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- EMDB-34835: cryoEM structure of glutamate dehydrogenase from Thermococcus pro... -

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Basic information

Entry
Database: EMDB / ID: EMD-34835
TitlecryoEM structure of glutamate dehydrogenase from Thermococcus profundus in complex with NADP
Map data
Sample
  • Complex: Hexamer of glutamate dehydrogenase in the presence of NADP
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
KeywordsComplex / Coenzyme / NADP / OXIDOREDUCTASE
Function / homology
Function and homology information


glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / amino acid metabolic process
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
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsWakabayashi T / Oide M / Kato T / Nakasako M
Funding support Japan, 14 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)jp13480214 Japan
Japan Society for the Promotion of Science (JSPS)jp19204042 Japan
Japan Society for the Promotion of Science (JSPS)jp22244054 Japan
Japan Society for the Promotion of Science (JSPS)jp21H01050 Japan
Japan Society for the Promotion of Science (JSPS)jp26800227 Japan
Japan Society for the Promotion of Science (JSPS)jp18J11653 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15076210 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp20050030 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp22018027 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp23120525 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp25120725 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15H01647 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp17H05891 Japan
Japan Science and TechnologyJPMJPR22E2 Japan
CitationJournal: FEBS J / Year: 2023
Title: Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron microscopy.
Authors: Taiki Wakabayashi / Mao Oide / Takayuki Kato / Masayoshi Nakasako /
Abstract: The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron ...The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron microscopy to investigate the ligand-binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer-forming core domain and one nucleotide-binding domain (NAD domain), which spontaneously opens and closes the active-site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active-site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active-site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.
History
DepositionNov 22, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34835.png

Downloads & links

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Map

FileDownload / File: emd_34835.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.986 Å
Density
Contour LevelBy AUTHOR: 0.0065
Minimum - Maximum-0.025018575 - 0.046458587
Average (Standard dev.)0.00009453535 (±0.0013602229)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 252.416 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34835_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34835_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34835_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamer of glutamate dehydrogenase in the presence of NADP

EntireName: Hexamer of glutamate dehydrogenase in the presence of NADP
Components
  • Complex: Hexamer of glutamate dehydrogenase in the presence of NADP
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Supramolecule #1: Hexamer of glutamate dehydrogenase in the presence of NADP

SupramoleculeName: Hexamer of glutamate dehydrogenase in the presence of NADP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermococcus profundus (archaea)
Molecular weightTheoretical: 264 KDa

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Macromolecule #1: Glutamate dehydrogenase

MacromoleculeName: Glutamate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: glutamate dehydrogenase [NAD(P)+]
Source (natural)Organism: Thermococcus profundus (archaea)
Molecular weightTheoretical: 46.758477 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVEIDPFEMA VKQLERAAQY MDISEEALEW LKKPMRIVEV SVPIEMDDGS VKVFTGFRVQ HNWARGPTKG GIRWHPAETL STVKALATW MTWKVAVVDL PYGGGKGGII VNPKELSERE QERLARAYIR AVYDVIGPWT DIPAPDVYTN PKIMGWMMDE Y ETIMRRKG ...String:
MVEIDPFEMA VKQLERAAQY MDISEEALEW LKKPMRIVEV SVPIEMDDGS VKVFTGFRVQ HNWARGPTKG GIRWHPAETL STVKALATW MTWKVAVVDL PYGGGKGGII VNPKELSERE QERLARAYIR AVYDVIGPWT DIPAPDVYTN PKIMGWMMDE Y ETIMRRKG PAFGVITGKP LSIGGSLGRG TATAQGAIFT IREAAKALGI DLKGKKIAVQ GYGNAGYYTA KLAKEQLGMT VV AVSDSRG GIYNPDGLDP DEVLKWKREH GSVKDFPGAT NITNEELLEL EVDVLAPAAI EEVITEKNAD NIKAKIVAEV ANG PVTPEA DDILREKGIL QIPDFLCNAG GVTVSYFEWV QNINGYYWTE EEVREKLDKK MTKAFWEVYN THKDKNIHMR DAAY VVAVS RVYQAMKDRG WVKK

UniProtKB: Glutamate dehydrogenase

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Macromolecule #2: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

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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

Concentration0.0036 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
3.0 mMNADPNicotinamide adenine dinucleotide phosphatenicotinamide adenine dinucleotide phosphate
135.0 mMNaClSodium chloridesodium chloride
15.0 mMTristris(hydroxymethyl)aminomethane
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / 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

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 0.5 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 10389 / Average electron dose: 1.2 e/Å2

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Image processing

Particle selectionNumber selected: 3037558
Startup modelType of model: INSILICO MODEL
Details: The startup model was generated by de novo 3D model generation in Relion.
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0)
Final 3D classificationAvg.num./class: 80000
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 94360
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8hj9:
cryoEM structure of glutamate dehydrogenase from Thermococcus profundus in complex with NADP

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