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- EMDB-3861: Cryo-EM map of calcium-free mTMEM16A chloride channel at 4.06 A r... -

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Basic information

Entry
Database: EMDB / ID: EMD-3861
TitleCryo-EM map of calcium-free mTMEM16A chloride channel at 4.06 A resolution
Map dataNone
Sample
  • Complex: mTMEM16A in absence of calcium ions
    • Protein or peptide: Anoctamin-1
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 / voltage-gated chloride channel activity / Stimuli-sensing channels / chloride transport ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / voltage-gated chloride channel activity / Stimuli-sensing channels / 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 / cell projection / establishment of localization in cell / regulation of membrane potential / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / cellular response to heat / 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: 4.06 Å
AuthorsPaulino C / Kalienkova V / Lam KM / Neldner Y / Dutzler R
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
University of ZurichFK-16-036 Switzerland
European Research Council339116AnoBest Switzerland
CitationJournal: Nature / Year: 2017
Title: Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.
Authors: Cristina Paulino / Valeria Kalienkova / Andy K M Lam / Yvonne Neldner / Raimund Dutzler /
Abstract: The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca concentration. The protein is broadly expressed and ...The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca concentration. The protein is broadly expressed and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16), and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.
History
DepositionSep 8, 2017-
Header (metadata) releaseSep 20, 2017-
Map releaseDec 20, 2017-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.036
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5oyg
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3861.png

Downloads & links

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Map

FileDownload / File: emd_3861.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.365 Å
Density
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum-0.055024542 - 0.12597124
Average (Standard dev.)0.00002115085 (±0.0042695184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3651.3651.365
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z349.440349.440349.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0550.1260.000

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Supplemental data

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Mask #1

Fileemd_3861_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: None

Fileemd_3861_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryo-EM half map 2 of the ion channel...

Fileemd_3861_half_map_1.map
Annotationcryo-EM half map 2 of the ion channel TMEM16A from mouse in absence of calcium ions
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryo-EM half map 1 of the ion channel...

Fileemd_3861_half_map_2.map
Annotationcryo-EM half map 1 of the ion channel TMEM16A from mouse in absence of calcium ions
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 in absence of calcium ions

EntireName: mTMEM16A in absence of calcium ions
Components
  • Complex: mTMEM16A in absence of calcium ions
    • Protein or peptide: Anoctamin-1

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Supramolecule #1: mTMEM16A in absence of calcium ions

SupramoleculeName: mTMEM16A in absence of calcium ions / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: calcium-activated chloride channel
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

Concentration3.3 mg/mL
BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Name: Hepes / Details: 20 mM Hepes 150 mM NaCl <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: 288 K / Instrument: FEI VITROBOT MARK IV / Details: 2 ul sample volume 2-4 sec blotting time.
Detailsfull-length (wild-type isoform ac) deglycosylated mTMEM16A in absence of CaCl2

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV / 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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-100 / Number grids imaged: 3 / Number real images: 5063 / Average exposure time: 15.0 sec. / Average electron dose: 80.0 e/Å2
Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). The dataset in absence of calcium ions was obtained at a pixel size of 0.6825A in super- ...Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). The dataset in absence of calcium ions was obtained at a pixel size of 0.6825A in super-resolution mode, a defocus range of -0.5 to -3.9 um, an exposure time of 15 sec and a sub-frame exposure time of 150 ms (100 frames) with an electron dose at the specimen level of 0.75-0.8 e-/A2/frame. The total accumulated dose on the specimen level was approximately 80 e-/A2.
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: 36630 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36630
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 1.365 A), motion correction and dose-weighting of frames were performed by MotionCor2
Particle selectionNumber selected: 1349821
Details: For the dataset collected in absence of calcium ions a total of 5063 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.365A) ...Details: For the dataset collected in absence of calcium ions a total of 5063 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.365A) and subjected to motion correction and dose-weighting of frames by MotionCor2. The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1. Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, From a total of 4,738 images (final pixel size 1.365 A) 1,349,821 particles were extracted with a box size of 256 pixels after auto-picking and initial round of 2D classification. Initial rounds of 2D and 3D classification, resulted in a set of 467,286 particles which where subjected to particle polishing, as described above, as well as final rounds of 3D classification. The final polished and auto-refined map was calculated from 195,465 particles derived from 4726 images with a resolution of 4.86 A before masking and 4.06 A after masking and was sharpened using an isotropic b-factor of -116 A2
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
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:

5oyb

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1b1) / Number images used: 195465
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 21b1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b1)
Final 3D classificationSoftware - Name: RELION (ver. 2.1b1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-5oyg:
Structure of calcium-free mTMEM16A chloride channel at 4.06 A resolution

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