EMDB-25492: Cryo-EM structure of the human NALCN channelosome NALCN-FAM155A-C...

Basic information

• Entry: Database: EMDB / ID: EMD-25492
• Title: Cryo-EM structure of the human NALCN channelosome NALCN-FAM155A-CaM-UNC79-UNC80 complex conformation 1
• Map data: combined map of focused refinements used for model refinements

Sample

• Complex: Pentameric complex of the leak channel NALCN with FAM155A, Calmodulin, UNC79 and UNC80
  • Protein or peptide: Sodium leak channel non-selective protein,Enhanced green fluorescent protein
  • Protein or peptide: Transmembrane protein FAM155A
  • Protein or peptide: Calmodulin-1
  • Protein or peptide: UNC79,Protein unc-79 homolog,Protein unc-79 homolog
  • Protein or peptide: Protein unc-80 homolog
• 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

• Keywords: ion channel / calmodulin / HEAT repeat protein / MEMBRANE PROTEIN

Function / homology

Function:

monoatomic cation homeostasis / positive regulation of synaptic transmission, cholinergic / leak channel activity / regulation of resting membrane potential / cation channel complex / 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 / voltage-gated sodium channel activity / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / CaMK IV-mediated phosphorylation of CREB / negative regulation of high voltage-gated calcium channel activity / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / Activation of RAC1 downstream of NMDARs / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / presynaptic endocytosis / sodium channel activity / regulation of cardiac muscle cell action potential / positive regulation of ryanodine-sensitive calcium-release channel activity / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of ryanodine-sensitive calcium-release channel activity / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / calcineurin-mediated signaling / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / calcium ion import across plasma membrane / Long-term potentiation / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / protein phosphatase activator activity / regulation of ryanodine-sensitive calcium-release channel activity / DARPP-32 events / monoatomic ion channel complex / Smooth Muscle Contraction / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / presynaptic cytosol / calcium channel inhibitor activity / cellular response to interferon-beta / monoatomic cation channel activity / Protein methylation / Activation of AMPK downstream of NMDARs / Ion homeostasis / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / regulation of calcium-mediated signaling / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / titin binding / voltage-gated potassium channel complex / potassium ion transmembrane transport / sperm midpiece / substantia nigra development / calcium channel complex / sodium ion transmembrane transport / calyx of Held / FCERI mediated Ca+2 mobilization / Ras activation upon Ca2+ influx through NMDA receptor / FCGR3A-mediated IL10 synthesis / adenylate cyclase activator activity / regulation of heart rate / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / bioluminescence / protein serine/threonine kinase activator activity / VEGFR2 mediated cell proliferation / sarcomere / regulation of cytokinesis / VEGFR2 mediated vascular permeability / positive regulation of synaptic transmission, GABAergic / generation of precursor metabolites and energy / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of receptor signaling pathway via JAK-STAT / spindle microtubule / RAF activation / Transcriptional activation of mitochondrial biogenesis / calcium ion transmembrane transport / Stimuli-sensing channels / cellular response to type II interferon / long-term synaptic potentiation / response to calcium ion / RAS processing / spindle pole / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants

Domain/homology:

UNC79 / Cation channel complex component UNC80, N-terminal / Protein UNC80, central region / Protein UNC80, C-terminal / Cation-channel complex subunit UNC-79 / UNC80 N-terminal / Protein UNC80 central region / Protein UNC80 C-terminal 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. / EF-hand domain / Ion transport domain / Ion transport protein / EF-hand domain pair / Armadillo-type fold

Component:

Enhanced green fluorescent protein / NALCN channel auxiliary factor 1 / Calmodulin-1 / Sodium leak channel NALCN / Protein unc-80 homolog / Protein unc-79 homolog

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.1 Å
• Authors: Kschonsak M / Chua HC

Funding support

1 items

Organization	Grant number	Country
Not funded

• Citation: Journal: 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 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. 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

Deposition	Nov 22, 2021	-
Header (metadata) release	Dec 29, 2021	-
Map release	Dec 29, 2021	-
Update	Oct 9, 2024	-
Current status	Oct 9, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_25492.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: combined map of focused refinements used for model refinements
• Voxel size: X=Y=Z: 1.1732 Å

Density

Contour Level	By AUTHOR: 7.9 / Movie #1: 9
Minimum - Maximum	-6.452718 - 68.236879999999999
Average (Standard dev.)	1.9775281 (±1.6830304)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 400 400 400 Spacing 400 400 400
Cell	A=B=C: 469.28 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.1732	1.1732	1.1732
M x/y/z	400	400	400
origin x/y/z	0.000	0.000	0.000
length x/y/z	469.280	469.280	469.280
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	320	320	320
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	400	400	400
D min/max/mean	-6.453	68.237	1.978

Supplemental data

Additional map: map (non-sharpened) of focused NALCN-FAM region containing the...

