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- EMDB-43994: Cryo-EM structure of human Cav3.2 with ML218 -

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Basic information

Entry
Database: EMDB / ID: EMD-43994
TitleCryo-EM structure of human Cav3.2 with ML218
Map data
Sample
  • Complex: Voltage-dependent T-type calcium channel subunit alpha-1H
    • Protein or peptide: Voltage-dependent T-type calcium channel subunit alpha-1H
  • Ligand: 3,5-dichloro-N-{[(1R,5S,6r)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl]methyl}benzamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: 1-O-OCTADECYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: [(2~{R})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-pentadecanoyloxy-propyl] pentadecanoate
KeywordsCav3.2 / voltage gated calcium channel / cryo-EM / TRANSPORT PROTEIN / ML218
Function / homology
Function and homology information


low voltage-gated calcium channel activity / aldosterone biosynthetic process / cortisol biosynthetic process / positive regulation of acrosome reaction / voltage-gated monoatomic ion channel activity / high voltage-gated calcium channel activity / cellular response to potassium ion / myoblast fusion / NCAM1 interactions / calcium ion import ...low voltage-gated calcium channel activity / aldosterone biosynthetic process / cortisol biosynthetic process / positive regulation of acrosome reaction / voltage-gated monoatomic ion channel activity / high voltage-gated calcium channel activity / cellular response to potassium ion / myoblast fusion / NCAM1 interactions / calcium ion import / inorganic cation transmembrane transport / voltage-gated calcium channel complex / regulation of heart contraction / muscle organ development / calcium ion import across plasma membrane / Smooth Muscle Contraction / cellular response to hormone stimulus / muscle contraction / regulation of membrane potential / scaffold protein binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Voltage-dependent calcium channel, T-type, alpha-1 subunit / Voltage-gated cation channel calcium and sodium / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Voltage-dependent T-type calcium channel subunit alpha-1H
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsFan X / Huang J / Yan N
Funding support France, 1 items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)LT000754/2020-L France
CitationJournal: Cell Res / Year: 2024
Title: Structural basis for human Ca3.2 inhibition by selective antagonists.
Authors: Jian Huang / Xiao Fan / Xueqin Jin / Chen Lyu / Qinmeng Guo / Tao Liu / Jiaofeng Chen / Amaël Davakan / Philippe Lory / Nieng Yan /
Abstract: The Ca3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Ca3.2 alone ...The Ca3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Ca3.2 alone and in complex with four T-type calcium channel selective antagonists with overall resolutions ranging from 2.8 Å to 3.2 Å. The four compounds display two binding poses. ACT-709478 and TTA-A2 both place their cyclopropylphenyl-containing ends in the central cavity to directly obstruct ion flow, meanwhile extending their polar tails into the IV-I fenestration. TTA-P2 and ML218 project their 3,5-dichlorobenzamide groups into the II-III fenestration and place their hydrophobic tails in the cavity to impede ion permeation. The fenestration-penetrating mode immediately affords an explanation for the state-dependent activities of these antagonists. Structure-guided mutational analysis identifies several key residues that determine the T-type preference of these drugs. The structures also suggest the role of an endogenous lipid in stabilizing drug binding in the central cavity.
History
DepositionMar 8, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43994.png

Downloads & links

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Map

FileDownload / File: emd_43994.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 265.216 Å		1.04 Å/pix.
x 256 pix.
= 265.216 Å		1.04 Å/pix.
x 256 pix.
= 265.216 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.036 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-2.0337589 - 2.43299
Average (Standard dev.)-0.000026272506 (±0.04922172)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 265.216 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43994_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43994_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Voltage-dependent T-type calcium channel subunit alpha-1H

EntireName: Voltage-dependent T-type calcium channel subunit alpha-1H
Components
  • Complex: Voltage-dependent T-type calcium channel subunit alpha-1H
    • Protein or peptide: Voltage-dependent T-type calcium channel subunit alpha-1H
  • Ligand: 3,5-dichloro-N-{[(1R,5S,6r)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl]methyl}benzamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: 1-O-OCTADECYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: [(2~{R})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-pentadecanoyloxy-propyl] pentadecanoate

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Supramolecule #1: Voltage-dependent T-type calcium channel subunit alpha-1H

SupramoleculeName: Voltage-dependent T-type calcium channel subunit alpha-1H
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Voltage-dependent T-type calcium channel subunit alpha-1H

