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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-1
KeywordsTMEM16 family / ion channel / membrane protein / cryo-EM
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: 6.6 Å
AuthorsPaulino C / Neldner Y
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
European Research Council339116 Switzerland
University of ZurichForschungskredit FK-16-036 Switzerland
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-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
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-1

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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)
Molecular weightTheoretical: 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

UniProtKB: Anoctamin-1

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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 / Pretreatment - Time: 30 sec.
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
TemperatureMin: 80.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: +10 eV
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 - 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.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.8000000000000003 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 37037
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

DetailsFourier cropping (final pixel size 1.35 A), motion correction and dose-weighting of frames were performed by MotionCor2
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.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wit

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
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wit


Chain - Source name: PDB / Chain - Initial model type: experimental model
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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