• File: emd_25492_additional_1.map
• Annotation: map (non-sharpened) of focused NALCN-FAM region containing the FAM155A, CaM and NALCN regions

Additional map: halfmap 2 of focused UNC-crossover region containing the...

• File: emd_25492_additional_10.map
• Annotation: halfmap 2 of focused UNC-crossover region containing the central regions of UNC79 and UNC80

Additional map: halfmap 1 of focused UNC-crossover region containing the...

• File: emd_25492_additional_11.map
• Annotation: halfmap 1 of focused UNC-crossover region containing the central regions of UNC79 and UNC80

Additional map: sharpened map of focused UNC-CN region containing the...

• File: emd_25492_additional_12.map
• Annotation: sharpened map of focused UNC-CN region containing the C-terminus of UNC79 and the N-terminus of UNC80 used for generating the combined map

Additional map: map (non-sharpened) of focused UNC-CN region containing the...

• File: emd_25492_additional_13.map
• Annotation: map (non-sharpened) of focused UNC-CN region containing the C-terminus of UNC79 and the N-terminus of UNC80

Additional map: halfmap 2 of focused UNC-CN region containing the...

• File: emd_25492_additional_14.map
• Annotation: halfmap 2 of focused UNC-CN region containing the C-terminus of UNC79 and the N-terminus of UNC80

Additional map: halfmap 1 of focused UNC-CN region containing the...

• File: emd_25492_additional_15.map
• Annotation: halfmap 1 of focused UNC-CN region containing the C-terminus of UNC79 and the N-terminus of UNC80

Additional map: sharpened map of focused NALCN-FAM region containing the...

• File: emd_25492_additional_16.map
• Annotation: sharpened map of focused NALCN-FAM region containing the FAM155A, CaM and NALCN regions used for generating the combined map

Additional map: halfmap 1 of focused NALCN-FAM region containing the...

• File: emd_25492_additional_17.map
• Annotation: halfmap 1 of focused NALCN-FAM region containing the FAM155A, CaM and NALCN regions

Additional map: halfmap 2 of focused NALCN-FAM region containing the...

• File: emd_25492_additional_2.map
• Annotation: halfmap 2 of focused NALCN-FAM region containing the FAM155A, CaM and NALCN regions

Additional map: map (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM used for alignment...

• File: emd_25492_additional_3.map
• Annotation: map (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM used for alignment of focused maps

Additional map: sharpened map of focused UNC-NC region containing the...

• File: emd_25492_additional_4.map
• Annotation: sharpened map of focused UNC-NC region containing the N-terminus of UNC79 and the C-terminus of UNC80 used for generating the combined map

Additional map: map (non-sharpened) of focused UNC-NC region containing the...

• File: emd_25492_additional_5.map
• Annotation: map (non-sharpened) of focused UNC-NC region containing the N-terminus of UNC79 and the C-terminus of UNC80

Additional map: halfmap 2 of focused UNC-NC region containing the...

• File: emd_25492_additional_6.map
• Annotation: halfmap 2 of focused UNC-NC region containing the N-terminus of UNC79 and the C-terminus of UNC80

Additional map: halfmap 1 of focused UNC-NC region containing the...

• File: emd_25492_additional_7.map
• Annotation: halfmap 1 of focused UNC-NC region containing the N-terminus of UNC79 and the C-terminus of UNC80

Additional map: sharpened map of focused UNC-crossover region containing the...

• File: emd_25492_additional_8.map
• Annotation: sharpened map of focused UNC-crossover region containing the central regions of UNC79 and UNC80 used for generating the combined map

Additional map: map (non-sharpened) of focused UNC-crossover region containing the...

• File: emd_25492_additional_9.map
• Annotation: map (non-sharpened) of focused UNC-crossover region containing the central regions of UNC79 and UNC80

Half map: halfmap 1 (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM

• File: emd_25492_half_map_1.map
• Annotation: halfmap 1 (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM

Half map: halfmap 2 (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM

• File: emd_25492_half_map_2.map
• Annotation: halfmap 2 (non-sharpened) of overall NALCN-FAM-UNC79-UNC80-CAM

Sample components

Entire : Pentameric complex of the leak channel NALCN with FAM155A, Calmod...

