246541546055	0.0203016400974	0.000535661535531	0.446276830958	0.00433913749949	0.124649296061	0.471609122262	0.00533211338987	0.330480220877	29.6392282837	23.4426462357	29.7968671854
8	2.11801736107	0.0486322203053	0.042328461368	3.89099473327	0.0671225611902	2.69510176221	-0.00933630130414	0.157241072247	-0.0230333836471	-0.315221607469	-0.0222449689522	0.258721061835	0.0545434254796	-0.0684071373013	0.0297401139019	0.347439537657	0.00822642124625	0.0439786051998	0.299498713109	-0.0514489136713	0.293311365918	24.4563512422	35.65619893	17.5362863281

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid -1 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 300 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 301 through 396 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid -1 through 32 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 33 through 300 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 301 through 331 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 332 through 396 )

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