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- EMDB-9847: Cryo-EM structure of glutamate dehydrogenase from Thermococcus pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-9847
TitleCryo-EM structure of glutamate dehydrogenase from Thermococcus profundus
Map data
SampleHexamer of glutamate dehydrogenase
Function / homology
Function and homology information


glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase [NAD(P)+] activity / cellular amino acid metabolic process
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 / NAD(P)-binding domain superfamily
Glutamate dehydrogenase
Biological speciesThermococcus profundus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsOide M / Kato T / Oroguchi T / Nakasako M
Funding support Japan, 14 items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp25120725 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp23120525 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp17H05891 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15H01647 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp26104535 Japan
Japan Society for the Promotion of Sciencejp13480214 Japan
Japan Society for the Promotion of Sciencejp22244054 Japan
Japan Society for the Promotion of Sciencejp19204042 Japan
Japan Society for the Promotion of Sciencejp17H04854 Japan
Japan Society for the Promotion of Sciencejp26800227 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp15076210 Japan
Japan Society for the Promotion of Science18J11653 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp22018027 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)jp20050030 Japan
CitationJournal: 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.
Validation ReportPDB-ID: 6jnc

SummaryFull reportAbout validation report
History
DepositionMar 11, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6jnc
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6jnc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9847.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 224 pix.
= 194.88 Å
0.87 Å/pix.
x 224 pix.
= 194.88 Å
0.87 Å/pix.
x 224 pix.
= 194.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.19825408 - 0.301604
Average (Standard dev.)0.0016217193 (±0.01365891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 194.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z194.880194.880194.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.1980.3020.002

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Supplemental data

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Sample components

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Entire Hexamer of glutamate dehydrogenase

EntireName: Hexamer of glutamate dehydrogenase / Number of components: 1

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Component #1: protein, Hexamer of glutamate dehydrogenase

ProteinName: Hexamer of glutamate dehydrogenase / Recombinant expression: No
MassTheoretical: 280 kDa
SourceSpecies: Thermococcus profundus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 10 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 BASE (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 146327
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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