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- EMDB-22154: CryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rec... -

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Basic information

Entry
Database: EMDB / ID: EMD-22154
TitleCryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Map dataCryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Sample
  • Complex: G protein-gated inwardly rectifying potassium channel (GIRK2)
    • Protein or peptide: G protein-activated inward rectifier potassium channel 2
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: POTASSIUM IONPotassium
  • Ligand: SODIUM IONSodium
Function / homology
Function and homology information


G-protein activated inward rectifier potassium channel activity / monoatomic ion channel complex
Similarity search - Function
Potassium channel, inwardly rectifying, Kir3.2 / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / Immunoglobulin E-set
Similarity search - Domain/homology
G protein-activated inward rectifier potassium channel 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMathiharan YK / Glaaser IW / Skiniotis G / Slesinger PA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA)AA018734 United States
CitationJournal: Cell Rep / Year: 2021
Title: Structural insights into GIRK2 channel modulation by cholesterol and PIP.
Authors: Yamuna Kalyani Mathiharan / Ian W Glaaser / Yulin Zhao / Michael J Robertson / Georgios Skiniotis / Paul A Slesinger /
Abstract: G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, ...G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, which is elevated in the brains of people with neurodegenerative diseases such as Alzheimer's dementia and Parkinson's disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. In this study, we present cryo- electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and phosphatidylinositol 4,5-bisphosphate (PIP). The structures reveal that CHS binds near PIP in lipid-facing hydrophobic pockets of the transmembrane domain. Our structural analysis suggests that CHS stabilizes PIP interaction with the channel and promotes engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of one of the CHS binding pockets eliminates cholesterol-dependent potentiation of GIRK2. Elucidating the structural mechanisms underlying cholesterol modulation of GIRK2 channels could facilitate the development of therapeutics for treating neurological diseases. VIDEO ABSTRACT.
History
DepositionJun 14, 2020-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateSep 1, 2021-
Current statusSep 1, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xev
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22154.png

Downloads & links

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Map

FileDownload / File: emd_22154.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.0121 / Movie #1: 0.0121
Minimum - Maximum-0.021421747 - 0.060670067
Average (Standard dev.)0.00012061937 (±0.0024212222)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 224.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z224.400224.400224.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.0210.0610.000

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Supplemental data

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Sample components

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Entire : G protein-gated inwardly rectifying potassium channel (GIRK2)

EntireName: G protein-gated inwardly rectifying potassium channel (GIRK2)
Components
  • Complex: G protein-gated inwardly rectifying potassium channel (GIRK2)
    • Protein or peptide: G protein-activated inward rectifier potassium channel 2
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: POTASSIUM IONPotassium
  • Ligand: SODIUM IONSodium

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Supramolecule #1: G protein-gated inwardly rectifying potassium channel (GIRK2)

SupramoleculeName: G protein-gated inwardly rectifying potassium channel (GIRK2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: CryoEM structure of G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Komagataella pastoris (fungus)

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Macromolecule #1: G protein-activated inward rectifier potassium channel 2

MacromoleculeName: G protein-activated inward rectifier potassium channel 2
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 39.061957 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MAKRKIQRYV RKDGKCNVHH GNVRETYRYL TDIFTTLVDL KWRFNLLIFV MVYTVTWLFF GMIWWLIAYI RGDMDHIEDP SWTPCVTNL NGFVSAFLFS IETETTIGYG YRVITDKCPE GIILLLIQSV LGSIVNAFMV GCMFVKISQP KKRAETLVFS T HAVISMRD ...String:
MAKRKIQRYV RKDGKCNVHH GNVRETYRYL TDIFTTLVDL KWRFNLLIFV MVYTVTWLFF GMIWWLIAYI RGDMDHIEDP SWTPCVTNL NGFVSAFLFS IETETTIGYG YRVITDKCPE GIILLLIQSV LGSIVNAFMV GCMFVKISQP KKRAETLVFS T HAVISMRD GKLCLMFRVG DLRNSHIVEA SIRAKLIKSK QTSEGEFIPL NQTDINVGYY TGDDRLFLVS PLIISHEINQ QS PFWEISK AQLPKEELEI VVILEGMVEA TGMTCQARSS YITSEILWGY RFTPVLTLED GFYEVDYNSF HETYETSTPS LSA KELAEL ANRAESNSLE VLFQ

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Macromolecule #2: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 2 / Number of copies: 4 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 13 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #5: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 5 / Number of copies: 4
Molecular weightTheoretical: 22.99 Da

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
150.0 mMKClPotassium Chloride
20.0 mMC4H10O2S2DTT
3.0 mMC9H15O6PTCEP
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
0.025 %C24H46O11DDM
0.0025 %C31H50O4CHS
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsCryoEM structure G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 83.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 297174
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3sya

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 44990
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3sya

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6xev:
CryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2

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