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- PDB-3hbx: Crystal structure of GAD1 from Arabidopsis thaliana -

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Basic information

Entry
Database: PDB / ID: 3hbx
TitleCrystal structure of GAD1 from Arabidopsis thaliana
ComponentsGlutamate decarboxylase 1
KeywordsLYASE / Calmodulin-binding / Decarboxylase / Pyridoxal phosphate
Function / homology
Function and homology information


glutamate decarboxylase / glutamate decarboxylase activity / glutamate metabolic process / pyridoxal phosphate binding / molecular adaptor activity / calmodulin binding
Similarity search - Function
MYOD Basic-Helix-Loop-Helix Domain, subunit B - #50 / Aspartate Aminotransferase, domain 1 - #160 / Glutamate decarboxylase / MYOD Basic-Helix-Loop-Helix Domain, subunit B / Pyridoxal phosphate-dependent decarboxylase / Pyridoxal-dependent decarboxylase conserved domain / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / Pyridoxal phosphate-dependent transferase, major domain ...MYOD Basic-Helix-Loop-Helix Domain, subunit B - #50 / Aspartate Aminotransferase, domain 1 - #160 / Glutamate decarboxylase / MYOD Basic-Helix-Loop-Helix Domain, subunit B / Pyridoxal phosphate-dependent decarboxylase / Pyridoxal-dependent decarboxylase conserved domain / Aspartate Aminotransferase, domain 1 / Aspartate Aminotransferase; domain 2 / Type I PLP-dependent aspartate aminotransferase-like (Major domain) / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase / Few Secondary Structures / Irregular / Alpha-Beta Complex / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Glutamate decarboxylase 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.672 Å
AuthorsGut, H. / Dominici, P. / Pilati, S. / Gruetter, M.G. / Capitani, G.
CitationJournal: J Mol Biol / Year: 2009
Title: A common structural basis for pH- and calmodulin-mediated regulation in plant glutamate decarboxylase.
Authors: Heinz Gut / Paola Dominici / Stefania Pilati / Alessandra Astegno / Maxim V Petoukhov / Dmitri I Svergun / Markus G Grütter / Guido Capitani /
Abstract: Glutamate decarboxylase (Gad) catalyzes glutamate to gamma-aminobutyrate conversion. Plant Gad is a approximately 340 kDa hexamer, involved in development and stress response, and regulated by pH and ...Glutamate decarboxylase (Gad) catalyzes glutamate to gamma-aminobutyrate conversion. Plant Gad is a approximately 340 kDa hexamer, involved in development and stress response, and regulated by pH and binding of Ca(2+)/calmodulin (CaM) to the C-terminal domain. We determined the crystal structure of Arabidopsis thaliana Gad1 in its CaM-free state, obtained a low-resolution structure of the calmodulin-activated Gad complex by small-angle X-ray scattering and identified the crucial residues, in the C-terminal domain, for regulation by pH and CaM binding. CaM activates Gad1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa Gad1-CaM complex with an unusual 1:3 stoichiometry. The complex is loosely packed: thanks to the flexible linkers connecting the enzyme core with the six C-terminal regulatory domains, the CaM molecules retain considerable positional and orientational freedom with respect to Gad1. The complex thus represents a prototype for a novel CaM-target interaction mode. Thanks to its two levels of regulation, both targeting the C-terminal domain, Gad can respond flexibly to different kinds of cellular stress occurring at different pH values.
History
DepositionMay 5, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 1, 2017Group: Refinement description / Category: software
Revision 1.3Sep 6, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Nov 22, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate decarboxylase 1
B: Glutamate decarboxylase 1
C: Glutamate decarboxylase 1
D: Glutamate decarboxylase 1
E: Glutamate decarboxylase 1
F: Glutamate decarboxylase 1


