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

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Basic information

Entry
Database: PDB / ID: 5k12
TitleCryo-EM structure of glutamate dehydrogenase at 1.8 A resolution
ComponentsGlutamate dehydrogenase 1, mitochondrial
KeywordsOXIDOREDUCTASE / glutamate dehydrogenase / small metabolic complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.8 Å
AuthorsMerk, A. / Bartesaghi, A. / Banerjee, S. / Falconieri, V. / Rao, P. / Earl, L. / Milne, J. / Subramaniam, S.
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2016Group: Database references
Revision 1.2Nov 23, 2016Group: Other
Revision 1.3Jul 18, 2018Group: Data collection / Experimental preparation
Category: em_imaging_optics / em_sample_support / em_software
Item: _em_imaging_optics.energyfilter_name / _em_sample_support.grid_type / _em_software.name
Revision 1.4Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Glutamate dehydrogenase 1, mitochondrial
B: Glutamate dehydrogenase 1, mitochondrial
C: Glutamate dehydrogenase 1, mitochondrial
D: Glutamate dehydrogenase 1, mitochondrial
E: Glutamate dehydrogenase 1, mitochondrial
F: Glutamate dehydrogenase 1, mitochondrial


Theoretical massNumber of molelcules
Total (without water)369,6536
Polymers369,6536
Non-polymers00
Water19,5641086
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24350 Å2
ΔGint-106 kcal/mol
Surface area70270 Å2

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Components

#1: Protein
Glutamate dehydrogenase 1, mitochondrial / / GDH 1


Mass: 61608.910 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
References: UniProt: P00366, glutamate dehydrogenase [NAD(P)+]
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1086 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Glutamate dehydrogenase / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.334 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Buffer solutionpH: 6.8
Buffer componentConc.: 100 nM / Name: Potassium phosphate
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: Plunged into liquiq ethane (FEI VITROBOT MARK IV)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 215000 X / Calibrated magnification: 78000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79.8 K / Temperature (min): 79.6 K
Image recordingAverage exposure time: 0.4 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 232
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 38 / Used frames/image: 3-9

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Processing

SoftwareName: PHENIX / Version: 1.10_2155: / Classification: refinement
EM software
IDNameVersionCategory
4FREALIGN9.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11FREALIGN9.1initial Euler assignment
12FREALIGN9.1final Euler assignment
14FREALIGN9.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 45388
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21818
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.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1NR7

1nr7


Accession code: 1NR7 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 1.8 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00923748
ELECTRON MICROSCOPYf_angle_d1.20332052
ELECTRON MICROSCOPYf_dihedral_angle_d8.74214244
ELECTRON MICROSCOPYf_chiral_restr0.073456
ELECTRON MICROSCOPYf_plane_restr0.0084188

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