• Entire: Name: Pentameric complex of the leak channel NALCN with FAM155A, Calmodulin, UNC79 and UNC80

Components

• Complex: Pentameric complex of the leak channel NALCN with FAM155A, Calmodulin, UNC79 and UNC80
  • Protein or peptide: Sodium leak channel non-selective protein,Enhanced green fluorescent protein
  • Protein or peptide: Transmembrane protein FAM155A
  • Protein or peptide: Calmodulin-1
  • Protein or peptide: UNC79,Protein unc-79 homolog,Protein unc-79 homolog
  • Protein or peptide: Protein unc-80 homolog
• 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

Supramolecule #1: Pentameric complex of the leak channel NALCN with FAM155A, Calmod...

• Supramolecule: Name: Pentameric complex of the leak channel NALCN with FAM155A, Calmodulin, UNC79 and UNC80; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5; Details: Calmodulin was not overexpressed but co-purified from Expi293 cells
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Sodium leak channel non-selective protein,Enhanced green fluoresc...

• Macromolecule: Name: Sodium leak channel non-selective protein,Enhanced green fluorescent protein; type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 234.128797 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

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 GSLVPRGSSG ENLYFQG SS GMVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPD H MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS HNVYIMADK QKNGIKVNFK IRHNIEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSK LSKDPNEKRD HMVLLEFVTA AGITLGMDEL YKSGGDYKD DDDKGSGSAW SHPQFEKGGG SGGGSGGSAW SHPQFEK

UniProtKB: Sodium leak channel NALCN, Enhanced green fluorescent protein

Macromolecule #2: Transmembrane protein FAM155A

• Macromolecule: Name: Transmembrane protein FAM155A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 54.205004 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

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

UniProtKB: NALCN channel auxiliary factor 1

Macromolecule #3: Calmodulin-1

• Macromolecule: Name: Calmodulin-1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 16.852545 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREA FRVFDKDGNG YISAAELRHV MTNLGEKLTD EEVDEMIREA DIDGDGQVNY EEFVQMMTAK