Theoretical massNumber of molelcules
Total (without water)344,1746
Polymers344,1746
Non-polymers00
Water5,657314
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)118.098, 118.098, 200.576
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number145
Space group name H-MP32
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
31
41
51
61
12
22
32
42
52
62

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection details
111chain A and backbone and resseq 300:314
211chain B and backbone and resseq 300:314
311chain C and backbone and resseq 300:314
411chain D and backbone and resseq 300:314
511chain E and backbone and resseq 300:314
611chain F and backbone and resseq 300:314
112chain A and (resseq 12:111 or resseq 117:148 or resseq...
212chain B and (resseq 12:111 or resseq 117:148 or resseq...
312chain C and (resseq 12:111 or resseq 117:148 or resseq...
412chain D and (resseq 12:111 or resseq 117:148 or resseq...
512chain E and (resseq 12:111 or resseq 117:148 or resseq...
612chain F and (resseq 12:111 or resseq 117:148 or resseq...

NCS ensembles :
ID
1
2

NCS oper:
IDCodeMatrixVector
1given(0.318302, 0.947977, 0.004831), (0.947233, -0.317841, -0.041559), (-0.037861, 0.017805, -0.999124)-54.366699, 75.451401, -1.46108
2given(-0.503232, 0.864004, 0.015946), (-0.864137, -0.503243, -0.003575), (0.004936, -0.015578, 0.999866)-48.508598, 88.459702, 1.23684
3given(-0.979238, -0.202103, 0.015704), (-0.202182, 0.979341, -0.003624), (-0.014647, -0.006724, -0.99987)13.9204, 1.35622, 0.689961
4given(-0.497458, -0.867314, 0.017358), (0.867356, -0.497632, -0.007499), (0.015142, 0.011325, 0.999821)52.357201, 86.293503, -0.800898
5given(0.667823, -0.744318, 0.001814), (-0.744144, -0.667715, -0.020149), (0.016208, 0.012106, -0.999795)43.750401, 97.965698, -1.02356
6given(0.317723, 0.948059, -0.015386), (0.948068, -0.317896, -0.010433), (-0.014782, -0.011273, -0.999827)-54.468102, 75.690903, 0.624706
7given(-0.50029, 0.865677, 0.017686), (-0.865653, -0.500512, 0.011547), (0.018848, -0.009533, 0.999777)-48.814899, 88.352501, 0.401869
8given(-0.979966, -0.199142, 0.002942), (-0.199138, 0.97997, 0.001718), (-0.003225, 0.001098, -0.999994)13.7739, 1.29489, 0.050583
9given(-0.499153, -0.866332, 0.017746), (0.866352, -0.499351, -0.0091), (0.016745, 0.010832, 0.999801)52.1954, 86.476402, -0.712213
10given(0.664267, -0.74726, -0.018753), (-0.74729, -0.664464, 0.0068), (-0.017542, 0.009497, -0.999801)44.003899, 97.825996, -0.54785

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Components

#1: Protein
Glutamate decarboxylase 1 / GAD 1


Mass: 57362.293 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At5g17330, GAD, GAD1, GDH1, MKP11.30, MKP11_18 / Plasmid: pET12b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 pLysS / References: UniProt: Q42521, glutamate decarboxylase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 314 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.35 Å3/Da / Density % sol: 47.58 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 2 uL protein solution (1.9 mg/ml Gad1, 50 mM Hepes pH 7.2, 150 mM NaCl and 10 uM PLP) + 1 uL reservoir solution (100 mM Na acetate pH 5.5, 720 mM Na formate, 9 % PEG 8000 and 9 % PEG 1000), ...Details: 2 uL protein solution (1.9 mg/ml Gad1, 50 mM Hepes pH 7.2, 150 mM NaCl and 10 uM PLP) + 1 uL reservoir solution (100 mM Na acetate pH 5.5, 720 mM Na formate, 9 % PEG 8000 and 9 % PEG 1000), VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.90002 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Feb 26, 2005 / Details: DYNAMICALLY BENDABLE MIRROR
RadiationMonochromator: LN2 COOLED FIXED-EXIT SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.90002 Å / Relative weight: 1
ReflectionResolution: 2.65→30 Å / Num. obs: 85232 / % possible obs: 96.4 % / Observed criterion σ(I): -3 / Redundancy: 2 % / Biso Wilson estimate: 45.7 Å2 / Rsym value: 0.077 / Net I/σ(I): 8
Reflection shellResolution: 2.65→2.74 Å / Mean I/σ(I) obs: 1.7 / Rsym value: 0.459 / % possible all: 95.1

