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- PDB-9hqp: Cryo-EM structure of mouse TMEM16F-YFP purified and plunged using... -

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Basic information

Entry
Database: PDB / ID: 9hqp
TitleCryo-EM structure of mouse TMEM16F-YFP purified and plunged using MISO (microfluidic isolation)
ComponentsAnoctamin-6,Yellow Fluorescent Protein
KeywordsMEMBRANE PROTEIN / lipid scramblase
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 / intracellularly calcium-gated chloride channel activity / plasma membrane phospholipid scrambling / negative regulation of cell volume / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsDe Gieter, S. / Eluru, G. / Schenck, S. / Stroobants, A. / Efremov, R.G. / Brunner, J.D.
Funding support Belgium, European Union, 3items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Anoctamin-6,Yellow Fluorescent Protein
B: Anoctamin-6,Yellow Fluorescent Protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)281,4144
Polymers281,3342
Non-polymers802
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Anoctamin-6,Yellow Fluorescent Protein / Small-conductance calcium-activated nonselective cation channel / SCAN channel / Transmembrane protein 16F


Mass: 140666.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Aequorea victoria (jellyfish)
Gene: Ano6, Tmem16f / Production host: Homo sapiens (human) / References: UniProt: Q6P9J9
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Scramblase 16F with c-terminal venus GFP / Type: CELL / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7.6
Details: 150 mM NaCl, 30 mM HEPES-Na, pH 7.6, 0.0063% GDN, 10 mM D-(+)-biotin
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1700 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2PHENIX1.19_4092model refinement
10cryoSPARC4.6.2.initial Euler assignment
11cryoSPARC4.6.2.final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69117 / Symmetry type: POINT
RefinementHighest resolution: 3.51 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310695
ELECTRON MICROSCOPYf_angle_d0.64314533
ELECTRON MICROSCOPYf_dihedral_angle_d4.8061396
ELECTRON MICROSCOPYf_chiral_restr0.0421612
ELECTRON MICROSCOPYf_plane_restr0.0051782

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