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- EMDB-8194: Cryo-EM structure of glutamate dehydrogenase at 1.8 A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-8194
TitleCryo-EM structure of glutamate dehydrogenase at 1.8 A resolution
Map dataGlutamate dehydrogenase at 1.8 A resolution
Sample
  • Complex: Glutamate dehydrogenase
    • Protein or peptide: Glutamate dehydrogenase 1, mitochondrial
  • Ligand: water
Keywordsglutamate dehydrogenase / small metabolic complex / OXIDOREDUCTASE
Function / homology
Function and homology information


glutamate dehydrogenase [NAD(P)+] activity / glutamate catabolic process / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / glutamine metabolic process / mitochondrial inner membrane / GTP binding / endoplasmic reticulum ...glutamate dehydrogenase [NAD(P)+] activity / glutamate catabolic process / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / glutamine metabolic process / mitochondrial inner membrane / GTP binding / endoplasmic reticulum / mitochondrion / ATP binding / identical protein binding
Similarity search - Function
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 1, mitochondrial
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.8 Å
AuthorsMerk A / Bartesaghi A
CitationJournal: Cell / Year: 2016
Title: Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.
Authors: Alan Merk / Alberto Bartesaghi / Soojay Banerjee / Veronica Falconieri / Prashant Rao / Mindy I Davis / Rajan Pragani / Matthew B Boxer / Lesley A Earl / Jacqueline L S Milne / Sriram Subramaniam /
Abstract: Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. ...Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
History
DepositionMay 17, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseJun 8, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k12
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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  • Supplemental map + atomic model
  • Surface view with fitted model
  • Atomic models: PDB-5k12
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8194.png

Downloads & links

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Map

FileDownload / File: emd_8194.map.gz / Format: CCP4 / Size: 48.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlutamate dehydrogenase at 1.8 A resolution
Voxel sizeX=Y=Z: 0.637 Å
Density
Contour LevelBy AUTHOR: 0.0085 / Movie #1: 0.0085
Minimum - Maximum-0.014285645 - 0.03361448
Average (Standard dev.)-0.0001064329 (±0.002899618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 149.058 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6370.6370.637
M x/y/z234234234
origin x/y/z0.0000.0000.000
length x/y/z149.058149.058149.058
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS234234234
D min/max/mean-0.0140.034-0.000

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Supplemental data

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Additional map: Map sharpened using a B-factor of -90

Fileemd_8194_additional.map
AnnotationMap sharpened using a B-factor of -90
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Glutamate dehydrogenase

EntireName: Glutamate dehydrogenase
Components
  • Complex: Glutamate dehydrogenase
    • Protein or peptide: Glutamate dehydrogenase 1, mitochondrial
  • Ligand: water

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Supramolecule #1: Glutamate dehydrogenase

SupramoleculeName: Glutamate dehydrogenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 334 KDa

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Macromolecule #1: Glutamate dehydrogenase 1, mitochondrial

MacromoleculeName: Glutamate dehydrogenase 1, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: glutamate dehydrogenase [NAD(P)+]
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 61.60891 KDa
SequenceString: MYRYLGEALL LSRAGPAALG SASADSAALL GWARGQPAAA PQPGLVPPAR RHYSEAAADR EDDPNFFKMV EGFFDRGASI VEDKLVEDL KTRETEEQKR NRVRSILRII KPCNHVLSLS FPIRRDDGSW EVIEGYRAQH SQHRTPCKGG IRYSTDVSVD E VKALASLM ...String:
MYRYLGEALL LSRAGPAALG SASADSAALL GWARGQPAAA PQPGLVPPAR RHYSEAAADR EDDPNFFKMV EGFFDRGASI VEDKLVEDL KTRETEEQKR NRVRSILRII KPCNHVLSLS FPIRRDDGSW EVIEGYRAQH SQHRTPCKGG IRYSTDVSVD E VKALASLM TYKCAVVDVP FGGAKAGVKI NPKNYTDNEL EKITRRFTME LAKKGFIGPG VDVPAPDMST GEREMSWIAD TY ASTIGHY DINAHACVTG KPISQGGIHG RISATGRGVF HGIENFINEA SYMSILGMTP GFGDKTFVVQ GFGNVGLHSM RYL HRFGAK CITVGESDGS IWNPDGIDPK ELEDFKLQHG TILGFPKAKI YEGSILEVDC DILIPAASEK QLTKSNAPRV KAKI IAEGA NGPTTPEADK IFLERNIMVI PDLYLNAGGV TVSYFEWLKN LNHVSYGRLT FKYERDSNYH LLMSVQESLE RKFGK HGGT IPIVPTAEFQ DRISGASEKD IVHSGLAYTM ERSARQIMRT AMKYNLGLDL RTAAYVNAIE KVFRVYNEAG VTFT

UniProtKB: Glutamate dehydrogenase 1, mitochondrial

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 1086 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration3 mg/mL
BufferpH: 6.8 / Component - Concentration: 100.0 nM / Component - Name: Potassium phosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: Plunged into liquiq ethane (FEI VITROBOT MARK IV).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 78000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 215000
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 79.6 K / Max: 79.8 K
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 3-9 / Number real images: 232 / Average exposure time: 0.4 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 45388
Startup modelType of model: OTHER
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.10)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.10)
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.10)
Details: The primary map in this entry corresponds to theuncorrected reconstruction. A version sharpened using a B-factor of -90 is provided as additional volume data.
Number images used: 21818

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Atomic model buiding 1

Initial modelPDB ID:

1nr7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5k12:
Cryo-EM structure of glutamate dehydrogenase at 1.8 A resolution

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