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- EMDB-7096: Cryo-EM structure of the TMEM16A calcium-activated chloride chann... -

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Basic information

Entry
Database: EMDB / ID: EMD-7096
TitleCryo-EM structure of the TMEM16A calcium-activated chloride channel in LMNG
Map datasharpened map
Sample
  • Cell: TMEM16A Calcium-activated chloride channel
    • Protein or peptide: Anoctamin-1
  • Ligand: CALCIUM ION
KeywordsChloride channel / TMEM16 family / 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: 3.8 Å
AuthorsDang S / Feng S
Funding support United States, 9 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 United States
National Institutes of Health/Office of the DirectorS100D0020054 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS069229 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35NS097227 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL080050 United States
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01DC007664 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA196276 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111126 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)K99DA041500 United States
CitationJournal: Nature / Year: 2017
Title: Cryo-EM structures of the TMEM16A calcium-activated chloride channel.
Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / ...Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / Yuh Nung Jan / Daniel L Minor / Yifan Cheng / Lily Yeh Jan /
Abstract: Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the ...Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.
History
DepositionOct 28, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 27, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bgj
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd_7096.png

Downloads & links

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Map

FileDownload / File: emd_7096.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 256 pix.
= 261.12 Å		1.02 Å/pix.
x 256 pix.
= 261.12 Å		1.02 Å/pix.
x 256 pix.
= 261.12 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.02 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.5 / Movie #1: 5.5
Minimum - Maximum-25.658325000000001 - 36.385463999999999
Average (Standard dev.)-0.000000003603186 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z261.120261.120261.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-25.65836.385-0.000

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Supplemental data

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Additional map: unsharpened map

Fileemd_7096_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_7096_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_7096_half_map_2.map
Annotationhalf map 2
Projections & Slices
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Sample components

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Entire : TMEM16A Calcium-activated chloride channel

EntireName: TMEM16A Calcium-activated chloride channel
Components
  • Cell: TMEM16A Calcium-activated chloride channel
    • Protein or peptide: Anoctamin-1
  • Ligand: CALCIUM ION

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Supramolecule #1: TMEM16A Calcium-activated chloride channel

SupramoleculeName: TMEM16A Calcium-activated chloride channel / type: cell / 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 / Details: Calcium ions / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 105.603984 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 EEDHPRAEYE ARVLEKSLRK ESRNKETDKV KLTWR DRFP AYFTNLVSII FMIAVTFAIV LGVIIYRIST AAALAMNSSP SVRSNIRVTV TATAVIINLV VIILLDEVYG CIARWL TKI EVPKTEKSFE ERLTFKAFLL KFVNSYTPIF YVAFFKGRFV GRPGDYVYIF RSFRMEECAP GGCLMELCIQ LSIIMLG KQ LIQNNLFEIG IPKMKKFIRY LKLRRQSPSD REEYVKRKQR YEVDFNLEPF AGLTPEYMEM IIQFGFVTLF VASFPLAP L FALLNNIIEI RLDAKKFVTE LRRPVAIRAK DIGIWYNILR GVGKLAVIIN AFVISFTSDF IPRLVYLYMY SQNGTMHGF VNHTLSSFNV SDFQNGTAPN DPLDLGYEVQ ICRYKDYREP PWSEHKYDIS KDFWAVLAAR LAFVIVFQNL VMFMSDFVDW VIPDIPKDI SQQIHKEKVL MVELSNSLEV LFQ

UniProtKB: Anoctamin-1

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / 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

Concentration0.5 mg/mL
BufferpH: 9
Component:
ConcentrationFormulaName
10.0 mMTrisNO3tris nitrate
150.0 mMKNO3potassium nitrate
1.0 mMCaCl2calcium chloride
0.02 mMLMNGLauryl Maltose Neopentyl Glycol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.027 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: blot 6-8 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 7882 / Average exposure time: 8.0 sec. / Average electron dose: 80.0 e/Å2
Details: 5326 images for TMEM16A alone in LMNG, 2556 images for TMEM16A with Fab bound in LMNG
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 1.9000000000000001 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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

Particle selectionNumber selected: 927414
Startup modelType of model: EMDB MAP
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1beta) / Number images used: 251851
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1beta1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1beta1)
Final 3D classificationNumber classes: 7 / Avg.num./class: 48839 / Software - Name: RELION (ver. 2.1beta1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6bgj:
Cryo-EM structure of the TMEM16A calcium-activated chloride channel in LMNG

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