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- PDB-5oyb: Structure of calcium-bound mTMEM16A chloride channel at 3.75 A re... -

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Basic information

Entry
Database: PDB / ID: 5oyb
TitleStructure of calcium-bound mTMEM16A chloride channel at 3.75 A resolution
ComponentsAnoctamin-1Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / TMEM16 family / ion channel / cryo-EM
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsPaulino, C. / Kalienkova, V. / Lam, K.M. / Neldner, Y. / Dutzler, R.
Funding support Switzerland, 2items
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, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2017Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jan 10, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 17, 2018Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _em_software.version / _pdbx_audit_support.funding_organization
Revision 1.4Jul 31, 2019Group: Data collection / Refinement description / Category: refine
Revision 1.5Dec 4, 2019Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name
Revision 1.6Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: Anoctamin-1
B: Anoctamin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)222,2786
Polymers222,1182
Non-polymers1604
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1570 Å2
ΔGint-18 kcal/mol
Surface area80120 Å2
MethodPISA

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Components

#1: Protein Anoctamin-1 / Calcium-dependent chloride channel / Transmembrane protein 16A


Mass: 111058.992 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano1, Tmem16a / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8BHY3
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: mTMEM16A with calcium ions bound / Type: COMPLEX / Details: calcium-activated chloride channel / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.110916 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: stabel mTMEM16A cell line (Flp-In System)
Buffer solutionpH: 7.5 / Details: 20 mM Hepes 150 mM NaCl 0.5 mM CaCl2 <0.12% digitonin
Buffer componentConc.: 20 mM / Name: Hepes
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: full-length (wild-type isoform ac) deglycosylated mTMEM16A in presence of 0.5mM CaCl2
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K / Details: 2 ul sample volume 2-4 sec blotting time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 46511 X / Calibrated magnification: 46511 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 4343
Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). For the dataset in presence of calcium ions, cryo-EM images were collected at a pixel size of 0. ...Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). For the dataset in presence of calcium ions, cryo-EM images were collected at a pixel size of 0.5375A in super-resolution mode, a defocus range of -0.5 to -3.0 um, an exposure time of 10 sec and a sub-frame exposure time of 125 ms (80 frames) with an approximate electron dose of 1 e-/A2/frame. An extensive effort was made for this dataset to screen different areas within the grid and only regions that provided an estimated resolution of the CTF fit of better than 4A were selected for data collection. The total accumulated dose on the specimen level was approximately 80 e-/A2.
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 80 / Used frames/image: 1-80

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1b1particle selection
2SerialEMimage acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9RELION21b1initial Euler assignment
10RELION2.1b1final Euler assignment
11RELION2.1b1classification
12RELION2.1b13D reconstruction
13PHENIXmodel refinement
14REFMAC5model refinement
Image processingDetails: Fourier cropping (final pixel size 1.075 A), motion correction and dose-weighting of frames were performed by MotionCor2
CTF correctionDetails: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 629679
Details: For the dataset collected in presence of calcium ions a total of 4,342 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1. ...Details: For the dataset collected in presence of calcium ions a total of 4,342 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.075A) and subjected to motion correction and dose-weighting of frames by MotionCor248. The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.149. Images showing a strong drift, higher defocus than -3.0 um or a bad CTF estimation were discarded, resulting in 2,997 images used for further analysis with the software package RELION2.1b150. Particles were picked automatically using 2D class averages from the previously obtained TMEM16A cryo-EM map as reference26 providing an initial set of 629,679 particles. After extraction with a box size of 300 pixels, false positives were eliminated manually or through a first round of 2D classification, resulting in 368,162 particles that were further subjected to several rounds of 2D classification to remove particles belonging to low-abundance classes. The remaining 252,577 particles were sorted during 3D Classification, a C2 symmetry was imposed and the low-resolution TMEM16A cryo-EM map (EMD-3658) was used as initial model. The best class, comprising 147,368 particles from a total of 2012 images, was subjected to auto-refinement and particle polishing in RELION, with a running average window of 5, a standard deviation of 1 pixel on translations and 200 pixels on particle distance. The final polished and auto-refined map used for model building had a resolution of 4.6A before masking and 3.75A after masking and was sharpened using an isotropic b-factor of -1063A2
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147368 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 3.75→129 Å / Cor.coef. Fo:Fc: 0.51 / ESU R: 5.717
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.9955 --
obs0.9955 52099 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 126.79 Å2
Baniso -1Baniso -2Baniso -3
1--2.2 Å221.46 Å2-0.69 Å2
2--14.76 Å2-0.64 Å2
3----12.57 Å2
Refinement stepCycle: 1 / Total: 11770
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01912072
ELECTRON MICROSCOPYr_bond_other_d00.0211368
ELECTRON MICROSCOPYr_angle_refined_deg1.2771.95416336
ELECTRON MICROSCOPYr_angle_other_deg3.646326250
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.21251426
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.37122.975558
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.612152114
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.5381584
ELECTRON MICROSCOPYr_chiral_restr0.1070.21796
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02113146
ELECTRON MICROSCOPYr_gen_planes_other0.0040.022698
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.34812.6285734
ELECTRON MICROSCOPYr_mcbond_other4.34712.6265733
ELECTRON MICROSCOPYr_mcangle_it7.97718.9087150
ELECTRON MICROSCOPYr_mcangle_other7.97718.917151
ELECTRON MICROSCOPYr_scbond_it4.11313.1766338
ELECTRON MICROSCOPYr_scbond_other4.11313.1786339
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other7.56419.5079187
ELECTRON MICROSCOPYr_long_range_B_refined13.65713880
ELECTRON MICROSCOPYr_long_range_B_other13.65613881
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.75→3.847 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.991 3880 -
Rfree-0 -
obs--100 %

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