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- PDB-8ffq: Wildtype rabbit TRPV5 into nanodiscs in the presence of PI(4,5)P2... -

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Basic information

Entry
Database: PDB / ID: 8ffq
TitleWildtype rabbit TRPV5 into nanodiscs in the presence of PI(4,5)P2 and ruthenium red
ComponentsTransient receptor potential cation channel subfamily V member 5
KeywordsMEMBRANE PROTEIN / TRPV5 / TRP Channel / Ruthenium Red / PI(4 / 5)P2
Function / homology
Function and homology information


regulation of urine volume / calcium ion import across plasma membrane / calcium ion homeostasis / calcium ion transmembrane transport / calcium channel activity / calcium ion transport / protein homotetramerization / calmodulin binding / apical plasma membrane / metal ion binding ...regulation of urine volume / calcium ion import across plasma membrane / calcium ion homeostasis / calcium ion transmembrane transport / calcium channel activity / calcium ion transport / protein homotetramerization / calmodulin binding / apical plasma membrane / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 5 / Transient receptor potential cation channel subfamily V member 5/6 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily ...Transient receptor potential cation channel subfamily V member 5 / Transient receptor potential cation channel subfamily V member 5/6 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-CPL / ERGOSTEROL / Chem-R2R / Transient receptor potential cation channel subfamily V member 5
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsDe Jesus-Perez, J.J. / Fluck, E.C. / Pumroy, R.A. / Protopopova, A.D. / Rocereta, J.A. / Moiseenkova-Bell, V.Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144120 United States
CitationJournal: EMBO Rep / Year: 2024
Title: Molecular details of ruthenium red pore block in TRPV channels.
Authors: Ruth A Pumroy / José J De Jesús-Pérez / Anna D Protopopova / Julia A Rocereta / Edwin C Fluck / Tabea Fricke / Bo-Hyun Lee / Tibor Rohacs / Andreas Leffler / Vera Moiseenkova-Bell /
Abstract: Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ...Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.
History
DepositionDec 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 5
B: Transient receptor potential cation channel subfamily V member 5
C: Transient receptor potential cation channel subfamily V member 5
D: Transient receptor potential cation channel subfamily V member 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)341,90317
Polymers335,1384
Non-polymers6,76513
Water00
1


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

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 5 / TrpV5 / Epithelial calcium channel 1 / ECaC1 / Osm-9-like TRP channel 3 / OTRPC3


Mass: 83784.586 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: Trpv5, Ecac1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9XSM3
#2: Chemical
ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C28H44O
#3: Chemical ChemComp-R2R / ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)


Mass: 559.525 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H28N14O2Ru3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE


Mass: 758.060 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H80NO8P / Comment: phospholipid*YM
Has ligand of interestY

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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: Tetramer of wildtype rabbit TRPV5 into nanodiscs in complex with ruthenium red and PI(4,5)P2
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.33514 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 12412

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
4RELION4CTF correction
7Coot0.9.8.3model fitting
9PHENIX1.20.1model refinement
10cryoSPARC3.3.2initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6859887
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 368030 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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