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- PDB-8ehx: cryo-EM structure of TMEM63B in LMNG -

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Basic information

Entry
Database: PDB / ID: 8ehx
Titlecryo-EM structure of TMEM63B in LMNG
ComponentsCSC1-like protein 2
KeywordsMEMBRANE PROTEIN / ion channel / mechanosensitive / monomeric
Function / homology
Function and homology information


osmolarity-sensing monoatomic cation channel activity / mechanosensitive monoatomic ion channel activity / calcium-activated cation channel activity / sensory perception of sound / actin cytoskeleton / plasma membrane
Similarity search - Function
Calcium permeable stress-gated cation channel 1-like / CSC1/OSCA1-like, 7TM region / CSC1/OSCA1-like, cytosolic domain / CSC1/OSCA1-like, N-terminal transmembrane domain / Calcium-dependent channel, 7TM region, putative phosphate / Late exocytosis, associated with Golgi transport / Cytosolic domain of 10TM putative phosphate transporter
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZheng, W. / Fu, T.M. / Holt, J.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01DC013521 United States
CitationJournal: Neuron / Year: 2023
Title: TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels.
Authors: Wang Zheng / Shaun Rawson / Zhangfei Shen / Elakkiya Tamilselvan / Harper E Smith / Julia Halford / Chen Shen / Swetha E Murthy / Maximilian H Ulbrich / Marcos Sotomayor / Tian-Min Fu / Jeffrey R Holt /
Abstract: OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. ...OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.
History
DepositionSep 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 23, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CSC1-like protein 2


Theoretical massNumber of molelcules
Total (without water)96,0511
Polymers96,0511
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, Multiple biochemical evidence were obtained to support this monomeric assembly
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CSC1-like protein 2 / Transmembrane protein 63B


Mass: 96051.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM63B, C6orf110 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q5T3F8

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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: TMEM63B purified protein in LMNG / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
250 mMTris1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.8 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEM3.8.6image acquisition
4cryoSPARC3.3.2CTF correction
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198144 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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