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- PDB-7sx4: Human NALCN-FAM155A-UNC79-UNC80 channelosome with CaM bound, conf... -

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Basic information

Entry
Database: PDB / ID: 7sx4
TitleHuman NALCN-FAM155A-UNC79-UNC80 channelosome with CaM bound, conformation 2/2
Components
  • Calmodulin-1
  • Protein unc-80 homolog
  • Sodium leak channel non-selective protein,Enhanced green fluorescent protein
  • Transmembrane protein FAM155ATransmembrane protein
  • UNC79,Protein unc-79 homolog
KeywordsMEMBRANE PROTEIN / ion channel / calmodulin / HEAT repeat protein
Function / homology
Function and homology information


monoatomic cation homeostasis / positive regulation of synaptic transmission, cholinergic / leak channel activity / regulation of resting membrane potential / cation channel complex / sodium channel activity / voltage-gated sodium channel activity / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers ...monoatomic cation homeostasis / positive regulation of synaptic transmission, cholinergic / leak channel activity / regulation of resting membrane potential / cation channel complex / sodium channel activity / voltage-gated sodium channel activity / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / negative regulation of high voltage-gated calcium channel activity / monoatomic ion channel complex / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / positive regulation of ryanodine-sensitive calcium-release channel activity / Negative regulation of NMDA receptor-mediated neuronal transmission / regulation of cell communication by electrical coupling involved in cardiac conduction / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / calcium ion import across plasma membrane / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / Long-term potentiation / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / sodium ion transmembrane transport / DARPP-32 events / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / voltage-gated potassium channel complex / Activation of AMPK downstream of NMDARs / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / monoatomic cation channel activity / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Ion homeostasis / positive regulation of protein autophosphorylation / potassium ion transmembrane transport / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / monoatomic ion transmembrane transport / protein serine/threonine kinase activator activity / bioluminescence / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / generation of precursor metabolites and energy / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / positive regulation of synaptic transmission, GABAergic / calcium ion transmembrane transport / RAF activation / positive regulation of receptor signaling pathway via JAK-STAT / positive regulation of protein serine/threonine kinase activity / Transcriptional activation of mitochondrial biogenesis / Stimuli-sensing channels / spindle pole / cellular response to type II interferon / response to calcium ion
Similarity search - Function
Protein UNC80, C-terminal / Cation-channel complex subunit UNC-79 / Protein UNC80 C-terminal region / UNC79 / Cation channel complex component UNC80, N-terminal / Protein UNC80, central region / UNC80 N-terminal / Protein UNC80 central region / Sodium leak channel NALCN / Voltage-dependent channel domain superfamily ...Protein UNC80, C-terminal / Cation-channel complex subunit UNC-79 / Protein UNC80 C-terminal region / UNC79 / Cation channel complex component UNC80, N-terminal / Protein UNC80, central region / UNC80 N-terminal / Protein UNC80 central region / Sodium leak channel NALCN / Voltage-dependent channel domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair / Armadillo-type fold
Similarity search - Domain/homology
Chem-PEV / Chem-PGV / CHOLESTEROL HEMISUCCINATE / Enhanced green fluorescent protein / NALCN channel auxiliary factor 1 / Calmodulin-1 / Sodium leak channel NALCN / Protein unc-80 homolog / Protein unc-79 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKschonsak, M. / Chua, H.C. / Weidling, C. / Chakouri, N. / Noland, C.L. / Schott, K. / Chang, T. / Tam, C. / Patel, N. / Arthur, C.P. ...Kschonsak, M. / Chua, H.C. / Weidling, C. / Chakouri, N. / Noland, C.L. / Schott, K. / Chang, T. / Tam, C. / Patel, N. / Arthur, C.P. / Leitner, A. / Ben-Johny, M. / Ciferri, C. / Pless, S.A. / Payandeh, J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2022
Title: Structural architecture of the human NALCN channelosome.
Authors: Marc Kschonsak / Han Chow Chua / Claudia Weidling / Nourdine Chakouri / Cameron L Noland / Katharina Schott / Timothy Chang / Christine Tam / Nidhi Patel / Christopher P Arthur / Alexander ...Authors: Marc Kschonsak / Han Chow Chua / Claudia Weidling / Nourdine Chakouri / Cameron L Noland / Katharina Schott / Timothy Chang / Christine Tam / Nidhi Patel / Christopher P Arthur / Alexander Leitner / Manu Ben-Johny / Claudio Ciferri / Stephan Alexander Pless / Jian Payandeh /
Abstract: Depolarizing sodium (Na) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity. ...Depolarizing sodium (Na) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood. NALCN, UNC79 and UNC80 are essential in rodents, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.
History
DepositionNov 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 29, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Sodium leak channel non-selective protein,Enhanced green fluorescent protein
B: Transmembrane protein FAM155A
C: Calmodulin-1
D: UNC79,Protein unc-79 homolog
E: Protein unc-80 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)960,24312
Polymers956,8725
Non-polymers3,3727
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, identification of CaM, gel filtration, elutes as single peak on SEC
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein Sodium leak channel non-selective protein,Enhanced green fluorescent protein / CanIon / Voltage gated channel-like protein 1


Mass: 234128.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NALCN, VGCNL1, eGFP / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q8IZF0, UniProt: A0A7G8ZY66
#2: Protein Transmembrane protein FAM155A / Transmembrane protein


Mass: 54205.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAM155A / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: B1AL88
#3: Protein Calmodulin-1 /


Mass: 16852.545 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM1, CALM, CAM, CAM1 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: P0DP23
#4: Protein UNC79,Protein unc-79 homolog


Mass: 285174.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UNC79, UNC79, KIAA1409 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q9P2D8
#5: Protein Protein unc-80 homolog


Mass: 366510.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UNC80, C2orf21, KIAA1843 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q8N2C7

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Sugars , 1 types, 2 molecules

#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 5 molecules

#7: Chemical ChemComp-PEV / (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLETHANOLAMINE / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / Phosphatidylethanolamine


Mass: 720.012 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H78NO8P / Comment: POPE, phospholipid*YM
#8: Chemical ChemComp-PGV / (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE / PHOSPHATIDYLGLYCEROL / 2-VACCENOYL-1-PALMITOYL-SN-GLYCEROL-3-PHOSPHOGLYCEROL / Phosphatidylglycerol


Mass: 749.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
#9: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4

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Details

Has ligand of interestN

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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: Pentameric complex of the leak channel NALCN with FAM155A, Calmodulin, UNC79 and UNC80
Type: COMPLEX
Details: Calmodulin was not overexpressed but co-purified from Expi293 cells
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: Expi293
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mMHEPES1
SpecimenConc.: 1.65 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: NALCN was reconstituted into lipid nanodiscs and mildly crosslinkned with 0.05% EM grade glutaraldehyde for 10 min at room temperature. Cross-linking was quenched with 9 mM Tris pH 7.5
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3.5 sec blotting

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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 / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 64.009 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 26550
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEM3.7.11image acquisition
4CTFFIND4.1.13CTF correction
10cisTEM1.02initial Euler assignment
11cisTEM1.02final Euler assignment
12RELION3.1classification
13cisTEM1.023D reconstruction
CTF correctionDetails: selected micrographs with a CTF fit of 10.0 A or better
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3048401
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56037 / Num. of class averages: 1 / Symmetry type: POINT

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