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- PDB-8own: CryoEM structure of glutamate dehydrogenase isoform 2 from Arabid... -

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Basic information

Entry
Database: PDB / ID: 8own
TitleCryoEM structure of glutamate dehydrogenase isoform 2 from Arabidopsis thaliana in apo-form
ComponentsGlutamate dehydrogenase 2
KeywordsOXIDOREDUCTASE / glutamic acid / calcium / NAD cofactor / nitrogen metabolism
Function / homology
Function and homology information


glutamate dehydrogenase [NAD(P)+] activity / glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / plant-type vacuole / amino acid metabolic process / cobalt ion binding / copper ion binding / mitochondrion / zinc ion binding ...glutamate dehydrogenase [NAD(P)+] activity / glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / plant-type vacuole / amino acid metabolic process / cobalt ion binding / copper ion binding / mitochondrion / zinc ion binding / ATP binding / plasma membrane
Similarity search - Function
Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase ...Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Aminoacid dehydrogenase-like, N-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Glutamate dehydrogenase 2
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsGrzechowiak, M. / Ruszkowski, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2018/31/D/NZ1/03630 Poland
CitationJournal: Plant Physiol Biochem / Year: 2023
Title: Structural and functional studies of Arabidopsis thaliana glutamate dehydrogenase isoform 2 demonstrate enzyme dynamics and identify its calcium binding site.
Authors: Marta Grzechowiak / Joanna Sliwiak / Mariusz Jaskolski / Milosz Ruszkowski /
Abstract: Glutamate dehydrogenase (GDH) is an enzyme at the crossroad of plant nitrogen and carbon metabolism. GDH catalyzes the conversion of 2-oxoglutarate into glutamate (2OG → Glu), utilizing ammonia as ...Glutamate dehydrogenase (GDH) is an enzyme at the crossroad of plant nitrogen and carbon metabolism. GDH catalyzes the conversion of 2-oxoglutarate into glutamate (2OG → Glu), utilizing ammonia as cosubstrate and NADH as coenzyme. The GDH reaction is reversible, meaning that the NAD-dependent reaction (Glu → 2OG) releases ammonia. In Arabidopsis thaliana, three GDH isoforms exist, AtGDH1, AtGDH2, and AtGDH3. The subject of this work is AtGDH2. Previous reports have suggested that enzymes homologous to AtGDH2 contain a calcium-binding EF-hand motif located in the coenzyme binding domain. Here, we show that while AtGDH2 indeed does bind calcium, the binding occurs elsewhere and the region predicted to be the EF-hand motif has a completely different structure. As the true calcium binding site is > 20 Å away from the active site, it seems to play a structural, rather than catalytic role. We also performed comparative kinetic characterization of AtGDH1 and AtGDH2 using spectroscopic methods and isothermal titration calorimetry, to note that the isoenzymes generally exhibit similar behavior, with calcium having only a minor effect. However, the spatial and temporal changes in the gene expression profiles of the three AtGDH genes point to AtGDH2 as the most prevalent isoform.
History
DepositionApr 28, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate dehydrogenase 2
B: Glutamate dehydrogenase 2
C: Glutamate dehydrogenase 2
D: Glutamate dehydrogenase 2
E: Glutamate dehydrogenase 2
F: Glutamate dehydrogenase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,40412
Polymers270,1636
Non-polymers2406
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASNALAB1 - 410
d_21ens_1ASNALAA1 - 410
d_31ens_1ASNALAC1 - 410
d_41ens_1ASNALAD1 - 410
d_51ens_1ASNALAE1 - 410
d_61ens_1ASNALAF1 - 410

NCS oper:
IDCodeMatrixVector
1given(-0.50404048281, -0.863676836356, -0.00234819744618), (0.863673790032, -0.504045312904, 0.0024304222015), (-0.00328269727453, -0.000803045408209, 0.999994289492)136.675656319, 40.1654153184, 0.226447117655
2given(-0.496718606925, 0.867909315879, 0.00201120491468), (-0.86790908412, -0.49672143753, 0.0012787497733), (0.00210884743732, -0.00111036420948, 0.999997159923)33.3502544305, 137.917819584, 0.160462550495
3given(0.798990037693, 0.601328350549, -0.00437429926457), (0.601318706982, -0.799002006523, -0.00340678814246), (-0.0055436721839, 9.16418086282E-5, -0.999984629532)-23.8944766334, 72.8811672066, 123.536037077
4given(0.118381926502, -0.992965547234, 0.0022674842639), (-0.992967243763, -0.118384573002, -0.00107036897642), (0.00133127467278, -0.0021248252583, -0.999996856408)108.763661368, 122.650076825, 123.228656402
5given(-0.917894025588, 0.396810710008, 0.00343776286869), (0.396799478836, 0.917898560596, -0.00352222253032), (-0.0045531732119, -0.00186892450271, -0.999987887794)84.9043123926, -17.4390323231, 123.698001964

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Components

#1: Protein
Glutamate dehydrogenase 2 / / GDH 2


Mass: 45027.188 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: GDH2, At5g07440, T2I1_150 / Production host: Escherichia coli (E. coli)
References: UniProt: Q38946, glutamate dehydrogenase [NAD(P)+]
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.27 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMhepesC8H18N2O4S1
250 mMsodium chlorideNaClSodium chloride1
3100 mMpotassium chlorideKCl1
41 mMcalcium chlorideCaCl21
51 mMtris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 231 nm / Calibrated defocus max: 4623 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.1_3865refinement
PHENIX1.18.1_3865refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
13RELIONclassification
14RELION3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25528 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004919176
ELECTRON MICROSCOPYf_angle_d0.561625974
ELECTRON MICROSCOPYf_chiral_restr0.04282904
ELECTRON MICROSCOPYf_plane_restr0.00393396
ELECTRON MICROSCOPYf_dihedral_angle_d9.51072616
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints0.000711123888411
ens_1d_3BELECTRON MICROSCOPYNCS constraints0.000697454832535
ens_1d_4BELECTRON MICROSCOPYNCS constraints0.000705349276396
ens_1d_5BELECTRON MICROSCOPYNCS constraints0.0007083411767
ens_1d_6BELECTRON MICROSCOPYNCS constraints0.000703734819278

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