UniProtKB: Calmodulin-1

Macromolecule #4: UNC79,Protein unc-79 homolog,Protein unc-79 homolog

• Macromolecule: Name: UNC79,Protein unc-79 homolog,Protein unc-79 homolog / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 285.174938 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)LP YTMISTLATF PPFLHKDIIE YLSTSFLP M AILGSSRREG VPAHVNLSAS SMLMIAMQYT SNPVYHCQLL ECLMKYKQEV WKDLLYVIAY GPSQVKPPAV QMLFHYWPN LKPPGAISEY RGLQYTAWNP IHCQHIECHN AINKPAVKMC IDPSLSVALG DKPPPLYLCE ECSERIAGDH SEWLIDVLLP QAEISAICQ KKNCSSHVRR AVVTCFSAGC CGRHGNRPVR YCKRCHSNHH SNEVGAAAET HLYQTSPPPI NTRECGAEEL V CAVEAVIS LLKEAEFHAE QREHELNRRR QLGLSSSHHS LDNADFDNKD DDKHDQRLLS QFGIWFLVSL CTPSENTPTE SL ARLVAMV FQWFHSTAYM MDDEVGSLVE KLKPQFVTKW LKTVCDVRFD VMVMCLLPKP MEFARVGGYW DKSCSTVTQL KEG LNRILC LIPYNVINQS VWECIMPEWL EAIRTEVPDN QLKEFREVLS KMFDIELCPL PFSMEEMFGF ISCRFTGYPS SVQE QALLW LHVLSELDIM VPLQLLISMF SDGVNSVKEL ANQRKSRVSE LAGNLASRRV SVASDPGRRV QHNMLSPFHS PFQSP FRSP LRSPFRSPFK NFGHPGGRTI DFDCEDDEMN LNCFILMFDL LLKQMELQDD GITMGLEHSL SKDIISIINN VFQAPW GGS HTCQKDEKAI ECNLCQSSIL CYQLACELLE RLAPKEESRL VEPTDSLEDS LLSSRPEFII GPEGEEEENP ASKHGEN PG NCTEPVEHAA VKNDTERKFC YQQLPVTLRL IYTIFQEMAK FEEPDILFNM LNCLKILCLH GECLYIARKD HPQFLAYI Q DHMLIASLWR VVKSEFSQLS SLAVPLLLHA LSLPHGADIF WTIINGNFNS KDWKMRFEAV EKVAVICRFL DIHSVTKNH LLKYSLAHAF CCFLTAVEDV NPAVATRAGL LLDTIKRPAL QGLCLCLDFQ FDTVVKDRPT ILSKLLLLHF LKQDIPALSW EFFVNRFET LSLEAQLHLD CNKEFPFPTT ITAVRTNVAN LSDAALWKIK RARFARNRQK SVRSLRDSVK GPVESKRALS L PETLTSKI RQQSPENDNT IKDLLPEDAG IDHQTVHQLI TVLMKFMAKD ESSAESDISS AKAFNTVKRH LYVLLGYDQQ EG CFMIAPQ KMRLSTCFNA FIAGIAQVMD YNINLGKHLL PLVVQVLKYC SCPQLRHYFQ QPPRCSLWSL KPHIRQMWLK ALL VILYKY PYRDCDISKI LLHLIHITVN TLNAQYHSCK PHATAGPLYS DNSNISRYSE KEKGEIELAE YRETGALQDS LLHC VREES IPKKKLRSFK QKSLDIGNAD SLLFTLDEHR RKSCIDRCDI EKPPTQAAYI AQRPNDPGRS RQNSATRPDN SEIPE NPAM EGFPDARRPV IPEVRLNCME TFEVKVDSPV KPAPKEDLDL IDLSSDSTSG PEKHSILSTS DSDSLVFEPL PPLRIV ESD EEEETMNQGD DGPSGKNAAS SPSVPSHPSV LSLSTAPLVQ VSVEDCSKDF SSKDSGNNQS AGNTDSALIT LEDPMDA EG SSKPEELPEF SCGSPLTLKQ KRDLLQKSFA LPEMSLDDHP DPGTEGEKPG ELMPSSGAKT VLLKVPEDAE NPTESEKP D TSAESDTEQN PERKVEEDGA EESEFKIQIV PRQRKQRKIA VSAIQREYLD ISFNILDKLG EQKDPDPSTK GLSTLEMPR ESSSAPTLDA GVPETSSHSS ISTQYRQMKR GSLGVLTMSQ LMKRQLEHQS SAPHNISNWD TEQIQPGKRQ CNVPTCLNPD LEGQPLRMR GATKSSLLSA PSIVSMFVPA PEEFTDEQPT VMTDKCHDCG AILEEYDEET LGLAIVVLST FIHLSPDLAA P LLLDIMQS VGRLASSTTF SNQAESMMVP GNAAGVAKQF LRCIFHQLAP NGIFPQLFQS TIKDGTFLRT LASSLMDFNE LS SIAALSQ LLEGLNNKKN LPAGGAMIRC LENIATFMEA LPMDSPSSLW TTISNQFQTF FAKLPCVLPL KCSLDSSLRI MIC LLKIPS TNATRSLLEP FSKLLSFVIQ NAVFTLAYLV ELCGLCYRAF TKERDKFYLS RSVVLELLQA LKLKSPLPDT NLLL LVQFI CADAGTKLAE STILSKQMIA SVPGCGTAAM ECVRQYINEV LDFMADMHTL TKLKSHMKTC SQPLHEDTFG GHLKV GLAQ IAAMDISRGN HRDNKAVIRY LPWLYHPPSA MQQGPKEFIE CVSHIRLLSW LLLGSLTHNA VCPNASSPCL PIPLDA GSH VADHLIVILI GFPEQSKTSV LHMCSLFHAF IFAQLWTVYC EQSAVATNLQ NQNEFSFTAI LTALEFWSRV TPSILQL MA HNKVMVEMVC LHVISLMEAL QECNSTIFVK LIPMWLPMIQ SNIKHLSAGL QLRLQAIQNH VNHHSLRTLP GSGQSSAG L AALRKWLQCT QFKMAQVEIQ SSEAASQFYP LGGSGGSDYK DDDDKGNSDY KDDDDK

