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- EMDB-29047: Wildtype rat TRPV2 in nanodiscs bound to RR and 2-APB -

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Basic information

Entry
Database: EMDB / ID: EMD-29047
TitleWildtype rat TRPV2 in nanodiscs bound to RR and 2-APB
Map data
Sample
  • Complex: Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 2
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE
  • Ligand: 2-aminoethyl diphenylborinate
  • Ligand: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)
KeywordsTRPV2 / 2-APB / TRP Channel / Ruthenium Red / MEMBRANE PROTEIN
Function / homology
Function and homology information


growth cone membrane / TRP channels / response to temperature stimulus / positive regulation of calcium ion import / endomembrane system / positive regulation of axon extension / axonal growth cone / monoatomic cation channel activity / calcium channel activity / melanosome ...growth cone membrane / TRP channels / response to temperature stimulus / positive regulation of calcium ion import / endomembrane system / positive regulation of axon extension / axonal growth cone / monoatomic cation channel activity / calcium channel activity / melanosome / lamellipodium / positive regulation of cold-induced thermogenesis / cell body / negative regulation of cell population proliferation / axon / cell surface / identical protein binding / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / 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
Transient receptor potential cation channel subfamily V member 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsPumroy RA / Protopopova AD / Rocereta JA / De Jesus-Perez JJ / Fluck EC / Moiseenkova-Bell VY
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29047.png

Downloads & links

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Map

FileDownload / File: emd_29047.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 320 pix.
= 266.88 Å		0.83 Å/pix.
x 320 pix.
= 266.88 Å		0.83 Å/pix.
x 320 pix.
= 266.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-19.149374000000002 - 35.629759999999997
Average (Standard dev.)-0.000000000007882 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29047_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29047_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red

EntireName: Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red
Components
  • Complex: Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 2
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE
  • Ligand: 2-aminoethyl diphenylborinate
  • Ligand: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)

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Supramolecule #1: Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red

SupramoleculeName: Tetramer of wildtype rat TRPV2 in nanodiscs bound to ruthenium red
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 340 KDa

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Macromolecule #1: Transient receptor potential cation channel subfamily V member 2

MacromoleculeName: Transient receptor potential cation channel subfamily V member 2
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 86.798891 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MTSASSPPAF RLETSDGDEE GNAEVNKGKQ EPPPMESPFQ REDRNSSPQI KVNLNFIKRP PKNTSAPSQQ EPDRFDRDRL FSVVSRGVP EELTGLLEYL RWNSKYLTDS AYTEGSTGKT CLMKAVLNLQ DGVNACIMPL LQIDKDSGNP KLLVNAQCTD E FYQGHSAL ...String:
MTSASSPPAF RLETSDGDEE GNAEVNKGKQ EPPPMESPFQ REDRNSSPQI KVNLNFIKRP PKNTSAPSQQ EPDRFDRDRL FSVVSRGVP EELTGLLEYL RWNSKYLTDS AYTEGSTGKT CLMKAVLNLQ DGVNACIMPL LQIDKDSGNP KLLVNAQCTD E FYQGHSAL HIAIEKRSLQ CVKLLVENGA DVHLRACGRF FQKHQGTCFY FGELPLSLAA CTKQWDVVTY LLENPHQPAS LE ATDSLGN TVLHALVMIA DNSPENSALV IHMYDGLLQM GARLCPTVQL EEISNHQGLT PLKLAAKEGK IEIFRHILQR EFS GPYQPL SRKFTEWCYG PVRVSLYDLS SVDSWEKNSV LEIIAFHCKS PNRHRMVVLE PLNKLLQEKW DRLVSRFFFN FACY LVYMF IFTVVAYHQP SLDQPAIPSS KATFGESMLL LGHILILLGG IYLLLGQLWY FWRRRLFIWI SFMDSYFEIL FLLQA LLTV LSQVLRFMET EWYLPLLVLS LVLGWLNLLY YTRGFQHTGI YSVMIQKVIL RDLLRFLLVY LVFLFGFAVA LVSLSR EAR SPKAPEDNNS TVTEQPTVGQ EEEPAPYRSI LDASLELFKF TIGMGELAFQ EQLRFRGVVL LLLLAYVLLT YVLLLNM LI ALMSETVNHV ADNSWSIWKL QKAISVLEME NGYWWCRRKK HREGRLLKVG TRGDGTPDER WCFRVEEVNW AAWEKTLP T LSEDPSGPGI TGNKKNPTSK PGKNSASEED HLPLQVLQSP

UniProtKB: Transient receptor potential cation channel subfamily V member 2

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Macromolecule #2: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE

MacromoleculeName: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / type: ligand / ID: 2 / Number of copies: 4 / Formula: PEX
Molecular weightTheoretical: 522.632 Da
Chemical component information

ChemComp-PEX:
1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE

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Macromolecule #3: 2-aminoethyl diphenylborinate

MacromoleculeName: 2-aminoethyl diphenylborinate / type: ligand / ID: 3 / Number of copies: 4 / Formula: FZ4
Molecular weightTheoretical: 225.094 Da
Chemical component information

ChemComp-FZ4:
2-aminoethyl diphenylborinate

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Macromolecule #4: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)

MacromoleculeName: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)
type: ligand / ID: 4 / Number of copies: 1 / Formula: R2R
Molecular weightTheoretical: 559.525 Da
Chemical component information

ChemComp-R2R:
ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)

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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 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
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 K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 10643 / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 20.0 µm / Nominal defocus min: 5.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 690077
Startup modelType of model: EMDB MAP
EMDB ID:

20677

Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 65741
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7n0m


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8ffm:
Wildtype rat TRPV2 in nanodiscs bound to RR and 2-APB

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