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- EMDB-19364: Structure of heteromeric CALHM2/4 channel in complex with synthet... -

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Basic information

Entry
Database: EMDB / ID: EMD-19364
TitleStructure of heteromeric CALHM2/4 channel in complex with synthetic nanobodies SbC2 and SbC4
Map data
Sample
  • Complex: Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4
    • Protein or peptide: Calcium homeostasis modulator protein 4
    • Protein or peptide: Synthetic nanobody SbC4
    • Protein or peptide: Calcium homeostasis modulator protein 2
    • Protein or peptide: Synthetic nanobody SbC2
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
Keywordsion channel / large pore channels / sybody / CALHM / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of microglial cell activation / ATP export / calcium ion import / monoatomic cation channel activity / regulation of synaptic plasticity / positive regulation of apoptotic process / plasma membrane
Similarity search - Function
Calcium homeostasis modulator family / Calcium homeostasis modulator
Similarity search - Domain/homology
Calcium homeostasis modulator protein 4 / Calcium homeostasis modulator protein 2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsDrozdzyk K / Dutzler R
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Elife / Year: 2024
Title: Structural features of heteromeric channels composed of CALHM2 and CALHM4 paralogs.
Authors: Katarzyna Drożdżyk / Martina Peter / Raimund Dutzler /
Abstract: The CALHM proteins constitute a family of large pore channels that contains six closely related paralogs in humans. Two family members, CALHM1 and 3, have been associated with the release of ATP ...The CALHM proteins constitute a family of large pore channels that contains six closely related paralogs in humans. Two family members, CALHM1 and 3, have been associated with the release of ATP during taste sensation. Both proteins form heteromeric channels that activate at positive potential and decreased extracellular Ca concentration. Although the structures of several family members displayed large oligomeric organizations of different size, their function has in most cases remained elusive. Our previous study has identified the paralogs CALHM2, 4 and, 6 to be highly expressed in the placenta and defined their structural properties as membrane proteins exhibiting features of large pore channels with unknown activation properties (Drożdżyk et al., 2020). Here, we investigated whether these placental paralogs would form heteromers and characterized heteromeric complexes consisting of CALHM2 and CALHM4 subunits using specific binders as fiducial markers. Both proteins assemble with different stoichiometries with the largest population containing CALHM2 as the predominant component. In these oligomers, the subunits segregate and reside in their preferred conformation found in homomeric channels. Our study has thus revealed the properties that govern the formation of CALHM heteromers in a process of potential relevance in a cellular context.
History
DepositionJan 8, 2024-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19364.png

Downloads & links

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Map

FileDownload / File: emd_19364.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 280 pix.
= 364.56 Å		1.3 Å/pix.
x 280 pix.
= 364.56 Å		1.3 Å/pix.
x 280 pix.
= 364.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.302 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.8869132 - 1.5778674
Average (Standard dev.)0.0028823507 (±0.0355409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 364.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_19364_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19364_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4

EntireName: Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4
Components
  • Complex: Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4
    • Protein or peptide: Calcium homeostasis modulator protein 4
    • Protein or peptide: Synthetic nanobody SbC4
    • Protein or peptide: Calcium homeostasis modulator protein 2
    • Protein or peptide: Synthetic nanobody SbC2
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE

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Supramolecule #1: Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4

SupramoleculeName: Complex of heteromeric CALHM2/4 channel with synthetic nanobody SbC4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 0.541 kDa/nm

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Macromolecule #1: Calcium homeostasis modulator protein 4

MacromoleculeName: Calcium homeostasis modulator protein 4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.981656 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSCPTLNNIV SSLQRNGIFI NSLIAALTIG GQQLFSSSTF SCPCQVGKNF YYGSAFLVIP ALILLVAGFA LRSQMWTITG EYCCSCAPP YRRISPLECK LACLRFFSIT GRAVIAPLTW LAVTLLTGTY YECAASEFAS VDHYPMFDNV SASKREEILA G FPCCRSAP ...String:
MSCPTLNNIV SSLQRNGIFI NSLIAALTIG GQQLFSSSTF SCPCQVGKNF YYGSAFLVIP ALILLVAGFA LRSQMWTITG EYCCSCAPP YRRISPLECK LACLRFFSIT GRAVIAPLTW LAVTLLTGTY YECAASEFAS VDHYPMFDNV SASKREEILA G FPCCRSAP SDVILVRDEI ALLHRYQSQM LGWILITLAT IAALVSCCVA KCCSPLTSLQ HCYWTSHLQN ERELFEQAAE QH SRLLMMH RIKKLFGFIP GSEDVKHIRI PSCQDWKDIS VPTLLCMGDD LQGHYSFLGN RVDEDNEEDR SRGIELKPAL EVL FQ

UniProtKB: Calcium homeostasis modulator protein 4

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Macromolecule #2: Synthetic nanobody SbC4

MacromoleculeName: Synthetic nanobody SbC4 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.87738 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QGPSQVQLVE SGGGLVQAGG SLRLSCAASG FPVYYTHMRW YRQAPGKERE WVAAIYSKGA GTHYADSVKG RFTISRDNAK NTVYLQMNS LKPEDTAVYY CFVGVGNSYI GQGTQVTVSA

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Macromolecule #3: Calcium homeostasis modulator protein 2

MacromoleculeName: Calcium homeostasis modulator protein 2 / type: protein_or_peptide / ID: 3 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.099535 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSAALIAENF RFLSLFFKSK DVMIFNGLVA LGTVGSQELF SVVAFHCPCS PARNYLYGLA AIGVPALVLF IIGIILNNHT WNLVAECQH RRTKNCSAAP TFLLLSSILG RAAVAPVTWS VISLLRGEAY VCALSEFVDP SSLTAREEHF PSAHATEILA R FPCKENPD ...String:
MSAALIAENF RFLSLFFKSK DVMIFNGLVA LGTVGSQELF SVVAFHCPCS PARNYLYGLA AIGVPALVLF IIGIILNNHT WNLVAECQH RRTKNCSAAP TFLLLSSILG RAAVAPVTWS VISLLRGEAY VCALSEFVDP SSLTAREEHF PSAHATEILA R FPCKENPD NLSDFREEVS RRLRYESQLF GWLLIGVVAI LVFLTKCLKH YCSPLSYRQE AYWAQYRANE DQLFQRTAEV HS RVLAANN VRRFFGFVAL NKDDEELIAN FPVEGTQPRP QWNAITGVYL YRENQGLPLY SRLHKWAQGL AGNGAAPDNV EMA LLPSAL EVLFQ

UniProtKB: Calcium homeostasis modulator protein 2

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Macromolecule #4: Synthetic nanobody SbC2

MacromoleculeName: Synthetic nanobody SbC2 / type: protein_or_peptide / ID: 4 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.067451 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QGPSQVQLVE SGGGSVQAGG SLRLSCAASG NIRNISYLGW FRQAPGKERE GVAALWTTQG QTYYADSVKG RFTVSLDNAK NTVYLQMNS LKPEDTALYY CAAATSGQYN PLRGYHYNEY WGQGTQVTVS A

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Macromolecule #5: DIUNDECYL PHOSPHATIDYL CHOLINE

MacromoleculeName: DIUNDECYL PHOSPHATIDYL CHOLINE / type: ligand / ID: 5 / Number of copies: 6 / Formula: PLC
Molecular weightTheoretical: 622.834 Da
Chemical component information

ChemComp-PLC:
DIUNDECYL PHOSPHATIDYL CHOLINE / phospholipid*YM

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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: 7.6
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 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: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93191
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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