PDB-9fxd: Structure of indole-3-acetic acid-amido synthetase GH3.6 from A.t...

ID or keywords:

Entry

Database: PDB / ID: 9fxd

Title

Structure of indole-3-acetic acid-amido synthetase GH3.6 from A.thaliana in complex with AMP and aspartate

Components

Indole-3-acetic acid-amido synthetase GH3.6

Keywords

LIGASE / Enzyme complex / AMP / aspartate

Function / homology

Function and homology information

indole-3-acetic acid amido synthetase activity / unidimensional cell growth / Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases) / response to auxin / auxin-activated signaling pathway / cytoplasm
Similarity search - Function

Biological species

Arabidopsis thaliana (thale cress)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.736 Å

Authors

Kopecny, D. / Briozzo, P.

Funding support

Czech Republic,

France, 2items

Organization	Grant number	Country
Palacky University in Olomouc	IGA_PrF_2023_012	Czech Republic
Agence Nationale de la Recherche (ANR)	Jean d'Alembert fellowship as part of France 2030 program ANR-11-IDEX-0003	France

Citation

Journal: J.Exp.Bot. / Year: 2025
Title: Phenylacetic acid metabolism in land plants: novel pathways and metabolites.
Authors: Hladik, P. / Brunoni, F. / Zukauskaite, A. / Zatloukal, M. / Belicek, J. / Kopecny, D. / Briozzo, P. / Ferchaud, N. / Novak, O. / Pencik, A.

History

Deposition	Jul 1, 2024	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Apr 9, 2025	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	9fxd.cif.gz	681.2 KB	Display	PDBx/mmCIF format
PDB format	pdb9fxd.ent.gz	571.7 KB	Display	PDB format
PDBx/mmJSON format	9fxd.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/fx/9fxd ftp://data.pdbj.org/pub/pdb/validation_reports/fx/9fxd	HTTPS FTP

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Related structure data

Related structure data	9fwdC 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	#171 - Mar 2014 Neurotransmitter Transporters similarity (1) #129 - Sep 2010 Isocitrate Dehydrogenase similarity (1) #154 - Oct 2012 Citric Acid Cycle similarity (1) #24 - Dec 2001 Glycogen Phosphorylase similarity (1) #55 - Jul 2004 DNA Ligase similarity (1) #16 - Apr 2001 Aminoacyl-tRNA Synthetases similarity (1)

Deposited unit

A: Indole-3-acetic acid-amido synthetase GH3.6

B: Indole-3-acetic acid-amido synthetase GH3.6

hetero molecules

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

1

A: Indole-3-acetic acid-amido synthetase GH3.6

hetero molecules

defined by author
Evidence: gel filtration
70.5 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Indole-3-acetic acid-amido synthetase GH3.6

hetero molecules

defined by author
70.5 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	197.886, 197.886, 65.284
Angle α, β, γ (deg.)	90, 90, 120
Int Tables number	172
Space group name H-M	P6₄

#1: Protein	Indole-3-acetic acid-amido synthetase GH3.6 / Auxin-responsive GH3-like protein 6 / AtGH3-6 / Protein DWARF IN LIGHT 1 / DFL-1 Mass: 70001.453 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: N-terminal 6xHis-tag / Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: GH3.6, DFL1, At5g54510, F24B18.13 / Plasmid: pETM11 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 References: UniProt: Q9LSQ4, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)
#2: Chemical	ChemComp-AMP / ADENOSINE MONOPHOSPHATE Mass: 347.221 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C₁₀H₁₄N₅O₇P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#3: Chemical	ChemComp-ASP / ASPARTIC ACID Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₄H₇NO₄ / Feature type: SUBJECT OF INVESTIGATION
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 676 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y
Has protein modification	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: 2.77 Å³/Da / Density % sol: 55.62 %
Crystal grow	Temperature: 294 K / Method: vapor diffusion, hanging drop / pH: 6.5 Details: Starting drop: 100 mM MES, pH 6.5, 0.6 M NaCl, 18% PEG 4000, 10 mM AMP and 1 mM Asp; protein: AtGH3.6 at 7.1 mg/ml in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2 and 1% glycerol

