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- EMDB-41911: Mechanically activated ion channel OSCA3.1 in nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-41911
TitleMechanically activated ion channel OSCA3.1 in nanodiscs
Map dataMap postprocessed with LocalDeblur
Sample
  • Complex: OSCA3.1 dimer in nanodisc
    • Protein or peptide: CSC1-like protein ERD4
  • Ligand: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: [(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl] octadecanoate
  • Ligand: PALMITIC ACID
KeywordsMechanically activated ion channel / membrane protein
Function / homology
Function and homology information


plasmodesma / plant-type vacuole / calcium-activated cation channel activity / chloroplast envelope / mRNA binding / nucleus / 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
CSC1-like protein ERD4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsJojoa-Cruz S / Lee WH / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL143297 United States
CitationJournal: Elife / Year: 2024
Title: Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli.
Authors: Sebastian Jojoa-Cruz / Adrienne E Dubin / Wen-Hsin Lee / Andrew B Ward /
Abstract: The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the ...The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.
History
DepositionSep 12, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41911.png

Downloads & links

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Map

FileDownload / File: emd_41911.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap postprocessed with LocalDeblur
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.0036643825 - 0.1660746
Average (Standard dev.)-0.000013690941 (±0.0040864553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 206.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Map postprocessed with DeepEMhance

Fileemd_41911_additional_1.map
AnnotationMap postprocessed with DeepEMhance
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_41911_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_41911_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : OSCA3.1 dimer in nanodisc

EntireName: OSCA3.1 dimer in nanodisc
Components
  • Complex: OSCA3.1 dimer in nanodisc
    • Protein or peptide: CSC1-like protein ERD4
  • Ligand: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: [(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl] octadecanoate
  • Ligand: PALMITIC ACID

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Supramolecule #1: OSCA3.1 dimer in nanodisc

SupramoleculeName: OSCA3.1 dimer in nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 164 KDa

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Macromolecule #1: CSC1-like protein ERD4

MacromoleculeName: CSC1-like protein ERD4 / type: protein_or_peptide / ID: 1
Details: The last 10 residues (GTGTLEVLFQ) are leftover of a linker and protease site.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 81.878898 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEFGSFLVSL GTSFVIFVIL MLLFTWLSRK SGNAPIYYPN RILKGLEPWE GTSLTRNPFA WMREALTSSE QDVVNLSGVD TAVHFVFLS TVLGIFACSS LLLLPTLLPL AATDNNIKNT KNATDTTSKG TFSQLDNLSM ANITKKSSRL WAFLGAVYWI S LVTYFFLW ...String:
MEFGSFLVSL GTSFVIFVIL MLLFTWLSRK SGNAPIYYPN RILKGLEPWE GTSLTRNPFA WMREALTSSE QDVVNLSGVD TAVHFVFLS TVLGIFACSS LLLLPTLLPL AATDNNIKNT KNATDTTSKG TFSQLDNLSM ANITKKSSRL WAFLGAVYWI S LVTYFFLW KAYKHVSSLR AQALMSADVK PEQFAILVRD MPAPPDGQTQ KEFIDSYFRE IYPETFYRSL VATEN(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)K KKLARAEAIL AATNNRPTN KTGFCGLVGK QVD(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)AE KQQTAAVVFF TTRVAAASAA QSL HCQMVD KWTVTEAPEP RQLLWQNLNI KLFSRIIRQY FIYFFVAVTI LFYMIPIAFV SAITTLKNLQ RIIPFIKPVV EITA IRTVL ESFLPQIALI VFLAMLPKLL LFLSKAEGIP SQSHAIRAAS GKYFYFSVFN VFIGVTLAGT LFNTVKDIAK NPKLD MIIN LLATSLPKSA TFFLTYVALK FFIGYGLELS RIIPLIIFHL KKKYLCKTEA EVKEAWYPGD LSYATRVPGD MLILTI TFC YSVIAPLILI FGITYFGLGW LVLRNQALKV YVPSYESYGR MWPHIHQRIL AALFLFQVVM FGYLGAKTFF YTALVIP LI ITSLIFGYVC RQKFYGGFEH TALEVACREL KQSPDLEEIF RAYIPHSLSS HKPEEHEFKG AMSRYQDFNA IAGVGTGT L EVLFQ

UniProtKB: CSC1-like protein ERD4

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Macromolecule #2: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 2 / Formula: CPL
Molecular weightTheoretical: 758.06 Da
Chemical component information

ChemComp-CPL:
1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #3: [(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl...

MacromoleculeName: [(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl] octadecanoate
type: ligand / ID: 3 / Number of copies: 2 / Formula: 82T
Molecular weightTheoretical: 481.603 Da
Chemical component information

ChemComp-82T:
[(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl] octadecanoate

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Macromolecule #4: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 4 / Number of copies: 20 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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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

Concentration3.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaCl
25.0 mMTris HCl
1.0 mMDTT
GridMaterial: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Frames/image: 1-38 / Number grids imaged: 1 / Number real images: 8375 / Average electron dose: 50.0 e/Å2
Details: Only micrographs with a CTF estimate of 2.6A or better were used for processing
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm
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

Particle selectionNumber selected: 1913316
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 197944
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mgv


Chain - Chain ID: A / Chain - Source name: SwissModel / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8u53:
Mechanically activated ion channel OSCA3.1 in nanodiscs

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