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- EMDB-28154: cryo-EM structure of TMEM63B in LMNG -

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Basic information

Entry
Database: EMDB / ID: EMD-28154
Titlecryo-EM structure of TMEM63B in LMNG
Map data
Sample
  • Complex: TMEM63B purified protein in LMNG
    • Protein or peptide: CSC1-like protein 2
Keywordsion channel / mechanosensitive / monomeric / MEMBRANE PROTEIN
Function / homology
Function and homology information


alveolar lamellar body membrane / surfactant secretion / osmolarity-sensing monoatomic cation channel activity / mechanosensitive monoatomic cation channel activity / mechanosensitive monoatomic ion channel activity / calcium-activated cation channel activity / exocytosis / sensory perception of sound / actin cytoskeleton / early endosome membrane / plasma membrane
Similarity search - Function
CSC1/OSCA1-like, 7TM region / CSC1/OSCA1-like, cytosolic domain / CSC1/OSCA1-like, N-terminal transmembrane domain / Calcium permeable stress-gated cation channel 1-like / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28154.png

Downloads & links

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Map

FileDownload / File: emd_28154.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163
Minimum - Maximum-1.0110935 - 1.3349612
Average (Standard dev.)0.0004001488 (±0.023876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_28154_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28154_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 : TMEM63B purified protein in LMNG

EntireName: TMEM63B purified protein in LMNG
Components
  • Complex: TMEM63B purified protein in LMNG
    • Protein or peptide: CSC1-like protein 2

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Supramolecule #1: TMEM63B purified protein in LMNG

SupramoleculeName: TMEM63B purified protein in LMNG / 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 2

MacromoleculeName: CSC1-like protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 96.051203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLPFLLATLG TTALNNSNPK DYCYSARIRS TVLQGLPFGG VPTVLALDFM CFLALLFLFS ILRKVAWDYG RLALVTDADR LRRQERDRV EQEYVASAMH GDSHDRYERL TSVSSSVDFD QRDNGFCSWL TAIFRIKDDE IRDKCGGDAV HYLSFQRHII G LLVVVGVL ...String:
MLPFLLATLG TTALNNSNPK DYCYSARIRS TVLQGLPFGG VPTVLALDFM CFLALLFLFS ILRKVAWDYG RLALVTDADR LRRQERDRV EQEYVASAMH GDSHDRYERL TSVSSSVDFD QRDNGFCSWL TAIFRIKDDE IRDKCGGDAV HYLSFQRHII G LLVVVGVL SVGIVLPVNF SGDLLENNAY SFGRTTIANL KSGNNLLWLH TSFAFLYLLL TVYSMRRHTS KMRYKEDDLV KR TLFINGI SKYAESEKIK KHFEEAYPNC TVLEARPCYN VARLMFLDAE RKKAERGKLY FTNLQSKENV PTMINPKPCG HLC CCVVRG CEQVEAIEYY TKLEQKLKED YKREKEKVNE KPLGMAFVTF HNETITAIIL KDFNVCKCQG CTCRGEPRPS SCSE SLHIS NWTVSYAPDP QNIYWEHLSI RGFIWWLRCL VINVVLFILL FFLTTPAIII TTMDKFNVTK PVEYLNNPII TQFFP TLLL WCFSALLPTI VYYSAFFEAH WTRSGENRTT MHKCYTFLIF MVLLLPSLGL SSLDLFFRWL FDKKFLAEAA IRFECV FLP DNGAFFVNYV IASAFIGNAM DLLRIPGLLM YMIRLCLARS AAERRNVKRH QAYEFQFGAA YAWMMCVFTV VMTYSIT CP IIVPFGLMYM LLKHLVDRYN LYYAYLPAKL DKKIHSGAVN QVVAAPILCL FWLLFFSTMR TGFLAPTSMF TFVVLVIT I VICLCHVCFG HFKYLSAHNY KIEHTETDTV DPRSNGRPPT AAAVPKSAKY IAQVLQDSEV DGDGDGAPGS SGDEPPSSS SQDEELLMPP DALTDTDFQS CEDSLIENEI HQTRLEVLFQ

UniProtKB: CSC1-like protein 2

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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

Concentration2.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium 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 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.8 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 198144
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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