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- EMDB-6635: Glutamate dehydrogenase in complex with NADH, open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-6635
TitleGlutamate dehydrogenase in complex with NADH, open conformation
Map dataReconstruction of the BINARY complex of bovine glutamate dehydrogenase with NADH. This entry is the OPEN conformation.
Sample
  • Sample: Binary complex of bovine glutamate dehydrogenase with NADH
  • Protein or peptide: L-glutamate:NAD(P)+ oxidoreductase (deaminating)
  • Ligand: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate
Keywordsenzyme / glutamate metabolism / mitochondria
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 / amino acid metabolic process / glutamine metabolic process / mitochondrial inner membrane ...: / glutamate dehydrogenase [NAD(P)+] activity / glutamate catabolic process / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / amino acid metabolic process / glutamine metabolic process / mitochondrial inner membrane / oxidoreductase activity / mitochondrial matrix / nucleotide binding / GTP binding / endoplasmic reticulum / mitochondrion / ATP binding / identical protein binding
Similarity search - Function
NAD(P)-binding domain / 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 / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase ...NAD(P)-binding domain / 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 / 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/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) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBorgnia MJ / Banerjee S / Merk A / Matthies D / Bartesaghi A / Rao P / Pierson J / Earl LA / Falconieri V / Subramaniam S / Milne JLS
CitationJournal: Mol Pharmacol / Year: 2016
Title: Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.
Authors: Mario J Borgnia / Soojay Banerjee / Alan Merk / Doreen Matthies / Alberto Bartesaghi / Prashant Rao / Jason Pierson / Lesley A Earl / Veronica Falconieri / Sriram Subramaniam / Jacqueline L S Milne /
Abstract: Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping ...Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.
History
DepositionMar 24, 2016-
Header (metadata) releaseApr 27, 2016-
Map releaseApr 27, 2016-
UpdateMay 11, 2016-
Current statusMay 11, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jd2
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6635.png

Downloads & links

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Map

FileDownload / File: emd_6635.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the BINARY complex of bovine glutamate dehydrogenase with NADH. This entry is the OPEN conformation.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 256 pix.
= 173.056 Å		0.68 Å/pix.
x 256 pix.
= 173.056 Å		0.68 Å/pix.
x 256 pix.
= 173.056 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.676 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02894788 - 0.07431421
Average (Standard dev.)0.00093909 (±0.00543256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 173.056 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6760.6760.676
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z173.056173.056173.056
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0290.0740.001

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Supplemental data

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Sample components

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Entire : Binary complex of bovine glutamate dehydrogenase with NADH

EntireName: Binary complex of bovine glutamate dehydrogenase with NADH
Components
  • Sample: Binary complex of bovine glutamate dehydrogenase with NADH
  • Protein or peptide: L-glutamate:NAD(P)+ oxidoreductase (deaminating)
  • Ligand: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate

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Supramolecule #1000: Binary complex of bovine glutamate dehydrogenase with NADH

SupramoleculeName: Binary complex of bovine glutamate dehydrogenase with NADH
type: sample / ID: 1000
Details: The sample was largely monodisperse. Some chains of hexamers were observed. Image processing revealed the presence of two conformational states (see related entry EMD-6634).
Oligomeric state: one homohexamer of GDH binds 12 molecules of NADH
Number unique components: 2
Molecular weightTheoretical: 344 KDa

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Macromolecule #1: L-glutamate:NAD(P)+ oxidoreductase (deaminating)

MacromoleculeName: L-glutamate:NAD(P)+ oxidoreductase (deaminating) / type: protein_or_peptide / ID: 1
Name.synonym: Glutamate dehydrogenase 1, mitochondrial, glutamate dehydrogenase [NAD(P)+], GDH
Details: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion ...Details: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column.
Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow / Tissue: Liver / Organelle: Mitochondria / Location in cell: Mitochondrial matrix
Molecular weightTheoretical: 56 KDa
SequenceUniProtKB: Glutamate dehydrogenase 1, mitochondrial
GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, ...GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, mitochondrion, mitochondrial inner membrane, mitochondrial matrix, amino acid metabolic process, glutamate catabolic process, glutamine metabolic process, oxidoreductase activity, oxidoreductase activity, GO: 0055114, GO: 0055114, tricarboxylic acid metabolic process
InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation ...InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain, NAD(P)-binding domain

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Macromolecule #2: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]me...

MacromoleculeName: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate
type: ligand / ID: 2 / Name.synonym: NADH, Nicotinamide Adenine Dinucleotide / Number of copies: 12 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 1 KDa

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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

Concentration2.0 mg/mL
BufferpH: 6.8
Details: 100 mM potassium phosphate, 0.1% n-octyl glucopyranoside, 20 mM NADH
GridDetails: 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3-6 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: Gatan GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateNov 23, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1588 / Average electron dose: 45 e/Å2
Details: Every image is the average of 38 frames recorded by the direct electron detector.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 73964 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2, Relion / Number images used: 34716

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Atomic model buiding 1

Initial modelPDB ID:

3mw9
PDB Unreleased entry


Chain - Chain ID: A
SoftwareName: Chimera, Rosetta, PHENIX, Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jd2:
Glutamate dehydrogenase in complex with NADH, open conformation

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