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

SOLEIL

/ Beamline: PROXIMA 1 / Wavelength: 0.987 Å

Detector

Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 26, 2023

Radiation

Monochromator: Kirkpatrick-Baez pair of bi-morph mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.987 Å / Relative weight: 1

Reflection

Resolution: 1.736→98.943 Å / Num. obs: 96838 / % possible obs: 96 % / Redundancy: 15.22 %
Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last ...Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last updated 2020-04-13: http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v50.dic/Index/) and the actual quantities provided by MRFANA (https://github.com/githubgphl/MRFANA) from the autoPROC package (https://www.globalphasing.com/autoproc/). In general, the mmCIF categories here should provide items that are currently used in the PDB archive. If there are alternatives, the one recommended by the PDB developers has been selected. The distinction between *_all and *_obs quantities is not always clear: often only one version is actively used within the PDB archive (or is the one recommended by PDB developers). The intention of distinguishing between classes of reflections before and after some kind of observation criterion was applied, can in principle be useful - but such criteria change in various ways throughout the data processing steps (rejection of overloaded or too partial reflections, outlier/misfit rejections during scaling etc) and there is no retrospect computation of data scaling/merging statistics for the reflections used in the final refinement (where another observation criterion might have been applied). Typical data processing will usually only provide one version of statistics at various stages and these are given in the recommended item here, irrespective of the "_all" and "_obs" connotation, see e.g. the use of _reflns.pdbx_Rmerge_I_obs, _reflns.pdbx_Rrim_I_all and _reflns.pdbx_Rpim_I_all. Please note that all statistics related to "merged intensities" (or "merging") are based on inverse-variance weighting of the individual measurements making up a symmetry-unique reflection. This is standard for several decades now, even if some of the dictionary definitions seem to suggest that a simple "mean" or "average" intensity is being used instead. R-values are always given for all symmetry-equivalent reflections following Friedel's law, i.e. Bijvoet pairs are not treated separately (since we want to describe the overall mean intensity and not the mean I(+) and I(-) here). The Rrim metric is identical to the Rmeas R-value and only differs in name. _reflns.pdbx_number_measured_all is the number of measured intensities just before the final merging step (at which point no additional rejection takes place). _reflns.number_obs is the number of symmetry-unique observations, i.e. the result of merging those measurements via inverse-variance weighting. _reflns.pdbx_netI_over_sigmaI is based on the merged intensities (_reflns.number_obs) as expected. _reflns.pdbx_redundancy is synonymous with "multiplicity". The per-shell item _reflns_shell.number_measured_all corresponds to the overall value _reflns.pdbx_number_measured_all. The per-shell item _reflns_shell.number_unique_all corresponds to the overall value _reflns.number_obs. The per-shell item _reflns_shell.percent_possible_all corresponds to the overall value _reflns.percent_possible_obs. The per-shell item _reflns_shell.meanI_over_sigI_obs corresponds to the overall value given as _reflns.pdbx_netI_over_sigmaI. But be aware of the incorrect definition of the former in the current dictionary!
CC_1/2: 0.999 / CC_1/2 anomalous: -0.108 / R_merge(I) obs: 0.1607 / R_pim(I) all: 0.0425 / R_rim(I) all: 0.1663 / AbsDiff over sigma anomalous: 0.737 / B_aniso tensor eigenvalue 1: 39.1 Å² / B_aniso tensor eigenvalue 2: 39.1 Å² / B_aniso tensor eigenvalue 3: 21.6 Å² / B_aniso tensor eigenvector 1 ortho1: 1 / B_aniso tensor eigenvector 1 ortho2: 0 / B_aniso tensor eigenvector 1 ortho3: 0 / B_aniso tensor eigenvector 2 ortho1: 0 / B_aniso tensor eigenvector 2 ortho2: 1 / B_aniso tensor eigenvector 2 ortho3: 0 / B_aniso tensor eigenvector 3 ortho1: 0 / B_aniso tensor eigenvector 3 ortho2: 0 / B_aniso tensor eigenvector 3 ortho3: 1 / Aniso diffraction limit 1: 2.13 Å / Aniso diffraction limit 2: 2.13 Å / Aniso diffraction limit 3: 1.727 Å / Aniso diffraction limit axis 1 ortho1: 1 / Aniso diffraction limit axis 1 ortho2: 0 / Aniso diffraction limit axis 1 ortho3: 0 / Aniso diffraction limit axis 2 ortho1: 0 / Aniso diffraction limit axis 2 ortho2: 1 / Aniso diffraction limit axis 2 ortho3: 0 / Aniso diffraction limit axis 3 ortho1: 0 / Aniso diffraction limit axis 3 ortho2: 0 / Aniso diffraction limit axis 3 ortho3: 1 / Net I/σ(I): 11.26 / Num. measured all: 1474201 / Observed signal threshold: 1.2 / Orthogonalization convention: pdb / % possible anomalous: 96 / % possible ellipsoidal: 96 / % possible ellipsoidal anomalous: 96 / % possible spherical: 64.2 / % possible spherical anomalous: 64.2 / Redundancy anomalous: 7.76 / Signal type: local