UniProtKB: Protein unc-79 homolog

Macromolecule #5: Protein unc-80 homolog

• Macromolecule: Name: Protein unc-80 homolog / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 366.510594 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MVKRKSSEGQ EQDGGRGIPL PIQTFLWRQT SAFLRPKLGK QYEASCVSFE RVLVENKLHG LSPALSEAIQ SISRWELVQA ALPHVLHCT ATLLSNRNKL GHQDKLGVAE TKLLHTLHWM LLEAPQDCNN ERFGGTDRGS SWGGSSSAFI HQVENQGSPG Q PCQSSSND EEENNRRKIF QNSMATVELF VFLFAPLVHR IKESDLTFRL ASGLVIWQPM WEHRQPGVSG FTALVKPIRN II TAKRSSP INSQSRTCES PNQDARHLEG LQVVCETFQS DSISPKATIS GCHRGNSFDG SLSSQTSQER GPSHSRASLV IPP CQRSRY ATYFDVAVLR CLLQPHWSEE GTQWSLMYYL QRLRHMLEEK PEKPPEPDIP LLPRPRSSSM VAAAPSLVNT HKTQ DLTMK CNEEEKSLSS EAFSKVSLTN LRRSAVPDLS SDLGMNIFKK FKSRKEDRER KGSIPFHHTG KRRPRRMGVP FLLHE DHLD VSPTRSTFSF GSFSGLGEDR RGIEKGGWQT TILGKLTRRG SSDAATEMES LSARHSHSHH TLVSDLPDPS NSHGEN TVK EVRSQISTIT VATFNTTLAS FNVGYADFFN EHMRKLCNQV PIPEMPHEPL ACANLPRSLT DSCINYSYLE DTEHIDG TN NFVHKNGMLD LSVVLKAVYL VLNHDISSRI CDVALNIVEC LLQLGVVPCV EKNRKKSENK ENETLEKRPS EGAFQFKG V SGSSTCGFGG PAVSGAGDGG GEEGGGGDGG GGGGDGGGGG GGGGGPYEKN DKNQEKDEST PVSNHRLALT MLIKIVKSL GCAYGCGEGH RGLSGDRLRH QVFRENAQNC LTKLYKLDKM QFRQTMRDYV NKDSLNNVVD FLHALLGFCM EPVTDNKAGF GNNFTTVDN KSTAQNVEGI IVSAMFKSLI TRCASTTHEL HSPENLGLYC DIRQLVQFIK EAHGNVFRRV ALSALLDSAE K LAPGKKVE ENEQESKPAG SKRSEAGSIV DKGQVSSAPE ECRSFMSGRP SQTPEHDEQM QGANLGRKDF WRKMFKSQSA AS DTSSQSE QDTSECTTAH SGTTSDRRAR SRSRRISLRK KLKLPIGKRN WLKRSSLSGL ADGVEDLLDI SSVDRLSFIR QSS KVKFTS AVKLSEGGPG SGMENGRDEE ENFFKRLGCH SFDDHLSPNQ DGGKSKNVVN LGAIRQGMKR FQFLLNCCEP GTIP DASIL AAALDLEAPV VARAALFLEC ARFVHRCNRG NWPEWMKGHH VNITKKGLSR GRSPIVGNKR NQKLQWNAAK LFYQW GDAI GVRLNELCHG ESESPANLLG LIYDEETKRR LRKEDEEEDF LDDSTVNPSK CGCPFALKMA ACQLLLEITT FLRETF SCL PRPRTEPLVD LESCRLRLDP ELDRHRYERK ISFAGVLDEN EDSKDSLHSS SHTLKSDAGV EEKKEGSPWS ASEPSIE PE GMSNAGAEEN YHRNMSWLHV MILLCNQQSF ICTHVDYCHP HCYLHHSRSC ARLVRAIKLL YGDSVDSLRE SSNISSVA L RGKKQKECSD KSCLRTPSLK KRVSDANLEG KKDSGMLKYI RLQVMSLSPA PLSLLIKAAP ILTEEMYGDI QPAAWELLL SMDEHMAGAA AAMFLLCAVK VPEAVSDMLM SEFHHPETVQ RLNAVLKFHT LWRFRYQVWP RMEEGAQQIF KIPPPSINFT LPSPVLGMP SVPMFDPPWV PQCSGSVQDP INEDQSKSFS ARAVSRSHQR AEHILKNLQQ EEEKKRLGRE ASLITAIPIT Q EACYEPTC TPNSEPEEEV EEVTNLASRR LSVSPSCTSS TSHRNYSFRR GSVWSVRSAV SAEDEEHTTE HTPNHHVPQP PQ AVFPACI CAAVLPIVHL MEDGEVREDG VAVSAVAQQV LWNCLIEDPS TVLRHFLEKL TISNRQDELM YMLRKLLLNI GDF PAQTSH ILFNYLVGLI MYFVRTPCEW GMDAISATLT FLWEVVGYVE GLFFKDLKQT MKKEQCEVKL LVTASMPGTK TLVV HGQNE CDIPTQLPVH EDTQFEALLK ECLEFFNIPE SQSTHYFLMD KRWNLIHYNK TYVRDIYPFR RSVSPQLNLV HMHPE KGQE LIQKQVFTRK LEEVGRVLFL ISLTQKIPTA HKQSHVSMLQ EDLLRLPSFP RSAIDAEFSL FSDPQAGKEL FGLDTL QKS LWIQLLEEMF LGMPSEFPWG DEIMLFLNVF NGALILHPED SALLRQYAAT VINTAVHFNH LFSLSGYQWI LPTMLQV YS DYESNPQLRQ AIEFACHQFY ILHRKPFVLQ LFASVAPLLE FPDAANNGPS KGVSAQCLFD LLQSLEGETT DILDILEL V KAEKPLKSLD FCYGNEDLTF SISEAIKLCV TVVAYAPESF RSLQMLMVLE ALVPCYLQKL KRQTSQVETV PAAREEIAA TAALATSLQA LLYSVEVLTR PMTAPQMSRC DQGHKGTTTA NHTMSSGVNT RYQEQGAKLH FIRENLHLLE EGQGIPREEL DERIAREEF RRPRESLLNI CTEFYKHCGP RLKILQNLAG EPRVIALELL DVKSHMRLAE IAHSLLKLAP YDTQTMESRG L RRYIMEML PITDWTAEAV RPALILILKR LDRMFNKIHK MPTLRRQVEW EPASNLIEGV CLTLQRQPII SFLPHLRSLI NV CVNLVMG VVGPSSVADG LPLLHLSPYL SPPLPFSTAV VRLVALQIQA LKEDFPLSHV ISPFTNQERR EGMLLNLLIP FVL TVGSGS KDSPWLEQPE VQLLLQTVIN VLLPPRIIST SRSKNFMLES SPAHCSTPGD AGKDLRREGL AESTSQAAYL ALKV ILVCF ERQLGSQWYW LSLQVKEMAL RKVGGLALWD FLDFIVRTRI PIFVLLRPFI QCKLLAQPAE NHEELSARQH IADQL ERRF IPRPLCKSSL IAEFNSELKI LKEAVHSGSA YQGKTSISTV GTSTSAYRLS LATMSRSNTG TGTVWEQDSE PSQQAS QDT LSRTDEEDEE NDSISMPSVV SEQEAYLLSA IGRRRFSSHV SSMSVPQAEV GMLPSQSEPN VLDDSQGLAA EGSLSRV AS IQSEPGQQNL LVQQPLGRKR GLRQLRRPLL SRQKTQTEPR NRQGARLSTT RRSIQPKTKP SADQKRSVTF IEAQPEPA A APTDALPATG QLQGCSPAPS RKPEAMDEPV LTSSPAIVVA DLHSVSPKQS ENFPTEEGEK EEDTEAQGAT AHSPLSAQL SDPDDFTGLE TSSLLQHGDT VLHISEENGM ENPLLSSQFT FTPTELGKTD AVLDESHVGG SGGSDYKDDD DKGNSDYKDD DDK

