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- EMDB-22203: Cryo-EM structure of the sodium leak channel NALCN-FAM155A complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22203
TitleCryo-EM structure of the sodium leak channel NALCN-FAM155A complex
Map dataSharpened map used for model refinement
Sample
  • Complex: NALCN in complex with FAM155A
    • Protein or peptide: Sodium leak channel non-selective protein
    • Protein or peptide: Transmembrane protein FAM155ATransmembrane protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
Function / homology
Function and homology information


positive regulation of synaptic transmission, cholinergic / leak channel activity / regulation of resting membrane potential / sodium channel activity / voltage-gated sodium channel activity / monoatomic ion channel complex / calcium ion import across plasma membrane / sodium ion transmembrane transport / monoatomic cation channel activity / potassium ion transmembrane transport ...positive regulation of synaptic transmission, cholinergic / leak channel activity / regulation of resting membrane potential / sodium channel activity / voltage-gated sodium channel activity / monoatomic ion channel complex / calcium ion import across plasma membrane / sodium ion transmembrane transport / monoatomic cation channel activity / potassium ion transmembrane transport / monoatomic ion transmembrane transport / positive regulation of synaptic transmission, GABAergic / calcium ion transmembrane transport / Stimuli-sensing channels / plasma membrane
Similarity search - Function
Sodium leak channel NALCN / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
NALCN channel auxiliary factor 1 / Sodium leak channel NALCN
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKschonsak M / Chua HC / Noland CL / Weidling C / Clairfeuille T / Bahlke OO / Ameen AO / Li ZR / Arthur CP / Ciferri C ...Kschonsak M / Chua HC / Noland CL / Weidling C / Clairfeuille T / Bahlke OO / Ameen AO / Li ZR / Arthur CP / Ciferri C / Pless SA / 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 enhance electrical excitability. The ion channel responsible for the major background Na conductance in neurons is the Na leak channel, non- ...Persistently depolarizing sodium (Na) leak currents enhance electrical excitability. The ion channel responsible for the major background Na conductance in neurons is the Na leak channel, non-selective (NALCN). NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm. NALCN activity is under tight regulation and mutations in NALCN 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 structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). 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. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.
History
DepositionJun 22, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xiw
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22203.png

Downloads & links

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Map

FileDownload / File: emd_22203.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used for model refinement
Voxel sizeX=Y=Z: 0.824 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.17
Minimum - Maximum-0.6131234 - 0.92917466
Average (Standard dev.)-0.0042198454 (±0.038623333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 296.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8240.8240.824
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z296.640296.640296.640
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.6130.929-0.004

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Supplemental data

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Additional map: non-sharpened map

Fileemd_22203_additional.map
Annotationnon-sharpened 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 : NALCN in complex with FAM155A

EntireName: NALCN in complex with FAM155A
Components
  • Complex: NALCN in complex with FAM155A
    • Protein or peptide: Sodium leak channel non-selective protein
    • Protein or peptide: Transmembrane protein FAM155ATransmembrane protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water

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Supramolecule #1: NALCN in complex with FAM155A

SupramoleculeName: NALCN in complex with FAM155A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: NALCN, FAM155A, UNC80 and UNC79 co-expressed and NALCN-FAM155A co-purified
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Macromolecule #1: Sodium leak channel non-selective protein

