|Entry||Database: PDB / ID: 6jn9|
|Title||Cryo-EM structure of glutamate dehydrogenase from Thermococcus profundus|
|Keywords||OXIDOREDUCTASE / enzyme / multi-domain protein|
|Function / homology|
Function and homology information
glutamate dehydrogenase [NAD(P)+] activity / glutamate dehydrogenase [NAD(P)+] / cellular amino acid metabolic process
NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / NAD(P) binding domain of glutamate dehydrogenase / Leucine Dehydrogenase, chain A, domain 1 ...NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / NAD(P) binding domain of glutamate dehydrogenase / Leucine Dehydrogenase, chain A, domain 1 / NAD(P)-binding Rossmann-like Domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
|Biological species||Thermococcus profundus (archaea)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å|
|Authors||Oide, M. / Kato, T. / Oroguchi, T. / Nakasako, M.|
|Funding support|| Japan, 14items |
|Citation||Journal: FEBS J / Year: 2020|
Title: Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy.
Authors: Mao Oide / Takayuki Kato / Tomotaka Oroguchi / Masayoshi Nakasako /
Abstract: Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of ...Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. DATABASE: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Glutamate dehydrogenase
|#1: Protein|| |
Mass: 46758.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus profundus (archaea) / Gene: gdhA, gdh / Production host: Escherichia coli (E. coli)
References: UniProt: O74024, glutamate dehydrogenase [NAD(P)+]
|#2: Chemical|| ChemComp-TRS / |
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Hexamer of glutamate dehydrogenase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT|
|Source (natural)||Organism: Thermococcus profundus (archaea)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
|Microscopy||Model: JEOL CRYO ARM 200|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 10 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118299 / Symmetry type: POINT|
|Refinement||Stereochemistry target values: CDL v1.2|
|Refine LS restraints|
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