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- EMDB-48154: single particle cryo EM density of full length rat Kir4.1 -

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Basic information

Entry
Database: EMDB / ID: EMD-48154
Titlesingle particle cryo EM density of full length rat Kir4.1
Map datasingle particle cryo EM map of full length rat Kir4.1 in complex with a LC8 dimer
Sample
  • Complex: Complex between Kir4.1 and dynein light chain 1 dimer
    • Complex: Kir4.1
    • Complex: dynein light chain 1
    • Protein or peptide: rat Kir4.1
    • Protein or peptide: Dynein light chain 1
KeywordsPotassium ion channel Complex with auxiliary proteins / TRANSPORT PROTEIN
Function / homology
Function and homology information


glutamate reuptake / Potassium transport channels / deoxyribonuclease inhibitor activity / negative regulation of DNA strand resection involved in replication fork processing / intraciliary retrograde transport / L-glutamate import / response to blue light / motile cilium assembly / Activation of BIM and translocation to mitochondria / response to mineralocorticoid ...glutamate reuptake / Potassium transport channels / deoxyribonuclease inhibitor activity / negative regulation of DNA strand resection involved in replication fork processing / intraciliary retrograde transport / L-glutamate import / response to blue light / motile cilium assembly / Activation of BIM and translocation to mitochondria / response to mineralocorticoid / negative regulation of phosphorylation / central nervous system myelination / ciliary tip / Intraflagellar transport / regulation of resting membrane potential / oligodendrocyte development / cellular response to potassium ion / astrocyte projection / inward rectifier potassium channel activity / potassium ion homeostasis / regulation of monoatomic ion transmembrane transport / non-motile cilium assembly / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / dynein complex / membrane hyperpolarization / COPI-independent Golgi-to-ER retrograde traffic / cytoplasmic dynein complex / adult walking behavior / dynein intermediate chain binding / optic nerve development / Macroautophagy / ciliary base / potassium ion import across plasma membrane / enzyme inhibitor activity / microvillus / tertiary granule membrane / ficolin-1-rich granule membrane / monoatomic ion channel complex / potassium channel activity / response to glucocorticoid / COPI-mediated anterograde transport / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / potassium ion transmembrane transport / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / substantia nigra development / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / visual perception / Recruitment of mitotic centrosome proteins and complexes / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / AURKA Activation by TPX2 / regulation of membrane potential / establishment of localization in cell / RHO GTPases Activate Formins / regulation of long-term neuronal synaptic plasticity / potassium ion transport / kinetochore / HCMV Early Events / Aggrephagy / mitotic spindle / Separation of Sister Chromatids / Regulation of PLK1 Activity at G2/M Transition / presynapse / site of double-strand break / cell body / basolateral plasma membrane / microtubule / cytoskeleton / cilium / apical plasma membrane / signaling receptor binding / apoptotic process / centrosome / DNA damage response / Neutrophil degranulation / mitochondrion / ATP binding / identical protein binding / nucleus / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Potassium channel, inwardly rectifying, Kir1.2 / Dynein light chain, type 1/2, conserved site / Dynein light chain type 1 signature. / Dynein light chain, type 1/2 / Dynein light chain type 1 / Dynein light chain type 1 / Dynein light chain superfamily / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic ...Potassium channel, inwardly rectifying, Kir1.2 / Dynein light chain, type 1/2, conserved site / Dynein light chain type 1 signature. / Dynein light chain, type 1/2 / Dynein light chain type 1 / Dynein light chain type 1 / Dynein light chain superfamily / 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
ATP-sensitive inward rectifier potassium channel 10 / Dynein light chain 1, cytoplasmic
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.69 Å
AuthorsLee S-J / Mount J / Nichols CG
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R35 ML140024 United States
CitationJournal: J Biol Chem / Year: 2025
Title: Dynein light chains 1 and 2 are auxiliary proteins of pH-sensitive Kir4.1 channels.
Authors: Sun-Joo Lee / Jian Gao / Ellen Thompson / Jonathan Mount / Colin G Nichols /
Abstract: Inward rectifier Kir4.1 potassium channels are abundantly expressed in cells that are important for electrolyte homeostasis. Dysregulation of Kir4.1 underlies various neurological disorders. Here, ...Inward rectifier Kir4.1 potassium channels are abundantly expressed in cells that are important for electrolyte homeostasis. Dysregulation of Kir4.1 underlies various neurological disorders. Here, through biochemical and structural studies of full-length Kir4.1, we show that dynein light chain 1 and 2 proteins, also as known as LC8, copurify with Kir4.1 at stoichiometric levels. Direct interaction between Kir4.1 and LC8 is supported by in vitro binding assays and reiterated with native Kir4.1 proteins from mouse brain. Notably, we identify a LC8 binding motif in the unstructured N terminus of Kir4.1. Among Kir subtypes, the motif is unique to Kir4.1 and is highly conserved between Kir4.1 orthologs. Deletion of the predicted anchoring sequence (ΔTQT) resulted in loss of LC8 interaction with Kir4.1 N-terminal peptides as well as with full-length Kir4.1, suggesting that the binding site is necessary and sufficient for the interaction. Purified Kir4.1-ΔTQT mutant proteins exhibited normal channel activity in vitro, whereas WT proteins lost phosphoinositide-(4,5)-phosphate activation. Single-particle cryo-EM analysis of the full-length proteins revealed extremely heterogeneous particles, indicating deformation from the typical fourfold symmetric conformation. Additional electron density attached to the Kir4.1 tetramer, ascribed to an LC8 dimer, further supports the direct interaction between the two proteins. While the biological implications of this interaction await further elucidation, the strong conservation of the LC8 binding motif suggests its potential importance in the regulation of Kir4.1 channels.
History
DepositionDec 4, 2024-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48154.png

