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- EMDB-4748: CryoEM structure of calcium-free human TMEM16K / Anoctamin 10 in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4748
TitleCryoEM structure of calcium-free human TMEM16K / Anoctamin 10 in detergent (closed form)
Map dataRelion2 post-processed sharpened with -150A**2 b-factor
Sample
  • Organelle or cellular component: Anoctamin 10Calcium-dependent chloride channel
    • Protein or peptide: Anoctamin-10Calcium-dependent chloride channel
Function / homology
Function and homology information


intracellularly calcium-gated chloride channel activity / calcium-activated cation channel activity / chloride transport / chloride channel activity / monoatomic cation transport / chloride transmembrane transport / monoatomic ion transmembrane transport / Stimuli-sensing channels / Induction of Cell-Cell Fusion / intracellular membrane-bounded organelle ...intracellularly calcium-gated chloride channel activity / calcium-activated cation channel activity / chloride transport / chloride channel activity / monoatomic cation transport / chloride transmembrane transport / monoatomic ion transmembrane transport / Stimuli-sensing channels / Induction of Cell-Cell Fusion / intracellular membrane-bounded organelle / membrane / plasma membrane
Similarity search - Function
Anoctamin / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.14 Å
AuthorsPike ACW / Bushell SR / Shintre CA / Tessitore A / Chu A / Mukhopadhyay S / Shrestha L / Chalk R / Burgess-Brown NA / Love J ...Pike ACW / Bushell SR / Shintre CA / Tessitore A / Chu A / Mukhopadhyay S / Shrestha L / Chalk R / Burgess-Brown NA / Love J / Huiskonen JT / Edwards AM / Arrowsmith CH / Bountra C / Carpenter EP / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust106169/Z/14/Z United Kingdom
European Commission115766 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K.
Authors: Simon R Bushell / Ashley C W Pike / Maria E Falzone / Nils J G Rorsman / Chau M Ta / Robin A Corey / Thomas D Newport / John C Christianson / Lara F Scofano / Chitra A Shintre / Annamaria ...Authors: Simon R Bushell / Ashley C W Pike / Maria E Falzone / Nils J G Rorsman / Chau M Ta / Robin A Corey / Thomas D Newport / John C Christianson / Lara F Scofano / Chitra A Shintre / Annamaria Tessitore / Amy Chu / Qinrui Wang / Leela Shrestha / Shubhashish M M Mukhopadhyay / James D Love / Nicola A Burgess-Brown / Rebecca Sitsapesan / Phillip J Stansfeld / Juha T Huiskonen / Paolo Tammaro / Alessio Accardi / Elisabeth P Carpenter /
Abstract: Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic ...Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
History
DepositionMar 29, 2019-
Header (metadata) releaseMay 1, 2019-
Map releaseMay 1, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6r7z
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4748.png

Downloads & links

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Map

FileDownload / File: emd_4748.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelion2 post-processed sharpened with -150A**2 b-factor
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.01239343 - 0.038761023
Average (Standard dev.)0.0017136736 (±0.003567372)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 164.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z164.400164.400164.400
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0120.0390.002

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Supplemental data

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Mask #1

Fileemd_4748_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION2 refine3d halfmap1

Fileemd_4748_half_map_1.map
AnnotationRELION2 refine3d halfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION2 refine3d halfmap2

Fileemd_4748_half_map_2.map
AnnotationRELION2 refine3d halfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Anoctamin 10

EntireName: Anoctamin 10Calcium-dependent chloride channel
Components
  • Organelle or cellular component: Anoctamin 10Calcium-dependent chloride channel
    • Protein or peptide: Anoctamin-10Calcium-dependent chloride channel

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Supramolecule #1: Anoctamin 10

SupramoleculeName: Anoctamin 10 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 153 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: PFB-CT10HF-LIC

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Macromolecule #1: Anoctamin-10

MacromoleculeName: Anoctamin-10 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.276016 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKVTLSALDT SESSFTPLVV IELAQDVKEE TKEWLKNRII AKKKDGGAQL LFRPLLNKYE QETLENQNLY LVGASKIRML LGAEAVGLV KECNDNTMRA FTYRTRQNFK GFDDNNDDFL TMAECQFIIK HELENLRAKD EKMIPGYPQA KLYPGKSLLR R LLTSGIVI ...String:
MKVTLSALDT SESSFTPLVV IELAQDVKEE TKEWLKNRII AKKKDGGAQL LFRPLLNKYE QETLENQNLY LVGASKIRML LGAEAVGLV KECNDNTMRA FTYRTRQNFK GFDDNNDDFL TMAECQFIIK HELENLRAKD EKMIPGYPQA KLYPGKSLLR R LLTSGIVI QVFPLHDSEA LKKLEDTWYT RFALKYQPID SIRGYFGETI ALYFGFLEYF TFALIPMAVI GLPYYLFVWE DY DKYVIFA SFNLIWSTVI LELWKRGCAN MTYRWGTLLM KRKFEEPRPG FHGVLGINSI TGKEEPLYPS YKRQLRIYLV SLP FVCLCL YFSLYVMMIY FDMEVWALGL HENSGSEWTS VLLYVPSIIY AIVIEIMNRL YRYAAEFLTS WENHRLESAY QNHL ILKVL VFNFLNCFAS LFYIAFVLKD MKLLRQSLAT LLITSQILNQ IMESFLPYWL QRKHGVRVKR KVQALKADID ATLYE QVIL EKEMGTYLGT FDDYLELFLQ FGYVSLFSCV YPLAAAFAVL NNFTEVNSDA LKMCRVFKRP FSEPSANIGV WQLAFE TMS VISVVTNCAL IGMSPQVNAV FPESKADLIL IVVAVEHALL ALKFILAFAI PDKPRHIQMK LARLEFESLE ALKQQQM KL VTENLKEEPM ESGKEKATAE NLYFQ

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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

Concentration4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC8H18N2O4SNa HEPES
10.0 mMC14H20N2O10Na4EGTA
0.045 % (w/v)C23H44O11Undecyl b-D Maltopyranoside
0.0045 % (w/v)C31H50O4cholesteryl hemisuccinate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4.5sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2038 / Average exposure time: 8.0 sec. / Average electron dose: 56.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 143308
Details: Particles count after a single round of 2D classification
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.5)
Startup modelType of model: OTHER / Details: Ab-initio model calculated in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 10000 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 23607
FSC plot (resolution estimation)

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Atomic model buiding 1

Detailsphenix.real_space_refine with NCS constraints and model reference restraints (PDB id: TBC).
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 150 / Target criteria: Correlation coef
Output model

PDB-6r7z:
CryoEM structure of calcium-free human TMEM16K / Anoctamin 10 in detergent (closed form)

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