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- EMDB-3658: cryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution. -

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Basic information

Entry
Database: EMDB / ID: EMD-3658
Titlecryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution.
Map dataNone
Sample
  • Complex: mTMEM16A
    • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel
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: 6.6 Å
AuthorsPaulino C / Neldner Y / Lam KM / Kalienkova V / Brunner JD / Schenck S / Dutzler R
CitationJournal: Elife / Year: 2017
Title: Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.
Authors: Cristina Paulino / Yvonne Neldner / Andy Km Lam / Valeria Kalienkova / Janine Denise Brunner / Stephan Schenck / Raimund Dutzler /
Abstract: The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has ...The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.
History
DepositionApr 3, 2017-
Header (metadata) releaseApr 12, 2017-
Map releaseJun 7, 2017-
UpdateFeb 28, 2018-
Current statusFeb 28, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5nl2
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3658.png

Downloads & links

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Map

FileDownload / File: emd_3658.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.020843247 - 0.06946631
Average (Standard dev.)-0.000032889748 (±0.0031602522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0210.069-0.000

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Supplemental data

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Additional map: None

Fileemd_3658_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mTMEM16A

EntireName: mTMEM16A
Components
  • Complex: mTMEM16A
    • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel

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Supramolecule #1: mTMEM16A

SupramoleculeName: mTMEM16A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293 / Recombinant cell: stabel mTMEM16A cell line (Flp-In System)
Molecular weightExperimental: 110.916 KDa

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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

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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

Concentration3.08 mg/mL
BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Name: Hepes / Details: 20 mM Hepes 150 mM NaCl 0.5 mM CaCl2 <0.12% digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: 2.5 ul sample volume 1-5 sec blotting time.
Detailsfull-length (wild-type isoform ac) deglycosylated mTMEM16A

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.8 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 37037
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: +10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 5 / Number real images: 5503 / Average exposure time: 15.0 sec. / Average electron dose: 80.0 e/Å2
Details: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. ...Details: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. Images were recorded for 15 sec with an initial sub-frame exposure time of 300 ms (50 frames total) with a dose of 1.5 e-/A^2/frame, and later with a sub-frame exposure time of 150 ms (100 frames total) with a dose of 0.8 e-/A^2/frame, resulting in a total accumulated dose on the specimen level of approximately 80 e-/A^2.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 755348
Details: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using ...Details: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using the software package RELION1.4 and at a later stage RELION2.0. Approximately 4,000 particles were manually picked to generate templates for automated particle selection. Following automated picking in RELION, false positives were eliminated manually or through a first round of 2D classification resulting in 755,348 particles.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.5)
Details: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wit

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 213243
DetailsFourier cropping (final pixel size 1.35 A), motion correction and dose-weighting of frames were performed by MotionCor2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wit

RefinementProtocol: RIGID BODY FIT
Output model

PDB-5nl2:
cryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution.

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