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- PDB-8t57: Structure of mechanically activated ion channel OSCA2.3 in peptidiscs -

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Basic information

Entry
Database: PDB / ID: 8t57
TitleStructure of mechanically activated ion channel OSCA2.3 in peptidiscs
ComponentsCSC1-like protein HYP1
KeywordsMEMBRANE PROTEIN / mechanically activated ion channel
Function / homology
Function and homology information


mechanosensitive monoatomic ion channel activity / calcium-activated cation channel activity / 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
CHOLESTEROL / PALMITIC ACID / CSC1-like protein HYP1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsJojoa-Cruz, S. / Burendei, B. / 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: Structure / Year: 2024
Title: Structure of mechanically activated ion channel OSCA2.3 reveals mobile elements in the transmembrane domain.
Authors: Sebastian Jojoa-Cruz / Batuujin Burendei / Wen-Hsin Lee / Andrew B Ward /
Abstract: Members of the OSCA/TMEM63 family are mechanically activated ion channels and structures of some OSCA members have revealed the architecture of these channels and structural features that are ...Members of the OSCA/TMEM63 family are mechanically activated ion channels and structures of some OSCA members have revealed the architecture of these channels and structural features that are potentially involved in mechanosensation. However, these structures are all in a similar state and information about the motion of different elements of the structure is limited, preventing a deeper understanding of how these channels work. Here, we used cryoelectron microscopy to determine high-resolution structures of Arabidopsis thaliana OSCA1.2 and OSCA2.3 in peptidiscs. The structure of OSCA1.2 matches previous structures of the same protein in different environments. Yet, in OSCA2.3, the TM6a-TM7 linker adopts a different conformation that constricts the pore on its cytoplasmic side. Furthermore, coevolutionary sequence analysis uncovered a conserved interaction between the TM6a-TM7 linker and the beam-like domain (BLD). Our results reveal conformational heterogeneity and differences in conserved interactions between the TMD and BLD among members of the OSCA family.
History
DepositionJun 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CSC1-like protein HYP1
B: CSC1-like protein HYP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,34622
Polymers161,6962
Non-polymers5,64920
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CSC1-like protein HYP1 / OSCA2.3


Mass: 80848.078 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: HYP1, At3g01100, T4P13.21 / Plasmid: pEG / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8GUH7
#2: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O
#3: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C16H32O2
Has ligand of interestN

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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: OSCA2.3 in peptidisc / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.159 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEG
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMsodium chlorideNaClSodium chloride1
32 mMdithiothreitol1
SpecimenConc.: 3.6 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 FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5323
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 39

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2particle selection
2Leginonimage acquisition
4Gctf1.06CTF correctionFor CryoSPARC
5CTFFIND4CTF correctionFor RELION
8Coot0.9model fitting
10cryoSPARC2initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14Coot0.9model refinement
15PHENIX1.18.2-3874model refinement
16Rosetta3.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2683410
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180640 / 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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