MacromoleculeName: Sodium leak channel non-selective protein / type: protein_or_peptide / ID: 1
Details: TYR287 modeled with sulfonation (TYS) as the EM density indicates a post-translational modification of this residue.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 206.341641 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLKRKQSSRV EAQPVTDFGP DESLSDNADI LWINKPWVHS LLRICAIISV ISVCMNTPMT FEHYPPLQYV TFTLDTLLMF LYTAEMIAK MHIRGIVKGD SSYVKDRWCV FDGFMVFCLW VSLVLQVFEI ADIVDQMSPW GMLRIPRPLI MIRAFRIYFR F ELPRTRIT ...String:
MLKRKQSSRV EAQPVTDFGP DESLSDNADI LWINKPWVHS LLRICAIISV ISVCMNTPMT FEHYPPLQYV TFTLDTLLMF LYTAEMIAK MHIRGIVKGD SSYVKDRWCV FDGFMVFCLW VSLVLQVFEI ADIVDQMSPW GMLRIPRPLI MIRAFRIYFR F ELPRTRIT NILKRSGEQI WSVSIFLLFF LLLYGILGVQ MFGTFTYHCV VNDTKPGNVT WNSLAIPDTH CSPELEEGYQ CP PGFKCMD LEDLGLSRQE LGYSGFNEIG TSIFTVYEAA SQEGWVFLM(TYS) RAIDSFPRWR SYFYFITLIF FLAWLVKNV FIAVIIETFA EIRVQFQQMW GSRSSTTSTA TTQMFHEDAA GGWQLVAVDV NKPQGRAPAC LQKMMRSSVF HMFILSMVTV DVIVAASNY YKGENFRRQY DEFYLAEVAF TVLFDLEALL KIWCLGFTGY ISSSLHKFEL LLVIGTTLHV YPDLYHSQFT Y FQVLRVVR LIKISPALED FVYKIFGPGK KLGSLVVFTA SLLIVMSAIS LQMFCFVEEL DRFTTFPRAF MSMFQILTQE GW VDVMDQT LNAVGHMWAP VVAIYFILYH LFATLILLSL FVAVILDNLE LDEDLKKLKQ LKQSEANADT KEKLPLRLRI FEK FPNRPQ MVKISKLPSD FTVPKIRESF MKQFIDRQQQ DTCCLLRSLP TTSSSSCDHS KRSAIEDNKY IDQKLRKSVF SIRA RNLLE KETAVTKILR ACTRQRMLSG SFEGQPAKER SILSVQHHIR QERRSLRHGS NSQRISRGKS LETLTQDHSN TVRYR NAQR EDSEIKMIQE KKEQAEMKRK VQEEELRENH PYFDKPLFIV GREHRFRNFC RVVVRARFNA SKTDPVTGAV KNTKYH QLY DLLGLVTYLD WVMIIVTICS CISMMFESPF RRVMHAPTLQ IAEYVFVIFM SIELNLKIMA DGLFFTPTAV IRDFGGV MD IFIYLVSLIF LCWMPQNVPA ESGAQLLMVL RCLRPLRIFK LVPQMRKVVR ELFSGFKEIF LVSILLLTLM LVFASFGV Q LFAGKLAKCN DPNIIRREDC NGIFRINVSV SKNLNLKLRP GEKKPGFWVP RVWANPRNFN FDNVGNAMLA LFEVLSLKG WVEVRDVIIH RVGPIHGIYI HVFVFLGCMI GLTLFVGVVI ANFNENKGTA LLTVDQRRWE DLKSRLKIAQ PLHLPPRPDN DGFRAKMYD ITQHPFFKRT IALLVLAQSV LLSVKWDVED PVTVPLATMS VVFTFIFVLE VTMKIIAMSP AGFWQSRRNR Y DLLVTSLG VVWVVLHFAL LNAYTYMMGA CVIVFRFFSI CGKHVTLKML LLTVVVSMYK SFFIIVGMFL LLLCYAFAGV VL FGTVKYG ENINRHANFS SAGKAITVLF RIVTGEDWNK IMHDCMVQPP FCTPDEFTYW ATDCGNYAGA LMYFCSFYVI IAY IMLNLL VAIIVENFSL FYSTEEDQLL SYNDLRHFQI IWNMVDDKRE GVIPTFRVKF LLRLLRGRLE VDLDKDKLLF KHMC YEMER LHNGGDVTFH DVLSMLSYRS VDIRKSLQLE ELLAREQLEY TIEEEVAKQT IRMWLKKCLK RIRAKQQQSC SIIHS LRES QQQELSRFLN PPSIETTQPS EDTNANSQDN SMQPETSSQQ QLLSPTLSDR GGSRQDAADA GKPQRKFGQW RLPSAP KPI SHSVSSVNLR FGGRTTMKSV VCKMNPMTDA ASCGSEVKKW WTRQLTVESD ESGDDLLDIG GGSGGWSHPQ FEKGGGS GG GSGGSAWSHP QFEKGSGDYK DDDDKGNSDY KDDDDK

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Macromolecule #2: Transmembrane protein FAM155A

MacromoleculeName: Transmembrane protein FAM155A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.205004 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTRGAWMCRQ YDDGLKIWLA APRENEKPFI DSERAQKWRL SLASLLFFTV LLSDHLWFCA EAKLTRARDK EHQQQQRQQQ QQQQQQRQR QQQQQQRRQQ EPSWPALLAS MGESSPAAQA HRLLSASSSP TLPPSPGDGG GGGGKGNRGK DDRGKALFLG N SAKPVWRL ...String:
MTRGAWMCRQ YDDGLKIWLA APRENEKPFI DSERAQKWRL SLASLLFFTV LLSDHLWFCA EAKLTRARDK EHQQQQRQQQ QQQQQQRQR QQQQQQRRQQ EPSWPALLAS MGESSPAAQA HRLLSASSSP TLPPSPGDGG GGGGKGNRGK DDRGKALFLG N SAKPVWRL ETCYPQGASS GQCFTVENAD AVCARNWSRG AAGGDGQEVR SKHPTPLWNL SDFYLSFCNS YTLWELFSGL SS PNTLNCS LDVVLKEGGE MTTCRQCVEA YQDYDHHAQE KYEEFESVLH KYLQSEEYSV KSCPEDCKIV YKAWLCSQYF EVT QFNCRK TIPCKQYCLE VQTRCPFILP DNDEVIYGGL SSFICTGLYE TFLTNDEPEC CDVRREEKSN NPSKGTVEKS GSCH RTSLT VSSATRLCNS RLKLCVLVLI LLHTVLTASA AQNTAGLSFG GINTLEENST NEEGGSGGSD YKDDDDKGNS DYKDD DDK

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #4: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY...

MacromoleculeName: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 4 / Number of copies: 5 / Formula: PEV
Molecular weightTheoretical: 720.012 Da
Chemical component information

ChemComp-PEV:
(1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / POPE, phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #5: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE
type: ligand / ID: 5 / Number of copies: 2 / Formula: PGV
Molecular weightTheoretical: 749.007 Da
Chemical component information

ChemComp-PGV:
(1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE / phospholipid*YM / Phosphatidylglycerol

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Macromolecule #6: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 6 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration2.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
25.0 mMC8H18N2O4SHEPES
3.0 mMCaCl2calcium chloride
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailssample 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 15080 / Average exposure time: 10.0 sec. / Average electron dose: 49.967 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1778009
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Startup modelType of model: EMDB MAP
EMDB ID:

9024


Details: Map low-pass filtered to 20 angstrom resolution and density outside nanodisc deleted
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: 3D classification
Final 3D classificationNumber classes: 6 / Software - Name: cisTEM (ver. 1.02)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.02)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.02) / Number images used: 365512

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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