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- PDB-5k11: Cryo-EM structure of isocitrate dehydrogenase (IDH1) in inhibitor... -

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Entry
Database: PDB / ID: 5k11
TitleCryo-EM structure of isocitrate dehydrogenase (IDH1) in inhibitor-bound state
ComponentsIsocitrate dehydrogenase [NADP] cytoplasmic
KeywordsOXIDOREDUCTASE / 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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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.
Validation Report
SummaryFull reportAbout validation report
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

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Structure visualization

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Assembly

Deposited unit
A: Isocitrate dehydrogenase [NADP] cytoplasmic
B: Isocitrate dehydrogenase [NADP] cytoplasmic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,1604
Polymers92,6692
Non-polymers1,4912
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6700 Å2
ΔGint-46 kcal/mol
Surface area34930 Å2

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Components

#1: Protein Isocitrate dehydrogenase [NADP] cytoplasmic / IDH / Cytosolic NADP-isocitrate dehydrogenase / IDP / NADP(+)-specific ICDH / Oxalosuccinate decarboxylase


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+)
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H30N7O17P3

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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: Isocitrate dehydrogenase R132C mutant in complex with ML309
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.093 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: unidentified baculovirus / Plasmid: unknown
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTrisC4H12NO3Cl1
2150 mMsodium chlorideNaClSodium chloride1
310 mMmagnesium chlorideMgCl21
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Details: Plunged into liquid ethane (LEICA EM GP)

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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: 270000 X / Calibrated magnification: 101000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79.8 K / Temperature (min): 79.6 K
Image recordingAverage 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 opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 60 / Used frames/image: 0-29

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Processing

SoftwareName: PHENIX / Version: 1.10.1_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: 232343
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 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 buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementHighest resolution: 3.8 Å
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.016470
ELECTRON MICROSCOPYf_angle_d1.0728736
ELECTRON MICROSCOPYf_dihedral_angle_d8.5085312
ELECTRON MICROSCOPYf_chiral_restr0.059954
ELECTRON MICROSCOPYf_plane_restr0.0061104

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