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- PDB-8xcw: Cryo-EM structure of Glutamate dehydrogenase from Thermococcus pr... -

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Basic information

Entry
Database: PDB / ID: 8xcw
TitleCryo-EM structure of Glutamate dehydrogenase from Thermococcus profundus incorporating NADPH and AKG in the steady stage of reaction
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE / Complex / Coenzyme / NADPH / 2-oxoglutarate
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
2-OXOGLUTARIC ACID / Chem-NDP / Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsWakabayashi, T. / Oide, M. / Nakasako, M.
Funding support Japan, 14items
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: To Be Published
Title: CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase
Authors: Wakabayashi, T. / Oide, M. / Nakasako, M.
History
DepositionDec 10, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,6503
Polymers46,7581
Non-polymers8922
Water30617
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Glutamate dehydrogenase /


Mass: 46758.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus profundus (archaea) / Gene: gdhA / Production host: Escherichia coli (E. coli)
References: UniProt: O74024, glutamate dehydrogenase [NAD(P)+]
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-AKG / 2-OXOGLUTARIC ACID / Α-Ketoglutaric acid


Mass: 146.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H6O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 17 / Source method: isolated from a natural source / Formula: H2O
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: Hexamer of glutamate dehydrogenase in the presence of NADP and glutamate
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.280 MDa / Experimental value: YES
Source (natural)Organism: Thermococcus profundus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.5 mMnicotinamide adenine dinucleotide phosphateNADPNicotinamide adenine dinucleotide phosphate1
2100 mMglutamateGlutamic acidGlu1
35 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenConc.: 4.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
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 imaging

MicroscopyModel: JEOL CRYO ARM 300
Details: Grid information as following: Company/model: Quantifoil Cu 1.2/1.3 Material:Cu Grid mesh: 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 47000 nm / Nominal defocus min: 400 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7075

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Processing

EM software
IDNameVersionCategory
1RELION4.0betaparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION4.0betainitial Euler assignment
11RELION4.0betafinal Euler assignment
12RELION4.0betaclassification
13RELION4.0beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6334528
Details: Auto-picking based on templates from manually picked GDH particles.
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136459 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023299
ELECTRON MICROSCOPYf_angle_d0.4384481
ELECTRON MICROSCOPYf_dihedral_angle_d3.542450
ELECTRON MICROSCOPYf_chiral_restr0.042493
ELECTRON MICROSCOPYf_plane_restr0.004576

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