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- EMDB-52486: Cryo-EM structure of TRPC6 (complete particle) purified and plung... -

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Basic information

Entry
Database: EMDB / ID: EMD-52486
TitleCryo-EM structure of TRPC6 (complete particle) purified and plunged using MISO (micro-purification)
Map data
Sample
  • Complex: TRPC6 with C-terminal eGFP
KeywordsTRPC6 / MEMBRANE PROTEIN
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsDe Gieter S / Eluru G / Stroobants A / Shrestha B / Erbel P / Efremov RG
Funding support Belgium, European Union, 4 items
OrganizationGrant numberCountry
Other governmentG0H5916N
Research Foundation - Flanders (FWO)G054617N Belgium
European Research Council (ERC)726436European Union
Other governmentSRP+Ablynx fund+VIB POC
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
DepositionJan 8, 2025-
Header (metadata) releaseNov 5, 2025-
Map releaseNov 5, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52486.png

Downloads & links

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Map

FileDownload / File: emd_52486.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.72 Å/pix.
x 400 pix.
= 286.2 Å		0.72 Å/pix.
x 400 pix.
= 286.2 Å		0.72 Å/pix.
x 400 pix.
= 286.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7155 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033
Minimum - Maximum-0.11627246 - 0.19801126
Average (Standard dev.)0.00055669143 (±0.0057434994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 286.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52486_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_52486_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_52486_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : TRPC6 with C-terminal eGFP

EntireName: TRPC6 with C-terminal eGFP
Components
  • Complex: TRPC6 with C-terminal eGFP

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Supramolecule #1: TRPC6 with C-terminal eGFP

SupramoleculeName: TRPC6 with C-terminal eGFP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: unidentified (others)

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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: 7.5
Details: 150 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG and 0.0006% CHS 15 mM D-(+)-biotin
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: 2.8000000000000003 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: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 11204
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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