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- EMDB-8191: Cryo-EM structure of lactate dehydrogenase (LDH) in complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8191
TitleCryo-EM structure of lactate dehydrogenase (LDH) in complex with GSK2837808A
Map dataLactate dehydrogenase (LDH) in complex with GSK2837808A
Sample
  • Complex: Lactate dehydrogenase (LDH) in complex with GSK2837808A
    • Protein or peptide: L-lactate dehydrogenase B chain
  • Ligand: water
Keywordslactate dehydrogenase / small metabolic complex / small molecule inhibitor / OXIDOREDUCTASE
Function / homology
Function and homology information


Pyruvate metabolism / Pyruvate metabolism / L-lactate dehydrogenase / oxidoreductase complex / L-lactate dehydrogenase (NAD+) activity / pyruvate metabolic process / lactate metabolic process / mitochondrial inner membrane / membrane raft / mitochondrion ...Pyruvate metabolism / Pyruvate metabolism / L-lactate dehydrogenase / oxidoreductase complex / L-lactate dehydrogenase (NAD+) activity / pyruvate metabolic process / lactate metabolic process / mitochondrial inner membrane / membrane raft / mitochondrion / identical protein binding / cytosol
Similarity search - Function
L-lactate dehydrogenase / L-lactate dehydrogenase active site. / L-lactate dehydrogenase, active site / 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 domain superfamily
Similarity search - Domain/homology
L-lactate dehydrogenase B chain
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMerk A / Bartesaghi A
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, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseJun 8, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00574
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00574
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k0z
  • Surface level: 0.00574
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8191.png

Downloads & links

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Map

FileDownload / File: emd_8191.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLactate dehydrogenase (LDH) in complex with GSK2837808A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.5 Å/pix.
x 180 pix.
= 89.1 Å		0.5 Å/pix.
x 180 pix.
= 89.1 Å		0.5 Å/pix.
x 180 pix.
= 89.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.495 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00574 / Movie #1: 0.00574
Minimum - Maximum-0.009958491 - 0.020634288
Average (Standard dev.)0.0008012398 (±0.0030885048)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 89.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.4950.4950.495
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z89.10089.10089.100
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0100.0210.001

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Supplemental data

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Additional map: Map sharpened using a B-factor of -150

Fileemd_8191_additional.map
AnnotationMap sharpened using a B-factor of -150
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lactate dehydrogenase (LDH) in complex with GSK2837808A

EntireName: Lactate dehydrogenase (LDH) in complex with GSK2837808A
Components
  • Complex: Lactate dehydrogenase (LDH) in complex with GSK2837808A
    • Protein or peptide: L-lactate dehydrogenase B chain
  • Ligand: water

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Supramolecule #1: Lactate dehydrogenase (LDH) in complex with GSK2837808A

SupramoleculeName: Lactate dehydrogenase (LDH) in complex with GSK2837808A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 145 KDa

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Macromolecule #1: L-lactate dehydrogenase B chain

MacromoleculeName: L-lactate dehydrogenase B chain / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: L-lactate dehydrogenase
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 36.115656 KDa
Recombinant expressionOrganism: Bacteria (eubacteria)
SequenceString: ATLKEKLITP VAAGSTVPSN KITVVGVGQV GMACAISILG KGLCDELALV DVLEDKLKGE MMDLQHGSLF LQTHKIVADK DYAVTANSK IVVVTAGVRQ QEGESRLNLV QRNVNVFKFI IPQIVKYSPN CTILVVSNPV DILTYVTWKL SGLPKHRVIG S GCNLDTAR ...String:
ATLKEKLITP VAAGSTVPSN KITVVGVGQV GMACAISILG KGLCDELALV DVLEDKLKGE MMDLQHGSLF LQTHKIVADK DYAVTANSK IVVVTAGVRQ QEGESRLNLV QRNVNVFKFI IPQIVKYSPN CTILVVSNPV DILTYVTWKL SGLPKHRVIG S GCNLDTAR FRYLMAERLG IHPTSCHGWI LGEHGDSSVA VWSGVNVAGV SLQELNPAMG TDKDSENWKE VHKQVVESAY EV IRLKGYT NWAIGLSVAE LCETMLKNLY RVHSVSTLVK GTYGIENDVF LSLPCVLSAS GLTSVINQKL KDDEVAQLKK SAD TLWSIQ KDLKD

UniProtKB: L-lactate dehydrogenase B chain

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 41 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.5 mg/mL
BufferpH: 7.4 / Component - Name: PBS / Details: Phosphate-buffered saline
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP / Details: Plunged into liquid ethane (LEICE EM GP).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.6 K / Max: 79.8 K
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 0-29 / Number real images: 1707 / Average exposure time: 0.2 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 101000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 270000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 508402
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.10)
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.
Number images used: 50865
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.10)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.10)

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Atomic model buiding 1

Initial modelPDB ID:

1i0z


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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