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

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Basic information

Entry
Database: PDB / ID: 8u53
TitleMechanically activated ion channel OSCA3.1 in nanodiscs
ComponentsCSC1-like protein ERD4
KeywordsMEMBRANE PROTEIN / Mechanically activated ion channel
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
Chem-82T / Chem-CPL / PALMITIC ACID / CSC1-like protein ERD4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsJojoa-Cruz, S. / Lee, W.H. / Ward, A.B.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CSC1-like protein ERD4
B: CSC1-like protein ERD4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,36626
Polymers163,7582
Non-polymers7,60824
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration, homology
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CSC1-like protein ERD4 / OSCA3.1


Mass: 81878.898 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The last 10 residues (GTGTLEVLFQ) are leftover of a linker and protease site.
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: ERD4 / Plasmid: pcDNA3.1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9C8G5
#2: Chemical ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE


Mass: 758.060 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H80NO8P / Comment: phospholipid*YM
#3: Chemical ChemComp-82T / [(2R)-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-oxidanyl-propyl] octadecanoate


Mass: 481.603 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C23H48NO7P
#4: Chemical
ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C16H32O2
Has ligand of interestN
Sequence detailsResidues that could not be identified in the map are represented as UNK. The actual sequence of the ...Residues that could not be identified in the map are represented as UNK. The actual sequence of the protein construct is: MEFGSFLVSLGTSFVIFVILMLLFTWLSRKSGNAPIYYPNRILKGLEPWEGTSLTRNPFA WMREALTSSEQDVVNLSGVDTAVHFVFLSTVLGIFACSSLLLLPTLLPLAATDNNIKNTK NATDTTSKGTFSQLDNLSMANITKKSSRLWAFLGAVYWISLVTYFFLWKAYKHVSSLRAQ ALMSADVKPEQFAILVRDMPAPPDGQTQKEFIDSYFREIYPETFYRSLVATENSKVNKIW EKLEGYKKKLARAEAILAATNNRPTNKTGFCGLVGKQVDSIEYYTELINESVAKLETEQK AVLAEKQQTAAVVFFTTRVAAASAAQSLHCQMVDKWTVTEAPEPRQLLWQNLNIKLFSRI IRQYFIYFFVAVTILFYMIPIAFVSAITTLKNLQRIIPFIKPVVEITAIRTVLESFLPQI ALIVFLAMLPKLLLFLSKAEGIPSQSHAIRAASGKYFYFSVFNVFIGVTLAGTLFNTVKD IAKNPKLDMIINLLATSLPKSATFFLTYVALKFFIGYGLELSRIIPLIIFHLKKKYLCKT EAEVKEAWYPGDLSYATRVPGDMLILTITFCYSVIAPLILIFGITYFGLGWLVLRNQALK VYVPSYESYGRMWPHIHQRILAALFLFQVVMFGYLGAKTFFYTALVIPLIITSLIFGYVC RQKFYGGFEHTALEVACRELKQSPDLEEIFRAYIPHSLSSHKPEEHEFKGAMSRYQDFNA IAGVGTGTLEVLFQ

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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: OSCA3.1 dimer in nanodisc / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.164 MDa / Experimental value: YES
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pcDNA3.1
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMNaCl1
225 mMTris HCl1
31 mMDTT1
SpecimenConc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 400 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8375
Details: Only micrographs with a CTF estimate of 2.6A or better were used for processing
Image scansMovie frames/image: 38 / Used frames/image: 1-38

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9cryoSPARC2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Cootmodel refinement
14PHENIXmodel refinement
15Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1913316
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 197944 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingAccession code: 6MGV / Source name: SwissModel / Type: in silico model

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