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- EMDB-52346: Cryo-EM structure of mouse TMEM16F-YFP purified and plunged using... -

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Basic information

Entry
Database: EMDB / ID: EMD-52346
TitleCryo-EM structure of mouse TMEM16F-YFP purified and plunged using MISO (microfluidic isolation)
Map data
Sample
  • Cell: Scramblase 16F with c-terminal venus GFP
    • Protein or peptide: Anoctamin-6,Yellow Fluorescent Protein
  • Ligand: CALCIUM ION
Keywordslipid scramblase / MEMBRANE PROTEIN
Function / homology
Function and homology information


calcium activated phospholipid scrambling / calcium activated galactosylceramide scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / positive regulation of potassium ion export across plasma membrane / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / cholinergic synapse / bone mineralization involved in bone maturation ...calcium activated phospholipid scrambling / calcium activated galactosylceramide scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / positive regulation of potassium ion export across plasma membrane / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / cholinergic synapse / bone mineralization involved in bone maturation / negative regulation of cell volume / intracellularly calcium-gated chloride channel activity / plasma membrane phospholipid scrambling / voltage-gated monoatomic ion channel activity / positive regulation of phagocytosis, engulfment / bleb assembly / Stimuli-sensing channels / calcium-activated cation channel activity / positive regulation of monocyte chemotaxis / chloride transport / dendritic cell chemotaxis / phospholipid translocation / regulation of postsynaptic membrane potential / positive regulation of bone mineralization / chloride channel complex / Neutrophil degranulation / chloride transmembrane transport / synaptic membrane / establishment of localization in cell / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / metal ion binding / identical protein 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) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsDe Gieter S / Eluru G / Schenck S / Stroobants A / Efremov RG / Brunner JD
Funding support Belgium, European Union, 3 items
OrganizationGrant numberCountry
Other governmentG0H5916N
Research Foundation - Flanders (FWO)G054617N Belgium
European Research Council (ERC)726436European Union
CitationJournal: Nat Methods / Year: 2025
Title: MISO: microfluidic protein isolation enables single-particle cryo-EM structure determination from a single cell colony.
Authors: Gangadhar Eluru / Steven De Gieter / Stephan Schenck / Annelore Stroobants / Binesh Shrestha / Paul Erbel / Janine D Brunner / Rouslan G Efremov /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) enables reconstruction of atomic-resolution 3D maps of proteins by visualizing thousands to millions of purified protein particles embedded in ...Single-particle cryogenic electron microscopy (cryo-EM) enables reconstruction of atomic-resolution 3D maps of proteins by visualizing thousands to millions of purified protein particles embedded in vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of a very sensitive analytical method for delivering high-resolution protein structure as a readout. In practice, millions of times more starting biological material is required to prepare cryo-EM grids. Here we show that using a micro isolation (MISO) method, which combines microfluidics-based protein purification with cryo-EM grid preparation, cryo-EM structures of soluble bacterial and eukaryotic membrane proteins can be solved starting from less than 1 µg of a target protein and progressing from cells to cryo-EM grids within a few hours. This scales down the amount of starting biological material hundreds to thousands of times, opening possibilities for the structural characterization of hitherto inaccessible proteins.
History
DepositionDec 16, 2024-
Header (metadata) releaseNov 26, 2025-
Map releaseNov 26, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52346.png

Downloads & links

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Map

FileDownload / File: emd_52346.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.7 Å/pix.
x 400 pix.
= 278. Å		0.7 Å/pix.
x 400 pix.
= 278. Å		0.7 Å/pix.
x 400 pix.
= 278. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.695 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.1925525 - 0.30663043
Average (Standard dev.)0.0003109353 (±0.005069578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 278.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52346_msk_1.map
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Half map: #2

Fileemd_52346_half_map_1.map
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Half map: #1

Fileemd_52346_half_map_2.map
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Sample components

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Entire : Scramblase 16F with c-terminal venus GFP

EntireName: Scramblase 16F with c-terminal venus GFP
Components
  • Cell: Scramblase 16F with c-terminal venus GFP
    • Protein or peptide: Anoctamin-6,Yellow Fluorescent Protein
  • Ligand: CALCIUM ION

