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- EMDB-28153: cryo-EM structure of TMEM63A in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-28153
Titlecryo-EM structure of TMEM63A in nanodisc
Map data
Sample
  • Complex: TMEM63A purified protein in nanodisc
    • Protein or peptide: CSC1-like protein 1
Keywordsion channel / mechanosensitive / monomeric / MEMBRANE PROTEIN
Function / homology
Function and homology information


osmolarity-sensing monoatomic cation channel activity / mechanosensitive monoatomic ion channel activity / calcium-activated cation channel activity / tertiary granule membrane / centriolar satellite / specific granule membrane / nucleic acid binding / lysosomal membrane / intracellular membrane-bounded organelle / Neutrophil degranulation ...osmolarity-sensing monoatomic cation channel activity / mechanosensitive monoatomic ion channel activity / calcium-activated cation channel activity / tertiary granule membrane / centriolar satellite / specific granule membrane / nucleic acid binding / lysosomal membrane / intracellular membrane-bounded organelle / Neutrophil degranulation / extracellular exosome / 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 / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZheng W / Fu TM / Holt JR
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseAug 23, 2023-
Map releaseAug 23, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28153.png

Downloads & links

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Map

FileDownload / File: emd_28153.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-1.1063843 - 1.7311482
Average (Standard dev.)0.0006425527 (±0.03594074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 217.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28153_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28153_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TMEM63A purified protein in nanodisc

EntireName: TMEM63A purified protein in nanodisc
Components
  • Complex: TMEM63A purified protein in nanodisc
    • Protein or peptide: CSC1-like protein 1

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Supramolecule #1: TMEM63A purified protein in nanodisc

SupramoleculeName: TMEM63A purified protein in nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: CSC1-like protein 1

MacromoleculeName: CSC1-like protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 93.200992 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MMDSPFLELW QSKAVSIREQ LGLGDRPNDS YCYNSAKNST VLQGVTFGGI PTVLLIDVSC FLFLILVFSI IRRRFWDYGR IALVSEADS ESRFQRLSST SSSGQQDFEN ELGCCPWLTA IFRLHDDQIL EWCGEDAIHY LSFQRHIIFL LVVVSFLSLC V ILPVNLSG ...String:
MMDSPFLELW QSKAVSIREQ LGLGDRPNDS YCYNSAKNST VLQGVTFGGI PTVLLIDVSC FLFLILVFSI IRRRFWDYGR IALVSEADS ESRFQRLSST SSSGQQDFEN ELGCCPWLTA IFRLHDDQIL EWCGEDAIHY LSFQRHIIFL LVVVSFLSLC V ILPVNLSG DLLDKDPYSF GRTTIANLQT DNDLLWLHTI FAVIYLFLTV GFMRHHTQSI KYKEENLVRR TLFITGLPRD AR KETVESH FRDAYPTCEV VDVQLCYNVA KLIYLCKEKK KTEKSLTYYT NLQVKTGQRT LINPKPCGQF CCCEVLGCEW EDA ISYYTR MKDRLLERIT EEERHVQDQP LGMAFVTFQE KSMATYILKD FNACKCQSLQ CKGEPQPSSH SRELYTSKWT VTFA ADPED ICWKNLSIQG LRWWLQWLGI NFTLFLGLFF LTTPSIILST MDKFNVTKPI HALNNPIISQ FFPTLLLWSF SALLP SIVY YSTLLESHWT KSGENQIMMT KVYIFLIFMV LILPSLGLTS LDFFFRWLFD KTSSEASIRL ECVFLPDQGA FFVNYV IAS AFIGNGMELL RLPGLILYTF RMIMAKTAAD RRNVKQNQAF QYEFGAMYAW MLCVFTVIVA YSITCPIIAP FGLIYIL LK HMVDRHNLYF VYLPAKLEKG IHFAAVNQAL AAPILCLFWL YFFSFLRLGM KAPATLFTFL VLLLTILVCL AHTCFGCF K HLSPLNYKTE EPASDKGSEA EAHMPPPFTP YVPRILNGLA SERTALSPQQ QQQQTYGAIH NISGTIPGQC LAQSATGSV AAAPQEATRL EVLFQ

UniProtKB: CSC1-like protein 1

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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

Concentration2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMTris
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.497 sec. / Average electron dose: 50.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 64399

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8ehw:
cryo-EM structure of TMEM63A in nanodisc

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