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- EMDB-14753: Cryo-EM map of 1PBC- and calcium-bound mTMEM16A(ac) chloride chan... -

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Basic information

Entry
Database: EMDB / ID: EMD-14753
TitleCryo-EM map of 1PBC- and calcium-bound mTMEM16A(ac) chloride channel at 2.85 A resolution
Map dataCryo-EM map of 1PBC- and calcium-bound mTMEM16A(ac) chloride channel at 2.85 A resolution
Sample
  • Organelle or cellular component: mouse TMEM16A ac splice variant in complex with 1PBC and calcium
    • Protein or peptide: Anoctamin-1
  • Ligand: CALCIUM ION
  • Ligand: 1-Hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile
Keywordscalcium-activated chloride channel / anoctamin-1 / MEMBRANE PROTEIN
Function / homology
Function and homology information


glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / mucus secretion / intracellularly calcium-gated chloride channel activity / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / chloride transport ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / mucus secretion / intracellularly calcium-gated chloride channel activity / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / chloride transport / chloride channel activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / detection of temperature stimulus involved in sensory perception of pain / chloride channel complex / chloride transmembrane transport / regulation of membrane potential / cell projection / establishment of localization in cell / presynaptic membrane / cellular response to heat / phospholipase C-activating G protein-coupled receptor signaling pathway / apical plasma membrane / external side of plasma membrane / signaling receptor binding / glutamatergic synapse / protein homodimerization activity / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Anoctamin, dimerisation domain / Anoctamin, dimerisation domain / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsLam AKM / Rutz S / Dutzler R
Funding supportEuropean Union, Switzerland, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)339116European Union
University of ZurichFK-18-048 Switzerland
CitationJournal: Nat Commun / Year: 2022
Title: Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC.
Authors: Andy K M Lam / Sonja Rutz / Raimund Dutzler /
Abstract: TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic ...TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic promises, its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A pocket located external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably also by its structural analogs. The binding of the blocker stabilizes an open-like conformation of the channel that involves a rearrangement of several pore helices. The expansion of the outer pore enhances blocker sensitivity and enables 1PBC to bind at a site within the transmembrane electric field. Our results define the mechanism of inhibition and gating and will facilitate the design of new, potent TMEM16A modulators.
History
DepositionApr 12, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14753.png

Downloads & links

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Map

FileDownload / File: emd_14753.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of 1PBC- and calcium-bound mTMEM16A(ac) chloride channel at 2.85 A resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 400 pix.
= 395.6 Å		0.99 Å/pix.
x 400 pix.
= 395.6 Å		0.99 Å/pix.
x 400 pix.
= 395.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.989 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.06701477 - 0.12930709
Average (Standard dev.)0.00006823132 (±0.0017000422)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 395.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14753_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_14753_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_14753_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mouse TMEM16A ac splice variant in complex with 1PBC and calcium

EntireName: mouse TMEM16A ac splice variant in complex with 1PBC and calcium
Components
  • Organelle or cellular component: mouse TMEM16A ac splice variant in complex with 1PBC and calcium
    • Protein or peptide: Anoctamin-1
  • Ligand: CALCIUM ION
  • Ligand: 1-Hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile

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Supramolecule #1: mouse TMEM16A ac splice variant in complex with 1PBC and calcium

SupramoleculeName: mouse TMEM16A ac splice variant in complex with 1PBC and calcium
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Anoctamin-1

MacromoleculeName: Anoctamin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 111.058992 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRVPEKYSTL PAEDRSVHIV NICAIEDLGY LPSEGTLLNS LSVDPDAECK YGLYFRDGKR KVDYILVYHH KRASGSRTLA RRGLQNDMV LGTRSVRQDQ PLPGKGSPVD AGSPEVPMDY HEDDKRFRRE EYEGNLLEAG LELENDEDTK IHGVGFVKIH A PWHVLCRE ...String:
MRVPEKYSTL PAEDRSVHIV NICAIEDLGY LPSEGTLLNS LSVDPDAECK YGLYFRDGKR KVDYILVYHH KRASGSRTLA RRGLQNDMV LGTRSVRQDQ PLPGKGSPVD AGSPEVPMDY HEDDKRFRRE EYEGNLLEAG LELENDEDTK IHGVGFVKIH A PWHVLCRE AEFLKLKMPT KKVYHISETR GLLKTINSVL QKITDPIQPK VAEHRPQTTK RLSYPFSREK QHLFDLTDRD SF FDSKTRS TIVYEILKRT TCTKAKYSMG ITSLLANGVY SAAYPLHDGD YEGDNVEFND RKLLYEEWAS YGVFYKYQPI DLV RKYFGE KVGLYFAWLG AYTQMLIPAS IVGVIVFLYG CATVDENIPS MEMCDQRYNI TMCPLCDKTC SYWKMSSACA TARA SHLFD NPATVFFSVF MALWAATFME HWKRKQMRLN YRWDLTGFEE EEEAVKDHPR AEYEARVLEK SLRKESRNKE TDKVK LTWR DRFPAYFTNL VSIIFMIAVT FAIVLGVIIY RISTAAALAM NSSPSVRSNI RVTVTATAVI INLVVIILLD EVYGCI ARW LTKIEVPKTE KSFEERLTFK AFLLKFVNSY TPIFYVAFFK GRFVGRPGDY VYIFRSFRME ECAPGGCLME LCIQLSI IM LGKQLIQNNL FEIGIPKMKK FIRYLKLRRQ SPSDREEYVK RKQRYEVDFN LEPFAGLTPE YMEMIIQFGF VTLFVASF P LAPLFALLNN IIEIRLDAKK FVTELRRPVA IRAKDIGIWY NILRGVGKLA VIINAFVISF TSDFIPRLVY LYMYSQNGT MHGFVNHTLS SFNVSDFQNG TAPNDPLDLG YEVQICRYKD YREPPWSEHK YDISKDFWAV LAARLAFVIV FQNLVMFMSD FVDWVIPDI PKDISQQIHK EKVLMVELFM REEQGKQQLL DTWMEKEKPR DVPCNNHSPT THPEAGDGSP VPSYEYHGDA L

UniProtKB: Anoctamin-1

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: 1-Hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile

MacromoleculeName: 1-Hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile
type: ligand / ID: 3 / Number of copies: 2 / Formula: JRF
Molecular weightTheoretical: 277.201 Da
Chemical component information

ChemComp-JRF:
1-Hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile

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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
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 61.0 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 / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7b5c

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 101813
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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