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Supramolecule #1: Scramblase 16F with c-terminal venus GFP

SupramoleculeName: Scramblase 16F with c-terminal venus GFP / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Anoctamin-6,Yellow Fluorescent Protein

MacromoleculeName: Anoctamin-6,Yellow Fluorescent Protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 140.666922 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSQMMTRKVL LNMELEEDDD EDGDIVLENF DQTIVCPTFG SLENQQDFRT PEFEEFNGKP DSLFFTDGQR RIDFILVYED ESKKENNKK GTNEKQKRKR QAYESNLICH GLQLEATRSV SDDKLVFVKV HAPWEVLCTY AEIMHIKLPL KPNDLKTRSP F GNLNWFTK ...String:
MSQMMTRKVL LNMELEEDDD EDGDIVLENF DQTIVCPTFG SLENQQDFRT PEFEEFNGKP DSLFFTDGQR RIDFILVYED ESKKENNKK GTNEKQKRKR QAYESNLICH GLQLEATRSV SDDKLVFVKV HAPWEVLCTY AEIMHIKLPL KPNDLKTRSP F GNLNWFTK VLRVNESVIK PEQEFFTAPF EKSRMNDFYI LDRDSFFNPA TRSRIVYFIL SRVKYQVMNN VNKFGINRLV SS GIYKAAF PLHDCRFNYE SEDISCPSER YLLYREWAHP RSIYKKQPLD LIRKYYGEKI GIYFAWLGYY TQMLLLAAVV GVA CFLYGY LDQDNCTWSK EVCDPDIGGQ ILMCPQCDRL CPFWRLNITC ESSKKLCIFD SFGTLIFAVF MGVWVTLFLE FWKR RQAEL EYEWDTVELQ QEEQARPEYE AQCNHVVINE ITQEEERIPF TTCGKCIRVT LCASAVFFWI LLIIASVIGI IVYRL SVFI VFSTTLPKNP NGTDPIQKYL TPQMATSITA SIISFIIIMI LNTIYEKVAI MITNFELPRT QTDYENSLTM KMFLFQ FVN YYSSCFYIAF FKGKFVGYPG DPVYLLGKYR SEECDPGGCL LELTTQLTII MGGKAIWNNI QEVLLPWVMN LIGRYKR VS GSEKITPRWE QDYHLQPMGK LGLFYEYLEM IIQFGFVTLF VASFPLAPLL ALVNNILEIR VDAWKLTTQF RRMVPEKA Q DIGAWQPIMQ GIAILAVVTN AMIIAFTSDM IPRLVYYWSF SIPPYGDHTY YTMDGYINNT LSVFNITDFK NTDKENPYI GLGNYTLCRY RDFRNPPGHP QEYKHNIYYW HVIAAKLAFI IVMEHIIYSV KFFISYAIPD VSKITKSKIK REKYLTQKLL HESHLKDLT KNMGIIAERI GGTVDNSVRP KLEALEVLFQ GPQGTMVSKG EELFTGVVPI LVELDGDVNG HKFSVSGEGE G DATYGKLT LKFICTTGKL PVPWPTLVTT LTYGVQCFSR YPDHMKQHDF FKSAMPEGYV QERTIFFKDD GNYKTRAEVK FE GDTLVNR IELKGIDFKE DGNILGHKLE YNYNSHNVYI MADKQKNGIK VNFKIRHNIE DGSVQLADHY QQNTPIGDGP VLL PDNHYL STQSALSKDP NEKRDHMVLL EFVTAAGITL GMDELYKGTE QKLISEEDLR GASMDEKTTG WRGGHVVEGL AGEL EQLRA RLEHHPQGQR EP

UniProtKB: Anoctamin-6

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

BufferpH: 7.6
Details: 150 mM NaCl, 30 mM HEPES-Na, pH 7.6, 0.0063% GDN, 10 mM D-(+)-biotin
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.0 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 69117
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2.)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2.)
FSC plot (resolution estimation)

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