[English] 日本語
Yorodumi
- PDB-8xco: Cryo-EM structure of Glutamate dehydrogenase from Thermococcus pr... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8xco
TitleCryo-EM structure of Glutamate dehydrogenase from Thermococcus profundus incorporating NADPH in the initial stage of reaction
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE / Complex / Coenzyme / NADPH
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
Chem-NDP / Glutamate dehydrogenase
Similarity search - Component
Biological speciesThermococcus profundus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 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: 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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,5042
Polymers46,7581
Non-polymers7451
Water25214
1


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

-
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


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
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 phosphateNADP1
2100 mMglutamateGlu1
315 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenConc.: 4.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: The sample solution kept at 277 K was flash-frozen 15-s after mixing GDH, NADP and glutamate solutions.

-
Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / 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: 7750

-
Processing

EM software
IDNameVersionCategory
1RELION4particle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5267415 / Details: auto-picking with LoG-fiter inplemented in Relion
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160610 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more