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- PDB-6xit: Cryo-EM structure of the G protein-gated inward rectifier K+ chan... -

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Basic information

Entry
Database: PDB / ID: 6xit
TitleCryo-EM structure of the G protein-gated inward rectifier K+ channel GIRK2 (Kir3.2) in complex with PIP2
ComponentsG protein-activated inward rectifier potassium channel 2
KeywordsMEMBRANE PROTEIN / G protein-coupled inwardly rectifying potassium channels / PIP2
Function / homology
Function and homology information


G-protein activated inward rectifier potassium channel activity / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / inward rectifier potassium channel activity / regulation of monoatomic ion transmembrane transport / parallel fiber to Purkinje cell synapse / monoatomic ion channel complex / neuronal cell body membrane / potassium ion import across plasma membrane ...G-protein activated inward rectifier potassium channel activity / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / inward rectifier potassium channel activity / regulation of monoatomic ion transmembrane transport / parallel fiber to Purkinje cell synapse / monoatomic ion channel complex / neuronal cell body membrane / potassium ion import across plasma membrane / potassium channel activity / G-protein alpha-subunit binding / negative regulation of insulin secretion / presynaptic membrane / axon / dendrite / cell surface / plasma membrane
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
: / Chem-PIO / G protein-activated inward rectifier potassium channel 2 / G protein-activated inward rectifier potassium channel 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNiu, Y. / Tao, X. / MacKinnon, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
CitationJournal: Elife / Year: 2020
Title: Cryo-EM analysis of PIP regulation in mammalian GIRK channels.
Authors: Yiming Niu / Xiao Tao / Kouki K Touhara / Roderick MacKinnon /
Abstract: G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of ...G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP concentration. We find that PIP shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form. In the docked form the cytoplasmic domain, to which G binds, becomes accessible to the cytoplasmic membrane surface where G resides. Furthermore, PIP binding reshapes the G binding surface on the cytoplasmic domain, preparing it to receive G. We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP-induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by G.
History
DepositionJun 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: G protein-activated inward rectifier potassium channel 2
B: G protein-activated inward rectifier potassium channel 2
C: G protein-activated inward rectifier potassium channel 2
D: G protein-activated inward rectifier potassium channel 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,35111
Polymers156,2484
Non-polymers3,1047
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Elution volume roughly equals to ~200 kDa molecular weight complex
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 39061.957 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kcnj6 / Production host: Komagataella pastoris (fungus) / References: UniProt: A0A338P6L0, UniProt: P48542*PLUS
#2: Chemical
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 / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C25H49O19P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
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: Homo-tetrameric assembly of G protein-activated inward rectifier potassium channel 2
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 150 mM KCl, 10 mM DTT, 1 mM DETA, 0.2% DM and 0.01% CHS
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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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 / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11349
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.0.6CTF correction
7UCSF Chimera1.14model fitting
8Coot0.9model fitting
9PHENIX1.15.2model fitting
11RELION3initial Euler assignment
12cryoSPARC2.9.0initial Euler assignment
13RELION3.1final Euler assignment
14RELION3classification
15RELION3.13D reconstruction
16PHENIX1.15.2model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155128 / Symmetry type: POINT
Atomic model buildingPDB-ID: 3SYA

3sya


Pdb chain-ID: A / Pdb chain residue range: 53-380

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