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- EMDB-38261: Cryo-EM structure of Glutamate dehydrogenase from Thermococcus pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-38261
TitleCryo-EM structure of Glutamate dehydrogenase from Thermococcus profundus incorporating NADPH and AKG in the steady stage of reaction
Map data
Sample
  • Complex: Hexamer of glutamate dehydrogenase in the presence of NADP and glutamate
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: 2-OXOGLUTARIC ACID
  • Ligand: water
KeywordsComplex / Coenzyme / NADPH / 2-oxoglutarate / 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 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: 2.83 Å
AuthorsWakabayashi T / Oide M / 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: Sci Rep / Year: 2024
Title: CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase.
Authors: Taiki Wakabayashi / Mao Oide / Masayoshi Nakasako /
Abstract: Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic ...Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.
History
DepositionDec 10, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38261.png

Downloads & links

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Map

FileDownload / File: emd_38261.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 256 pix.
= 192.512 Å		0.75 Å/pix.
x 256 pix.
= 192.512 Å		0.75 Å/pix.
x 256 pix.
= 192.512 Å

Surface

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.752 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.024791593 - 0.035770986
Average (Standard dev.)0.000013687894 (±0.0018152508)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 192.512 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_38261_msk_1.map
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Half map: #2

Fileemd_38261_half_map_1.map
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Half map: #1

Fileemd_38261_half_map_2.map
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Sample components

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Entire : Hexamer of glutamate dehydrogenase in the presence of NADP and gl...

EntireName: Hexamer of glutamate dehydrogenase in the presence of NADP and glutamate
Components
  • Complex: Hexamer of glutamate dehydrogenase in the presence of NADP and glutamate
    • Protein or peptide: Glutamate dehydrogenase
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: 2-OXOGLUTARIC ACID
  • Ligand: water

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

SupramoleculeName: Hexamer of glutamate dehydrogenase in the presence of NADP and glutamate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermococcus profundus (archaea)
Molecular weightTheoretical: 280 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: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
type: ligand / ID: 2 / Number of copies: 1 / Formula: NDP
Molecular weightTheoretical: 745.421 Da
Chemical component information

ChemComp-NDP:
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #3: 2-OXOGLUTARIC ACID

MacromoleculeName: 2-OXOGLUTARIC ACID / type: ligand / ID: 3 / Number of copies: 1 / Formula: AKG
Molecular weightTheoretical: 146.098 Da
Chemical component information

ChemComp-AKG:
2-OXOGLUTARIC ACID

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 14 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration4.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.5 mMNADPnicotinamide adenine dinucleotide phosphate
100.0 mMGluglutamate
5.0 mMTristris(hydroxymethyl)aminomethane
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: The sample solution kept at room temperature was flash-frozen 1-h after mixing GDH, NADP and glutamate solutions..

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
DetailsGrid information as following: Company/model: Quantifoil Cu 1.2/1.3 Material:Cu Grid mesh: 200
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 7075 / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 47.0 µm / Nominal defocus min: 0.4 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 6334528
Details: Auto-picking based on templates from manually picked GDH particles.
Startup modelType of model: INSILICO MODEL
Details: The startup model was generated by de novo 3D model generation in Relion.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0beta) / Number images used: 113612
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta)
Final 3D classificationNumber classes: 32 / Avg.num./class: 80000 / Software - Name: RELION (ver. 4.0beta)
Details: The final 3D classification involved a two-step process. In the first step, all particles were separated into eight classes. Subsequently, each of these classes was further classified into ...Details: The final 3D classification involved a two-step process. In the first step, all particles were separated into eight classes. Subsequently, each of these classes was further classified into four subclasses. This entire process was conducted following a symmetry expansion operation on all particles, in accordance with the D3 symmetry of GDH.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8xd0:
Cryo-EM structure of Glutamate dehydrogenase from Thermococcus profundus incorporating NADPH and AKG in the steady stage of reaction

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