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- EMDB-52345: Cryo-EM structure of bovine TMEM206-YFP purified and plunged usin... -

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Basic information

Entry
Database: EMDB / ID: EMD-52345
TitleCryo-EM structure of bovine TMEM206-YFP purified and plunged using MISO (micro-purification)
Map data
Sample
  • Complex: Bos taurus Proton-activated chloride channel with C-terminal venus GFP
    • Protein or peptide: Proton-activated chloride channel
KeywordspH-gated chloride channel / MEMBRANE PROTEIN
Function / homologypH-gated chloride channel activity / TMEM206 protein / TMEM206 protein family / chloride transport / chloride channel complex / plasma membrane / Proton-activated chloride channel
Function and homology information
Biological speciesBos taurus (domestic cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 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 5, 2025-
Map releaseNov 5, 2025-
UpdateNov 26, 2025-
Current statusNov 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52345.png

Downloads & links

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Map

FileDownload / File: emd_52345.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 200 pix.
= 278. Å		1.39 Å/pix.
x 200 pix.
= 278. Å		1.39 Å/pix.
x 200 pix.
= 278. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.39 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.315
Minimum - Maximum-2.5638433 - 3.2221053
Average (Standard dev.)0.0008707751 (±0.04919057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 278.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52345_msk_1.map
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Half map: #1

Fileemd_52345_half_map_1.map
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Half map: #2

Fileemd_52345_half_map_2.map
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Sample components

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Entire : Bos taurus Proton-activated chloride channel with C-terminal venus GFP

EntireName: Bos taurus Proton-activated chloride channel with C-terminal venus GFP
Components
  • Complex: Bos taurus Proton-activated chloride channel with C-terminal venus GFP
    • Protein or peptide: Proton-activated chloride channel

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Supramolecule #1: Bos taurus Proton-activated chloride channel with C-terminal venus GFP

SupramoleculeName: Bos taurus Proton-activated chloride channel with C-terminal venus GFP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (domestic cattle)

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Macromolecule #1: Proton-activated chloride channel

MacromoleculeName: Proton-activated chloride channel / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 30.375881 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LLIFIYLLLM AVAVFLVYQT IMDFREKLKH PVMSVSYKEV DRYDAPGIAL YPGQAQLLSC KHYYEVIPPL RSPGQPGDVN CTTQRVNYT DPFSNQTLKS ALIVRGPREV QKRELVFLQF RLNQSSEDFS AIDYLLFSSF QEFLQSPDRA GFMQACESAY S SWKFSGGF ...String:
LLIFIYLLLM AVAVFLVYQT IMDFREKLKH PVMSVSYKEV DRYDAPGIAL YPGQAQLLSC KHYYEVIPPL RSPGQPGDVN CTTQRVNYT DPFSNQTLKS ALIVRGPREV QKRELVFLQF RLNQSSEDFS AIDYLLFSSF QEFLQSPDRA GFMQACESAY S SWKFSGGF RTWVKMSLVE TKEEDGREAV EFRQETSVVN YIDQRPAAEK SAQLFFVVFE WKDPFIQKVQ DIITANPWNT IA LLCGAFL ALFKAAEFAK LSVKWMA

UniProtKB: Proton-activated chloride channel

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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: Au-flat 1.2/1.3 / Material: GOLD / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER
Details: MISO Chip: 3 ul single column chip filled with Pierce High-capacity streptavidin agarose resin Plunging: 80 nl.

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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: 2.7 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 87106
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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