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- EMDB-18774: Cryo-EM map of calcium-bound mTMEM16A(ac)-L647V/I733V chloride ch... -

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Basic information

Entry
Database: EMDB / ID: EMD-18774
TitleCryo-EM map of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel at 3.29 A resolution
Map dataCryo-EM map of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel at 3.29 A resolution
Sample
  • Organelle or cellular component: mouse TMEM16A ac splice variant with introduced mutations L647V and I733V in complex with calcium
    • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel
  • Ligand: CALCIUM IONCalcium
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 / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity / protein localization to membrane / 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 / monoatomic cation transport / 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 / nucleoplasm / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsLam AKM / Dutzler R
Funding supportEuropean Union, Switzerland, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)339116European Union
University of ZurichFK-18-048 Switzerland
CitationJournal: EMBO J / Year: 2023
Title: Mechanistic basis of ligand efficacy in the calcium-activated chloride channel TMEM16A.
Authors: Andy Km Lam / Raimund Dutzler /
Abstract: Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes ...Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.
History
DepositionOct 26, 2023-
Header (metadata) releaseDec 6, 2023-
Map releaseDec 6, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18774.png

Downloads & links

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Map

FileDownload / File: emd_18774.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel at 3.29 A resolution
Voxel sizeX=Y=Z: 0.659 Å
Density
Contour LevelBy AUTHOR: 0.014
Minimum - Maximum-0.057193745 - 0.06714081
Average (Standard dev.)0.00012563635 (±0.0017080036)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 237.23999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18774_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel

Fileemd_18774_half_map_1.map
AnnotationHalf-map 2 of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel

Fileemd_18774_half_map_2.map
AnnotationHalf-map 1 of calcium-bound mTMEM16A(ac)-L647V/I733V chloride channel
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 with introduced mutations L647V a...

EntireName: mouse TMEM16A ac splice variant with introduced mutations L647V and I733V in complex with calcium
Components
  • Organelle or cellular component: mouse TMEM16A ac splice variant with introduced mutations L647V and I733V in complex with calcium
    • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: mouse TMEM16A ac splice variant with introduced mutations L647V a...

SupramoleculeName: mouse TMEM16A ac splice variant with introduced mutations L647V and I733V in complex with 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: 111.030945 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 LGKQVIQNNL FEIGIPKMKK FIRYLKLRRQ SPSDREEYVK RKQRYEVDFN LEPFAGLTPE YMEMIIQFGF VTLFVASF P LAPLFALLNN IVEIRLDAKK 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: 4 / Formula: CA
Molecular weightTheoretical: 40.078 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-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.5 e/Å2
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:

7b5d

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 144934

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