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- PDB-9eio: Cryo-EM structure of the mutant KCa2.2_F244S channel -

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Basic information

Entry
Database: PDB / ID: 9eio
TitleCryo-EM structure of the mutant KCa2.2_F244S channel
Components
  • Calmodulin-1
  • Small conductance calcium-activated potassium channel protein 2
KeywordsTRANSPORT PROTEIN / Ion channel / Calmodulin binding protein. Membrane protein
Function / homology
Function and homology information


Ca2+ activated K+ channels / small conductance calcium-activated potassium channel activity / membrane repolarization during atrial cardiac muscle cell action potential / calcium-activated potassium channel activity / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / positive regulation of potassium ion transport / inward rectifier potassium channel activity / establishment of protein localization to membrane / regulation of potassium ion transmembrane transport ...Ca2+ activated K+ channels / small conductance calcium-activated potassium channel activity / membrane repolarization during atrial cardiac muscle cell action potential / calcium-activated potassium channel activity / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / positive regulation of potassium ion transport / inward rectifier potassium channel activity / establishment of protein localization to membrane / regulation of potassium ion transmembrane transport / nitric-oxide synthase binding / organelle localization by membrane tethering / regulation of synaptic vesicle exocytosis / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / presynaptic endocytosis / regulation of cardiac muscle cell action potential / negative regulation of ryanodine-sensitive calcium-release channel activity / alpha-actinin binding / regulation of synaptic vesicle endocytosis / regulation of neuronal synaptic plasticity / protein phosphatase activator activity / postsynaptic cytosol / adenylate cyclase binding / regulation of ryanodine-sensitive calcium-release channel activity / catalytic complex / calcium channel regulator activity / detection of calcium ion / regulation of cardiac muscle contraction / phosphatidylinositol 3-kinase binding / calcium channel inhibitor activity / smooth endoplasmic reticulum / presynaptic cytosol / cellular response to interferon-beta / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / titin binding / voltage-gated potassium channel complex / T-tubule / sperm midpiece / potassium ion transmembrane transport / calcium channel complex / calyx of Held / nitric-oxide synthase regulator activity / response to amphetamine / adenylate cyclase activator activity / regulation of heart rate / sarcomere / protein serine/threonine kinase activator activity / regulation of cytokinesis / spindle microtubule / positive regulation of receptor signaling pathway via JAK-STAT / modulation of chemical synaptic transmission / potassium ion transport / sarcolemma / Schaffer collateral - CA1 synapse / cellular response to type II interferon / spindle pole / Z disc / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / growth cone / postsynaptic membrane / dendritic spine / transmembrane transporter binding / calmodulin binding / protein domain specific binding / neuronal cell body / centrosome / calcium ion binding / protein kinase binding / glutamatergic synapse / cell surface / protein homodimerization activity / protein-containing complex / mitochondrion / nucleoplasm / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Calmodulin-binding domain / Potassium channel, calcium-activated, SK / SK, calmodulin-binding domain superfamily / Calmodulin binding domain / Calcium-activated SK potassium channel / Calmodulin binding domain / Potassium channel domain / Ion channel / : / EF-hand domain pair ...Calmodulin-binding domain / Potassium channel, calcium-activated, SK / SK, calmodulin-binding domain superfamily / Calmodulin binding domain / Calcium-activated SK potassium channel / Calmodulin binding domain / Potassium channel domain / Ion channel / : / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
: / Calmodulin-1 / Small conductance calcium-activated potassium channel protein 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsNam, Y.W. / Zhang, M.
Funding support United States, 2items
OrganizationGrant numberCountry
American Heart Association23AIREA1039423 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS101182-03 United States
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM structures of the small-conductance Ca-activated K2.2 channel.
Authors: Young-Woo Nam / Dohyun Im / Ana Santa Cruz Garcia / Marios L Tringides / Hai Minh Nguyen / Yan Liu / Razan Orfali / Alena Ramanishka / Grigore Pintilie / Chih-Chia Su / Meng Cui / Diomedes E ...Authors: Young-Woo Nam / Dohyun Im / Ana Santa Cruz Garcia / Marios L Tringides / Hai Minh Nguyen / Yan Liu / Razan Orfali / Alena Ramanishka / Grigore Pintilie / Chih-Chia Su / Meng Cui / Diomedes E Logothetis / Edward W Yu / Heike Wulff / K George Chandy / Miao Zhang /
Abstract: Small-conductance Ca-activated K (K2.1-K2.3) channels modulate neuronal and cardiac excitability. We report cryo-electron microscopy structures of the K2.2 channel in complex with calmodulin and Ca, ...Small-conductance Ca-activated K (K2.1-K2.3) channels modulate neuronal and cardiac excitability. We report cryo-electron microscopy structures of the K2.2 channel in complex with calmodulin and Ca, alone or bound to two small molecule inhibitors, at 3.18, 3.50, 2.99 and 2.97 angstrom resolution, respectively. Extracellular S3-S4 loops in β-hairpin configuration form an outer canopy over the pore with an aromatic box at the canopy's center. Each S3-S4 β-hairpin is tethered to the selectivity filter in the neighboring subunit by inter-subunit hydrogen bonds. This hydrogen bond network flips the aromatic residue (Tyr362) in the filter's GYG signature by 180°, causing the outer selectivity filter to widen and water to enter the filter. Disruption of the tether by a mutation narrows the outer selectivity filter, realigns Tyr362 to the position seen in other K channels, and significantly increases unitary conductance. UCL1684, a mimetic of the bee venom peptide apamin, sits atop the canopy and occludes the opening in the aromatic box. AP14145, an analogue of a therapeutic for atrial fibrillation, binds in the central cavity below the selectivity filter and induces closure of the inner gate. These structures provide a basis for understanding the small unitary conductance and pharmacology of K2.x channels.
History
DepositionNov 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Small conductance calcium-activated potassium channel protein 2
B: Small conductance calcium-activated potassium channel protein 2
C: Small conductance calcium-activated potassium channel protein 2
D: Small conductance calcium-activated potassium channel protein 2
E: Calmodulin-1
F: Calmodulin-1
G: Calmodulin-1
H: Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,34720
Polymers228,8708
Non-polymers47712
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Small conductance calcium-activated potassium channel protein 2 / SK2 / SKCa 2 / SKCa2 / KCa2.2


Mass: 41114.754 Da / Num. of mol.: 4 / Fragment: UNP residues 118-478
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Kcnn2 / Production host: Homo sapiens (human) / References: UniProt: P70604
#2: Protein
Calmodulin-1


Mass: 16102.626 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Calm1, Calm, Cam, Cam1, CaMI / Production host: Homo sapiens (human) / References: UniProt: P0DP29
#3: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: Rat mutant KCa2.2_F244S channel complex with calmodulin in the presence of calcium
Type: COMPLEX / Details: Mutant KCa2.2_F244S / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293s / Cell: HEK293s / Plasmid: pEGBacMam
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2300 nm / Nominal defocus min: 1300 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF ChimeraX1.8model fitting
13PHENIX1.21.1_5286:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81168 / Symmetry type: POINT
RefinementHighest resolution: 3.62 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214870
ELECTRON MICROSCOPYf_angle_d0.50920092
ELECTRON MICROSCOPYf_dihedral_angle_d4.4132049
ELECTRON MICROSCOPYf_chiral_restr0.0392369
ELECTRON MICROSCOPYf_plane_restr0.0022529

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