Reflection shell

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. measured all	Num. measured obs	Num. unique all	Num. unique obs	CC_1/2	CC_1/2 anomalous	R_pim(I) all	R_rim(I) all	AbsDiff over sigma anomalous	% possible anomalous	% possible ellipsoidal	% possible ellipsoidal anomalous	% possible spherical	% possible spherical anomalous	Redundancy anomalous	% possible all
5.537-98.943	15.26	0.0634	32.67	73913	73913	4842	4842	0.999	-0.179	0.0165	0.0656	0.627	99.9	100	99.9	100	99.9	8.14	100
4.377-5.537	14.29	0.0774	29.81	69158	69158	4841	4841	0.998	-0.213	0.0209	0.0803	0.703	100	100	100	100	100	7.44	100
3.814-4.377	13.46	0.0858	26.84	65204	65204	4843	4843	0.998	-0.143	0.0238	0.0891	0.729	100	100	100	100	100	6.94	100
3.462-3.814	14.4	0.1051	23.07	69707	69707	4842	4842	0.998	-0.059	0.0287	0.109	0.785	100	100	100	100	100	7.4	100
3.211-3.462	15.38	0.1327	19.58	74474	74474	4842	4842	0.997	-0.024	0.0351	0.1373	0.775	100	100	100	100	100	7.87	100
3.02-3.211	15.91	0.1764	15.89	77031	77031	4841	4841	0.995	-0.081	0.0458	0.1823	0.772	100	100	100	100	100	8.13	100
2.866-3.02	16.31	0.2198	13.01	78964	78964	4842	4842	0.994	-0.028	0.0563	0.227	0.774	100	100	100	100	100	8.32	100
2.74-2.866	16.49	0.2662	11.11	79819	79819	4841	4841	0.992	-0.036	0.0676	0.2747	0.765	100	100	100	100	100	8.41	100
2.634-2.74	16.63	0.3443	8.88	80559	80559	4843	4843	0.988	-0.063	0.087	0.3552	0.755	100	100	100	100	100	8.48	100
2.542-2.634	16.72	0.4048	7.64	80924	80924	4841	4841	0.983	-0.044	0.1018	0.4175	0.737	100	100	100	100	100	8.51	100
2.462-2.542	16.54	0.482	6.52	80172	80172	4847	4847	0.977	-0.001	0.1218	0.4972	0.746	100	100	100	100	100	8.43	100
2.391-2.462	14.9	0.5452	5.37	72060	72060	4836	4836	0.967	-0.036	0.1458	0.5646	0.728	99.9	99.9	99.9	99.9	99.9	7.59	99.9
2.327-2.391	15.52	0.6693	4.65	75145	75145	4841	4841	0.95	-0.02	0.1749	0.6919	0.741	100	100	100	100	100	7.91	100
2.27-2.327	15.33	0.7636	4.08	74330	74330	4848	4848	0.944	-0.019	0.2008	0.7899	0.731	100	100	100	100	100	7.81	100
2.218-2.27	14.37	0.8752	3.42	69521	69521	4837	4837	0.913	-0.016	0.2387	0.9075	0.74	100	100	100	100	100	7.31	100
2.17-2.218	15.18	1.0432	3.01	73495	73495	4843	4843	0.899	-0.006	0.2756	1.0793	0.732	99.6	99	99.6	99	99.6	7.7	99
2.121-2.17	15.67	1.2011	2.75	75855	75855	4840	4840	0.865	-0.028	0.3117	1.2412	0.722	92.2	90.4	92.2	88.7	91	7.87	90.4
2.058-2.121	15.4	1.2395	2.62	74609	74609	4844	4844	0.854	0.006	0.3239	1.2814	0.729	90	90	90	62.2	64	7.71	90
1.967-2.058	14.42	1.2408	2.45	69776	69776	4840	4840	0.824	0.015	0.3358	1.2858	0.725	84.3	84.3	84.3	37	38.2	7.21	84.3
1.736-1.967	12.28	1.4285	1.88	59485	59485	4844	4844	0.705	0.015	0.4209	1.49	0.721	70.3	70.3	70.3	10.3	10.6	6.14	70.3

