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- PDB-6xiw: Cryo-EM structure of the sodium leak channel NALCN-FAM155A complex -

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Basic information

Entry
Database: PDB / ID: 6xiw
TitleCryo-EM structure of the sodium leak channel NALCN-FAM155A complex
Components
  • Sodium leak channel non-selective protein
  • Transmembrane protein FAM155ATransmembrane protein
KeywordsMEMBRANE PROTEIN / ion channel / complex / cysteine rich domain
Function / homology
Function and homology information


leak channel activity / regulation of resting membrane potential / calcium ion import across plasma membrane / sodium channel activity / voltage-gated ion channel activity / sodium ion transmembrane transport / potassium ion transmembrane transport / regulation of ion transmembrane transport / cation channel activity / calcium ion transmembrane transport ...leak channel activity / regulation of resting membrane potential / calcium ion import across plasma membrane / sodium channel activity / voltage-gated ion channel activity / sodium ion transmembrane transport / potassium ion transmembrane transport / regulation of ion transmembrane transport / cation channel activity / calcium ion transmembrane transport / ion transmembrane transport / go:0005623: / integral component of membrane / plasma membrane
Transmembrane protein FAM155 / Sodium leak channel non-selective protein / Voltage-dependent channel domain superfamily / Ion transport domain
Transmembrane protein FAM155A / Sodium leak channel non-selective protein
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKschonsak, M. / Chua, H.C. / Noland, C.L. / Weidling, C. / Clairfeuille, T. / Bahlke, O.O. / Ameen, A.O. / Li, Z.R. / Arthur, C.P. / Ciferri, C. / Pless, S.A. / Payandeh, J.
CitationJournal: Nature / Year: 2020
Title: Structure of the human sodium leak channel NALCN.
Authors: Marc Kschonsak / Han Chow Chua / Cameron L Noland / Claudia Weidling / Thomas Clairfeuille / Oskar Ørts Bahlke / Aishat Oluwanifemi Ameen / Zhong Rong Li / Christopher P Arthur / Claudio ...Authors: Marc Kschonsak / Han Chow Chua / Cameron L Noland / Claudia Weidling / Thomas Clairfeuille / Oskar Ørts Bahlke / Aishat Oluwanifemi Ameen / Zhong Rong Li / Christopher P Arthur / Claudio Ciferri / Stephan Alexander Pless / Jian Payandeh /
Abstract: Persistently depolarizing sodium (Na) leak currents that enhance electrical excitability have been described for decades. The entity responsible for the major background Na conductance in neurons had ...Persistently depolarizing sodium (Na) leak currents that enhance electrical excitability have been described for decades. The entity responsible for the major background Na conductance in neurons had remained a mystery until characterization of NALCN (Na leak channel, non-selective). NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm. NALCN activity is under tight regulation and NALCN mutations cause severe neurological disorders and early death. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here, we investigate this essential leak channel and determined the NALCN structure in complex with FAM155A (Family with sequence similarity 155, member A). FAM155A forms an extracellular dome that shields the ion selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Clues to the modulation of NALCN activity are revealed by unusual voltage-sensor domains with asymmetric linkages to the pore. We discover a tightly closed pore gate where the vast majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6-gate and distinctive π-bulges. Our study provides a framework to demystify the physiology of NALCN and a foundation to discover treatments for NALCN channelopathies and other electrical disorders.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Sodium leak channel non-selective protein
B: Transmembrane protein FAM155A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)267,54714
Polymers260,5472
Non-polymers7,00112
Water905
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 206341.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: TYR287 modeled with sulfonation (TYS) as the EM density indicates a post-translational modification of this residue.
Source: (gene. exp.) Homo sapiens (human) / Gene: NALCN, VGCNL1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8IZF0
#2: Protein Transmembrane protein FAM155A / Transmembrane protein / 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 / Production host: Homo sapiens (human) / References: UniProt: B1AL88

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Sugars , 1 types, 2 molecules

#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / 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 , 4 types, 15 molecules

#4: 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.: 5 / Source method: obtained synthetically / Formula: C39H78NO8P / Comment: POPE, phospholipid*YM
#5: 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.: 2 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
#6: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: C31H50O4
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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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: NALCN in complex with FAM155A / Type: COMPLEX
Details: NALCN, FAM155A, UNC80 and UNC79 co-expressed and NALCN-FAM155A co-purified
Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mMHEPESC8H18N2O4S1
33 mMcalcium chlorideCaCl21
SpecimenConc.: 2.4 mg/ml
Details: sample was gently cross-linked with 0.05% EM-grade glutaraldehyde for 10 min at room temperature and quenched with 0.09 M TRIS pH 7.5
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 / Nominal magnification: 165000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderModel: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 49.967 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15080
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEM3.7.11image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.8.9model fitting
8ISOLDE1,0b5model fitting
9UCSF Chimera1.02model fitting
11RELION3.1initial Euler assignment3D classification
12cisTEM1.02final Euler assignment
13cisTEM1.02classification
14cisTEM1.023D reconstruction
15PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1778009
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 365512 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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