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- EMDB-17240: CryoEM structure of glutamate dehydrogenase isoform 2 from Arabid... -

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Basic information

Entry
Database: EMDB / ID: EMD-17240
TitleCryoEM structure of glutamate dehydrogenase isoform 2 from Arabidopsis thaliana in apo-form
Map data
Sample
  • Organelle or cellular component: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2
    • Protein or peptide: Glutamate dehydrogenase 2
  • Ligand: CALCIUM IONCalcium
Keywordsglutamic acid / calcium / NAD cofactor / nitrogen metabolism / OXIDOREDUCTASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsGrzechowiak M / Ruszkowski M
Funding support Poland, 1 items
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, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17240.png

Downloads & links

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Map

FileDownload / File: emd_17240.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 309.6 Å		0.86 Å/pix.
x 360 pix.
= 309.6 Å		0.86 Å/pix.
x 360 pix.
= 309.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.103302166 - 0.20072703
Average (Standard dev.)0.00013940419 (±0.007189136)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 309.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17240_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17240_half_map_2.map
Projections & Slices
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Sample components

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Entire : homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2

EntireName: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2
Components
  • Organelle or cellular component: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2
    • Protein or peptide: Glutamate dehydrogenase 2
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2

SupramoleculeName: homohexameric Arabidopsis thaliana glutamate dehydrogenase isoform 2
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 270 KDa

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Macromolecule #1: Glutamate dehydrogenase 2

MacromoleculeName: Glutamate dehydrogenase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: glutamate dehydrogenase [NAD(P)+]
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 45.027188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMNALAAT NRNFRHASRI LGLDSKIERS LMIPFREIKV ECTIPKDDGT LVSYIGFRVQ HDNARGPMKG GIRYHPEVDP DEVNALAQL MTWKTAVADI PYGGAKGGIG CSPRDLSLSE LERLTRVFTQ KIHDLIGIHT DVPAPDMGTN AQTMAWILDE Y SKFHGHSP ...String:
SNAMNALAAT NRNFRHASRI LGLDSKIERS LMIPFREIKV ECTIPKDDGT LVSYIGFRVQ HDNARGPMKG GIRYHPEVDP DEVNALAQL MTWKTAVADI PYGGAKGGIG CSPRDLSLSE LERLTRVFTQ KIHDLIGIHT DVPAPDMGTN AQTMAWILDE Y SKFHGHSP AVVTGKPIDL GGSLGREAAT GRGVVFATEA LLAEYGKSIQ GLTFVIQGFG NVGTWAAKLI HEKGGKVVAV SD ITGAIRN PEGIDINALI KHKDATGSLN DFNGGDAMNS DELLIHECDV LIPCALGGVL NKENAGDVKA KFIVEAANHP TDP DADEIL SKKGVIILPD IYANAGGVTV SYFEWVQNIQ GFMWEEEKVN LELQKYMTRA FHNIKTMCHT HSCNLRMGAF TLGV NRVAR ATQLRGWEA

UniProtKB: Glutamate dehydrogenase 2

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4Shepes
50.0 mMNaClSodium chloridesodium chloride
100.0 mMKClpotassium chloride
1.0 mMCaCl2calcium chloride
1.0 mMC9H15O6Ptris(2-carboxyethyl)phosphine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.623 µm / Calibrated defocus min: 0.231 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 25528
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8own:
CryoEM structure of glutamate dehydrogenase isoform 2 from Arabidopsis thaliana in apo-form

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