EMDB-3860: Cryo-EM map of calcium-bound mTMEM16A chloride channel at 3.75 A ...

Basic information

• Entry: Database: EMDB / ID: EMD-3860
• Title: Cryo-EM map of calcium-bound mTMEM16A chloride channel at 3.75 A resolution
• Map data: None

Sample

• Complex: mTMEM16A with calcium ions bound
  • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel
• Ligand: CALCIUM IONCalcium

Function / homology

Function:

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

Domain/homology:

: / Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin / Calcium-activated chloride channel

Component:

Anoctamin-1

• Biological species: Mus musculus (house mouse)
• Method: single particle reconstruction / cryo EM / Resolution: 3.75 Å
• Authors: Paulino C / Kalienkova V / Lam KM / Neldner Y / Dutzler R

Funding support

Switzerland, 2 items

Organization	Grant number	Country
University of Zurich	FK-16-036	Switzerland
European Research Council	339116AnoBest	Switzerland

• Citation: Journal: 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 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

Deposition	Sep 8, 2017	-
Header (metadata) release	Sep 20, 2017	-
Map release	Dec 20, 2017	-
Update	Dec 11, 2019	-
Current status	Dec 11, 2019	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3860.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: None
• Voxel size: X=Y=Z: 1.075 Å

Density

Contour Level	By AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum	-0.04136806 - 0.081163116
Average (Standard dev.)	0.00008710322 (±0.0029635872)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 300 300 300 Spacing 300 300 300
Cell	A=B=C: 322.5 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.075	1.075	1.075
M x/y/z	300	300	300
origin x/y/z	0.000	0.000	0.000
length x/y/z	322.500	322.500	322.500
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	300	300	300
D min/max/mean	-0.041	0.081	0.000

Supplemental data

Mask #1

• File: emd_3860_msk_1.map

Additional map: None

• File: emd_3860_additional.map
• Annotation: None

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

• File: emd_3860_half_map_1.map
• Annotation: cryo-EM half map 2 of the ion channel TMEM16A from mouse in presence of calcium ions

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

• File: emd_3860_half_map_2.map
• Annotation: cryo-EM half map 1 of the ion channel TMEM16A from mouse in presence of calcium ions

Sample components

Entire : mTMEM16A with calcium ions bound

• Entire: Name: mTMEM16A with calcium ions bound

Components

• Complex: mTMEM16A with calcium ions bound
  • Protein or peptide: Anoctamin-1Calcium-dependent chloride channel
• Ligand: CALCIUM IONCalcium

Supramolecule #1: mTMEM16A with calcium ions bound

• Supramolecule: Name: mTMEM16A with calcium ions bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: calcium-activated chloride channel
• Source (natural): Organism: Mus musculus (house mouse)
• Recombinant expression: Organism: Homo sapiens (human) / Recombinant strain: HEK293 / Recombinant cell: stabel mTMEM16A cell line (Flp-In System)
• Molecular weight: Experimental: 110.916 KDa

Macromolecule #1: Anoctamin-1

• Macromolecule: Name: Anoctamin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Mus musculus (house mouse)
• Molecular weight: Theoretical: 111.058992 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

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

Macromolecule #2: CALCIUM ION

• Macromolecule: Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
• Molecular weight: Theoretical: 40.078 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 3.5 mg/mL
• Buffer: pH: 7.5 / Component - Concentration: 20.0 mM / Component - Name: Hepes / Details: 20 mM Hepes 150 mM NaCl 0.5 mM CaCl2 <0.12% digitonin
• Grid: Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV / Details: 2 ul sample volume 2-4 sec blotting time.
• Details: full-length (wild-type isoform ac) deglycosylated mTMEM16A in presence of 0.5mM CaCl2

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 46511 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 46511
• Specialist optics: Energy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: +10 eV
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Temperature: Min: 80.0 K / Max: 100.0 K
• Image recording: Film 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: 1-80 / Number grids imaged: 3 / Number real images: 4343 / Average exposure time: 10.0 sec. / Average electron dose: 80.0 e/Ų; 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.
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number 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 1.075A) 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
• CTF correction: Software - Name: CTFFIND (ver. 4.1.8); Details: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

5nl2

• Initial angle assignment: Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 21b1)
• Final 3D classification: Software - Name: RELION (ver. 2.1b1)
• Final angle assignment: Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b1)
• Final reconstruction: Applied symmetry - Point group: C₂ (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1b1) / Number images used: 147368
• Details: Fourier cropping (final pixel size 1.075 A), motion correction and dose-weighting of frames were performed by MotionCor2

Atomic model buiding 1

• Refinement: Protocol: RIGID BODY FIT

Output model

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