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Processing

Software

Name	Version	Classification
MxCuBE		data collection
BUSTER	2.10.4	refinement
autoPROC	1.0.5 20211020	data processing
STARANISO		data scaling
Aimless	0.7.7	data scaling
autoPROC	1.0.5 20211020	data reduction
PHASER		phasing
Coot		model building
XDS		data reduction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.736→25.3 Å / Cor.coef. F_o:F_c: 0.942 / Cor.coef. F_o:F_c free: 0.934 / SU R Cruickshank DPI: 0.282 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.169 / SU R_free Blow DPI: 0.143 / SU R_free Cruickshank DPI: 0.143

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.2215	4781	-	RANDOM
R_work	0.1976	-	-	-
obs	0.1988	96781	64.3 %	-

Displacement parameters

B_iso mean: 33.44 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.8542 Å²	0 Å²	0 Å²
2-	-	0.8542 Å²	0 Å²
3-	-	-	-1.7083 Å²

Refine analyze

Luzzati coordinate error obs: 0.26 Å

Refinement step

Cycle: LAST / Resolution: 1.736→25.3 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	9280	0	64	676	10020

Refine LS restraints

Refine-ID	Type	Dev ideal	Number	Restraint function	Weight
X-RAY DIFFRACTION	t_bond_d	0.009	18775	HARMONIC	2
X-RAY DIFFRACTION	t_angle_deg	1.06	33950	HARMONIC	2
X-RAY DIFFRACTION	t_dihedral_angle_d		5604	SINUSOIDAL	2
X-RAY DIFFRACTION	t_gen_planes		2911	HARMONIC	5
X-RAY DIFFRACTION	t_it		18775	HARMONIC	10
X-RAY DIFFRACTION	t_chiral_improper_torsion		1250	SEMIHARMONIC	5
X-RAY DIFFRACTION	t_ideal_dist_contact		16757	SEMIHARMONIC	4
X-RAY DIFFRACTION	t_omega_torsion	3.83
X-RAY DIFFRACTION	t_other_torsion	15.73

LS refinement shell

Resolution: 1.74→1.89 Å

	R_factor	Num. reflection	% reflection
R_free	0.2183	109	-
R_work	0.2445	-	-
obs	0.243	1936	5.84 %

Refinement TLS params.

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	0.535	0.0529	0.2986	0.2434	0.1257	1.6896	0.0369	-0.0125	0.0749	-0.0125	-0.0339	-0.0751	0.0749	-0.0751	-0.0031	-0.2155	-0.043	0.0162	-0.2008	-0.0021	-0.2514	-39.2085	43.1193	-2.3321
2	0.2641	0.0545	-0.0552	0.3084	-0.3385	1.2736	0.0076	-0.0191	0.2059	-0.0191	0.0317	-0.1019	0.2059	-0.1019	-0.0392	-0.1597	-0.0546	-0.0019	-0.2499	0.0002	-0.245	-19.3212	88.2317	-22.4785

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	{ A\|* }
2	X-RAY DIFFRACTION	2	{ B\|* }

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