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- PDB-5k0z: Cryo-EM structure of lactate dehydrogenase (LDH) in inhibitor-bou... -

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Basic information

Entry
Database: PDB / ID: 5k0z
TitleCryo-EM structure of lactate dehydrogenase (LDH) in inhibitor-bound state
ComponentsL-lactate dehydrogenase B chain
KeywordsOXIDOREDUCTASE / lactate dehydrogenase / small metabolic complex / small molecule inhibitor
Function / homology
Function and homology information


Pyruvate metabolism / Pyruvate metabolism / L-lactate dehydrogenase / L-lactate dehydrogenase activity / carboxylic acid metabolic process / carbohydrate metabolic process / cytosol
Similarity search - Function
L-lactate dehydrogenase active site. / L-lactate dehydrogenase / L-lactate dehydrogenase, active site / L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2 / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / L-lactate/malate dehydrogenase / Lactate/malate dehydrogenase, N-terminal / Lactate/malate dehydrogenase, C-terminal / lactate/malate dehydrogenase, NAD binding domain / lactate/malate dehydrogenase, alpha/beta C-terminal domain ...L-lactate dehydrogenase active site. / L-lactate dehydrogenase / L-lactate dehydrogenase, active site / L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2 / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / L-lactate/malate dehydrogenase / Lactate/malate dehydrogenase, N-terminal / Lactate/malate dehydrogenase, C-terminal / lactate/malate dehydrogenase, NAD binding domain / lactate/malate dehydrogenase, alpha/beta C-terminal domain / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Alpha-Beta Complex / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
L-lactate dehydrogenase B chain
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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: L-lactate dehydrogenase B chain
B: L-lactate dehydrogenase B chain
C: L-lactate dehydrogenase B chain
D: L-lactate dehydrogenase B chain


Theoretical massNumber of molelcules
Total (without water)144,4634
Polymers144,4634
Non-polymers00
Water73941
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19870 Å2
ΔGint-128 kcal/mol
Surface area40870 Å2

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Components

#1: Protein
L-lactate dehydrogenase B chain / LDH-B


Mass: 36115.656 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: LDHB / Production host: Bacteria (eubacteria) / References: UniProt: P00337, L-lactate dehydrogenase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 41 / 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: Lactate dehydrogenase (LDH) in complex with GSK2837808A
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.145 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Bacteria (eubacteria) / Plasmid: unknown
Buffer solutionpH: 7.4 / Details: Phosphate-buffered saline
Buffer componentName: PBS
SpecimenConc.: 1.5 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 (LEICE 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: 2200 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: 0.2 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 1707
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

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: 508402
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50865 / Algorithm: FOURIER SPACE
Details: The primary map in this entry corresponds to the uncorrected reconstruction. A version sharpened using a B-factor of -150 is provided as additional volume data.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1I0Z

1i0z


Accession code: 1I0Z / Source name: PDB / Type: experimental model

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