UniProtKB: Protein unc-80 homolog

Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 2 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

Macromolecule #7: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY...

• Macromolecule: Name: (1S)-2-{[(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE; type: ligand / ID: 7 / Number of copies: 4 / Formula: PEV
• Molecular weight: Theoretical: 720.012 Da

Chemical component information

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

Macromolecule #8: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

• Macromolecule: Name: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE; type: ligand / ID: 8 / Number of copies: 2 / Formula: PGV
• Molecular weight: Theoretical: 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

Macromolecule #9: CHOLESTEROL HEMISUCCINATE

• Macromolecule: Name: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 9 / Number of copies: 3 / Formula: Y01
• Molecular weight: Theoretical: 486.726 Da

Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.65 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
200.0 mM	NaCl	sodium chloride
25.0 mM	HEPES

• Grid: Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: 3.5 sec blotting.
• 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

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 26550 / Average exposure time: 3.0 sec. / Average electron dose: 64.009 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 3048401
• Startup model: Type of model: OTHER / Details: ab initio 3D reconstruction within cisTEM
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.02) / Number images used: 132257
• Initial angle assignment: Type: RANDOM ASSIGNMENT / Software - Name: cisTEM (ver. 1.02)
• Final angle assignment: Type: OTHER / Software - Name: cisTEM (ver. 1.02); Details: Focused refinements were conducted after dividing the map into four distinct regions.
• Final 3D classification: Number classes: 8 / Software - Name: RELION (ver. 3.1)