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHENIX1.4_29refinement
PDB_EXTRACT3.005data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1PMM (E. coli GadB)

1pmm


Resolution: 2.672→29.091 Å / Occupancy max: 1 / Occupancy min: 0 / SU ML: 0.29 / Isotropic thermal model: ISOTROPIC / σ(F): 1.51 / Phase error: 28.16 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2213 2223 2.61 %
Rwork0.208 --
obs0.2084 85127 96.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 28.074 Å2 / ksol: 0.342 e/Å3
Displacement parametersBiso mean: 47.553 Å2
Baniso -1Baniso -2Baniso -3
1--8.548 Å2-0 Å20 Å2
2---8.548 Å20 Å2
3---17.097 Å2
Refinement stepCycle: LAST / Resolution: 2.672→29.091 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21142 0 0 314 21456
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221624
X-RAY DIFFRACTIONf_angle_d0.60629356
X-RAY DIFFRACTIONf_dihedral_angle_d14.3357978
X-RAY DIFFRACTIONf_chiral_restr0.0433240
X-RAY DIFFRACTIONf_plane_restr0.0023788
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A60X-RAY DIFFRACTIONPOSITIONAL
12B60X-RAY DIFFRACTIONPOSITIONAL0.019
13C60X-RAY DIFFRACTIONPOSITIONAL0.015
14D60X-RAY DIFFRACTIONPOSITIONAL0.012
15E60X-RAY DIFFRACTIONPOSITIONAL0.012
16F60X-RAY DIFFRACTIONPOSITIONAL0.017
21A3202X-RAY DIFFRACTIONPOSITIONAL
22B3202X-RAY DIFFRACTIONPOSITIONAL0.009
23C3202X-RAY DIFFRACTIONPOSITIONAL0.008
24D3202X-RAY DIFFRACTIONPOSITIONAL0.008
25E3202X-RAY DIFFRACTIONPOSITIONAL0.007
26F3202X-RAY DIFFRACTIONPOSITIONAL0.009
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.672-2.73030.3526970.30294931X-RAY DIFFRACTION91
2.7303-2.79370.34611260.2875189X-RAY DIFFRACTION97
2.7937-2.86350.3089970.27855333X-RAY DIFFRACTION98
2.8635-2.94090.26831130.27565350X-RAY DIFFRACTION98
2.9409-3.02730.30891170.26555346X-RAY DIFFRACTION99
3.0273-3.1250.26591440.2545278X-RAY DIFFRACTION98
3.125-3.23650.25771780.24155246X-RAY DIFFRACTION98
3.2365-3.36590.24061500.22265338X-RAY DIFFRACTION98
3.3659-3.51880.22691300.20785253X-RAY DIFFRACTION98
3.5188-3.7040.23071680.19935262X-RAY DIFFRACTION97
3.704-3.93560.20261330.17945190X-RAY DIFFRACTION97
3.9356-4.23860.17811670.17775158X-RAY DIFFRACTION96
4.2386-4.66360.17751500.16435057X-RAY DIFFRACTION95
4.6636-5.33480.17191660.16745014X-RAY DIFFRACTION94
5.3348-6.70770.21451480.1815062X-RAY DIFFRACTION94
6.7077-29.09270.17141390.16124897X-RAY DIFFRACTION91

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