|Entry||Database: PDB / ID: 5k11|
|Title||Cryo-EM structure of isocitrate dehydrogenase (IDH1) in inhibitor-bound state|
|Components||Isocitrate dehydrogenase [NADP] cytoplasmic|
|Keywords||OXIDOREDUCTASE / isocitrate dehydrogenase / small metabolic complex / small molecule inhibitor|
|Function / homology|
Function and homology information
regulation of phospholipid catabolic process / regulation of phospholipid biosynthetic process / NADPH regeneration / NADP metabolic process / isocitrate metabolic process / isocitrate dehydrogenase (NADP+) / isocitrate dehydrogenase (NADP+) activity / 2-oxoglutarate metabolic process / glyoxylate cycle / female gonad development ...regulation of phospholipid catabolic process / regulation of phospholipid biosynthetic process / NADPH regeneration / NADP metabolic process / isocitrate metabolic process / isocitrate dehydrogenase (NADP+) / isocitrate dehydrogenase (NADP+) activity / 2-oxoglutarate metabolic process / glyoxylate cycle / female gonad development / peroxisomal matrix / tricarboxylic acid cycle / glutathione metabolic process / response to steroid hormone / peroxisome / NAD binding / protein localization / tertiary granule lumen / NADP binding / secretory granule lumen / response to oxidative stress / ficolin-1-rich granule lumen / cadherin binding / neutrophil degranulation / magnesium ion binding / protein homodimerization activity / mitochondrion / extracellular exosome / extracellular region / identical protein binding / cytosol / cytoplasm
Isocitrate/isopropylmalate dehydrogenase / Isopropylmalate dehydrogenase-like domain / Isocitrate/isopropylmalate dehydrogenase, conserved site / Isocitrate dehydrogenase NADP-dependent / Isopropylmalate Dehydrogenase / Isopropylmalate Dehydrogenase / 3-Layer(aba) Sandwich / Alpha Beta
Isocitrate dehydrogenase [NADP] cytoplasmic
|Biological species||Homo sapiens (human)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å|
|Authors||Merk, A. / Bartesaghi, A. / Banerjee, S. / Falconieri, V. / Rao, P. / Earl, L. / Milne, J. / Subramaniam, S.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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A: Isocitrate dehydrogenase [NADP] cytoplasmic
B: Isocitrate dehydrogenase [NADP] cytoplasmic
Mass: 46334.742 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IDH1, PICD / Production host: unidentified baculovirus
References: UniProt: O75874, isocitrate dehydrogenase (NADP+)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Isocitrate dehydrogenase R132C mutant in complex with ML309|
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
|Molecular weight||Value: 0.093 MDa / Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Source (recombinant)||Organism: unidentified baculovirus / Plasmid: unknown|
|Buffer solution||pH: 7.5|
|Specimen||Conc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: LEICA EM GP / Cryogen name: ETHANE / Details: Plunged into liquid ethane (LEICA EM GP)|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 270000 X / Calibrated magnification: 101000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79.8 K / Temperature (min): 79.6 K|
|Image recording||Average exposure time: 0.2 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 820|
|EM imaging optics||Energyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV|
|Image scans||Movie frames/image: 60 / Used frames/image: 0-29|
|Software||Name: PHENIX / Version: 1.10.1_2155: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Num. of particles selected: 232343|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46483 / Algorithm: FOURIER SPACE|
Details: The primary map for this entry corresponds to the uncorrected reconstruction. A version sharpened using a B-factor of -180 is provided as additional volume data. The reconstruction, obtained ...Details: The primary map for this entry corresponds to the uncorrected reconstruction. A version sharpened using a B-factor of -180 is provided as additional volume data. The reconstruction, obtained without imposing symmetry, is also provided with this entry as additional volume data.
Symmetry type: POINT
|Atomic model building||Protocol: FLEXIBLE FIT / Space: REAL|
|Refinement||Highest resolution: 3.8 Å|
|Refine LS restraints|
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