MacromoleculeName: Voltage-dependent T-type calcium channel subunit alpha-1H
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 234.853609 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMTEGARA ADEVRVPLGA PPPGPAALVG ASPESPGAPG REAERGSEL GVSPSESPAA ERGAELGADE EQRVPYPALA ATVFFCLGQT TRPRSWCLRL VCNPWFEHVS MLVIMLNCVT L GMFRPCED ...String:
MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMTEGARA ADEVRVPLGA PPPGPAALVG ASPESPGAPG REAERGSEL GVSPSESPAA ERGAELGADE EQRVPYPALA ATVFFCLGQT TRPRSWCLRL VCNPWFEHVS MLVIMLNCVT L GMFRPCED VECGSERCNI LEAFDAFIFA FFAVEMVIKM VALGLFGQKC YLGDTWNRLD FFIVVAGMME YSLDGHNVSL SA IRTVRVL RPLRAINRVP SMRILVTLLL DTLPMLGNVL LLCFFVFFIF GIVGVQLWAG LLRNRCFLDS AFVRNNNLTF LRP YYQTEE GEENPFICSS RRDNGMQKCS HIPGRRELRM PCTLGWEAYT QPQAEGVGAA RNACINWNQY YNVCRSGDSN PHNG AINFD NIGYAWIAIF QVITLEGWVD IMYYVMDAHS FYNFIYFILL IIVGSFFMIN LCLVVIATQF SETKQRESQL MREQR ARHL SNDSTLASFS EPGSCYEELL KYVGHIFRKV KRRSLRLYAR WQSRWRKKVD PGWMGRLWVT FSGKLRRIVD SKYFSR GIM MAILVNTLSM GVEYHEQPEE LTNALEISNI VFTSMFALEM LLKLLACGPL GYIRNPYNIF DGIIVVISVW EIVGQAD GG LSVLRTFRLL RVLKLVRFLP ALRRQLVVLV KTMDNVATFC TLLMLFIFIF SILGMHLFGC KFSLKTDTGD TVPDRKNF D SLLWAIVTVF QILTQEDWNV VLYNGMASTS SWAALYFVAL MTFGNYVLFN LLVAILVEGF QAEGDANRSD TDEDKTSVH FEEDFHKLRE LQTTELKMCS LAVTPNGHLE GRGSLSPPLI MCTAATPMPT PKSSPFLDAA PSLPDSRRGS SSSGDPPLGD QKPPASLRS SPCAPWGPSG AWSSRRSSWS SLGRAPSLKR RGQCGERESL LSGEGKGSTD DEAEDGRAAP GPRATPLRRA E SLDPRPLR PAALPPTKCR DRDGQVVALP SDFFLRIDSH REDAAELDDD SEDSCCLRLH KVLEPYKPQW CRSREAWALY LF SPQNRFR VSCQKVITHK MFDHVVLVFI FLNCVTIALE RPDIDPGSTE RVFLSVSNYI FTAIFVAEMM VKVVALGLLS GEH AYLQSS WNLLDGLLVL VSLVDIVVAM ASAGGAKILG VLRVLRLLRT LRPLRVISRA PGLKLVVETL ISSLRPIGNI VLIC CAFFI IFGILGVQLF KGKFYYCEGP DTRNISTKAQ CRAAHYRWVR RKYNFDNLGQ ALMSLFVLSS KDGWVNIMYD GLDAV GVDQ QPVQNHNPWM LLYFISFLLI VSFFVLNMFV GVVVENFHKC RQHQEAEEAR RREEKRLRRL ERRRRSTFPS PEAQRR PYY ADYSPTRRSI HSLCTSHYLD LFITFIICVN VITMSMEHYN QPKSLDEALK YCNYVFTIVF VFEAALKLVA FGFRRFF KD RWNQLDLAIV LLSLMGITLE EIEMSAALPI NPTIIRIMRV LRIARVLKLL KMATGMRALL DTVVQALPQV GNLGLLFM L LFFIYAALGV ELFGRLECSE DNPCEGLSRH ATFSNFGMAF LTLFRVSTGD NWNGIMKDTL RECSREDKHC LSYLPALSP VYFVTFVLVA QFVLVNVVVA VLMKHLEESN KEAREDAELD AEIELEMAQG PGSARRVDAD RPPLPQESPG ARDAPNLVAR KVSVSRMLS LPNDSYMFRP VVPASAPHPR PLQEVEMETY GAGTPLGSVA SVHSPPAESC ASLQIPLAVS SPARSGEPLH A LSPRGTAR SPSLSRLLCR QEAVHTDSLE GKIDSPRDTL DPAEPGEKTP VRPVTQGGSL QSPPRSPRPA SVRTRKHTFG QR CVSSRPA APGGEEAEAS DPADEEVSHI TSSACPWQPT AEPHGPEASP VAGGERDLRR LYSVDAQGFL DKPGRADEQW RPS AELGSG EPGEAKAWGP EAEPALGARR KKKMSPPCIS VEPPAEDEGS ARPSAAEGGS TTLRRRTPSC EATPHRDSLE PTEG SGAGG DPAAKGERWG QASCRAEHLT VPSFAFEPLD LGVPSGDPFL DGSHSVTPES RASSSGAIVP LEPPESEPPM PVGDP PEKR RGLYLTVPQC PLEKPGSPSA TPAPGGGADD PV

UniProtKB: Voltage-dependent T-type calcium channel subunit alpha-1H, Voltage-dependent T-type calcium channel subunit alpha-1H

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Macromolecule #2: 3,5-dichloro-N-{[(1R,5S,6r)-3-(3,3-dimethylbutyl)-3-azabicyclo[3....

MacromoleculeName: 3,5-dichloro-N-{[(1R,5S,6r)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl]methyl}benzamide
type: ligand / ID: 2 / Number of copies: 1 / Formula: A1AHJ
Molecular weightTheoretical: 369.329 Da

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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

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: 1-O-OCTADECYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1-O-OCTADECYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 5 / Number of copies: 1 / Formula: LPE
Molecular weightTheoretical: 510.708 Da
Chemical component information

ChemComp-LPE:
1-O-OCTADECYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Macromolecule #6: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 6 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #7: [(2~{R})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-pentadecanoy...

MacromoleculeName: [(2~{R})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-pentadecanoyloxy-propyl] pentadecanoate
type: ligand / ID: 7 / Number of copies: 4 / Formula: JL3
Molecular weightTheoretical: 663.906 Da

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 126670
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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