Downloads & links

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Map

FileDownload / File: emd_48154.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsingle particle cryo EM map of full length rat Kir4.1 in complex with a LC8 dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 200 pix.
= 262.8 Å		1.31 Å/pix.
x 200 pix.
= 262.8 Å		1.31 Å/pix.
x 200 pix.
= 262.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.314 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.269
Minimum - Maximum-0.21949987 - 0.6768058
Average (Standard dev.)0.00076346117 (±0.04499641)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 262.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_48154_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_48154_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between Kir4.1 and dynein light chain 1 dimer

EntireName: Complex between Kir4.1 and dynein light chain 1 dimer
Components
  • Complex: Complex between Kir4.1 and dynein light chain 1 dimer
    • Complex: Kir4.1
    • Complex: dynein light chain 1
    • Protein or peptide: rat Kir4.1
    • Protein or peptide: Dynein light chain 1

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Supramolecule #1: Complex between Kir4.1 and dynein light chain 1 dimer

SupramoleculeName: Complex between Kir4.1 and dynein light chain 1 dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Native dynein light chain 1 was copurified with Kir4.1 overexpressed in HEK293 cells
Molecular weightTheoretical: 180 KDa

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Supramolecule #2: Kir4.1

SupramoleculeName: Kir4.1 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: dynein light chain 1

SupramoleculeName: dynein light chain 1 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: rat Kir4.1

MacromoleculeName: rat Kir4.1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPAAATSVAK VYYSQTTQTE SRPLVAPGIR RRRVLTKDGR SNVRMEHIAD KRFLYLKDLW TTFIDMQWRY KLLLFSATFA GTWFLFGVVW YLVAVAHGDL LELGPPANHT PCVVQVHTLT GAFLFSLESQ TTIGYGFRYI SEECPLAIVL LIAQLVLTTI LEIFITGTFL ...String:
GPAAATSVAK VYYSQTTQTE SRPLVAPGIR RRRVLTKDGR SNVRMEHIAD KRFLYLKDLW TTFIDMQWRY KLLLFSATFA GTWFLFGVVW YLVAVAHGDL LELGPPANHT PCVVQVHTLT GAFLFSLESQ TTIGYGFRYI SEECPLAIVL LIAQLVLTTI LEIFITGTFL AKIARPKKRA ETIRFSQHAV VAYHNGKLCL MIRVANMRKS LLIGCQVTGK LLQTHQTKEG ENIRLNQVNV TFQVDTASDS PFLILPLTFY HVVDETSPLK DLPLRSGEGD FELVLILSGT VESTSATCQV RTSYLPEEIL WGYEFTPAIS LSASGKYVAD FSLFDQVVKV ASPGGLRDST VRYGDPEKLK LEESLREQAE KEGSALSVRI SNV

UniProtKB: ATP-sensitive inward rectifier potassium channel 10

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Macromolecule #2: Dynein light chain 1

MacromoleculeName: Dynein light chain 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MCDRKAVIKN ADMSEEMQQD SVECATQALE KYNIEKDIAA HIKKEFDKKY NPTWHCIVGR NFGSYVTHET KHFIYFYLGQ VAILLFKSG

UniProtKB: Dynein light chain 1, cytoplasmic

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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

Concentration7 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMpotassium chlorideKCl
1.0 mMTCEP
1.0 mMEDTA
0.04 mMglyco-diosgenin

Details: diC8 PIP2 was added 30 min prior to vitrification.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.009300000000000001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 2 second blotting.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 13522 / Number real images: 296755 / Average exposure time: 4.28 sec. / Average electron dose: 48.42 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
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

Detailscounting
Particle selectionNumber selected: 4273763
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 296755
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final 3D classificationNumber classes: 6 / Avg.num./class: 150000 / Software - Name: cryoSPARC (ver. 4.3)
FSC plot (resolution estimation)

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