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- PDB-8de8: Cryo-EM structure of the zebrafish two pore domain K+ channel TRE... -

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Basic information

Entry
Database: PDB / ID: 8de8
TitleCryo-EM structure of the zebrafish two pore domain K+ channel TREK1 (K2P2.1) in DDM/POPA mixed micelles
ComponentsPotassium channel, subfamily K, member 2a
KeywordsMEMBRANE PROTEIN / ion channel / K2P / K2P2.1 / TREK1 / TREK-1 / POPA
Function / homologyChem-D21 / : / :
Function and homology information
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsSchmidpeter, P.A.M. / Nimigean, C.M. / Riegelhaupt, P.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)K08GM132781 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM145918 United States
CitationJournal: Nat Commun / Year: 2023
Title: Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1.
Authors: Philipp A M Schmidpeter / John T Petroff / Leila Khajoueinejad / Aboubacar Wague / Cheryl Frankfater / Wayland W L Cheng / Crina M Nimigean / Paul M Riegelhaupt /
Abstract: Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within ...Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.
History
DepositionJun 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Potassium channel, subfamily K, member 2a
A: Potassium channel, subfamily K, member 2a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,97510
Polymers71,1192
Non-polymers2,8568
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Potassium channel, subfamily K, member 2a


Mass: 35559.383 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: kcnk2a / Production host: Komagataella pastoris (fungus) / References: UniProt: X1WC65
#2: Chemical
ChemComp-D21 / (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec-9-enoate


Mass: 674.929 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C37H71O8P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K
Has ligand of interestY

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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: Potassium channel subfamily K member 2 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 8 / Details: 150mM KCl, 20mM TRIS, 0.25mM DDM, 0.1mg/ml POPA
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 63.32 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1.2particle selectiontemplate based picking
2Leginonimage acquisition
4CTFFIND4CTF correctionInitial Estimation
5RELION3.1.2CTF correctionCTF Refinement
8UCSF Chimera1.14.0model fittingModel was first docked into density using Chimera Fit in Map tool
9Coot0.8.9.1model fittingModel was manually adjusted to fit map using Coot
11RELION3.1.2initial Euler assignment
12RELION3.1.2final Euler assignment
14RELION3.1.23D reconstruction
15PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 492284
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 303950 / Symmetry type: POINT

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