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- EMDB-29048: RR-bound wildtype rabbit TRPV5 in nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-29048
TitleRR-bound wildtype rabbit TRPV5 in nanodiscs
Map data
Sample
  • Complex: Tetramer of wildtype rabbit TRPV5 in nanodiscs bound to ruthenium red
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 5
  • Ligand: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: ERGOSTEROL
KeywordsTRPV2 / TRPV5 / TRP Channel / Ruthenium Red / MEMBRANE PROTEIN
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 / identical protein 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 / identical protein binding / metal ion 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
Transient receptor potential cation channel subfamily V member 5
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsFluck EC / De Jesus-Perez JJ / Pumroy RA / Protopopova AD / Rocereta JA / 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_29048.png

Downloads & links

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Map

FileDownload / File: emd_29048.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.67186636 - 1.2530622
Average (Standard dev.)0.008945752 (±0.04763315)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29048_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_29048_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 rabbit TRPV5 in nanodiscs bound to ruthenium red

EntireName: Tetramer of wildtype rabbit TRPV5 in nanodiscs bound to ruthenium red
Components
  • Complex: Tetramer of wildtype rabbit TRPV5 in nanodiscs bound to ruthenium red
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 5
  • Ligand: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: ERGOSTEROL

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Supramolecule #1: Tetramer of wildtype rabbit TRPV5 in nanodiscs bound to ruthenium red

SupramoleculeName: Tetramer of wildtype rabbit TRPV5 in nanodiscs bound to ruthenium red
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 335.14 KDa

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Macromolecule #1: Transient receptor potential cation channel subfamily V member 5

MacromoleculeName: Transient receptor potential cation channel subfamily V member 5
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 83.784586 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGACPPKAKG PWAQLQKLLI SWPVGEQDWE QYRDRVNMLQ QERIRDSPLL QAAKENDLRL LKILLLNQSC DFQQRGAVGE TALHVAALY DNLEAATLLM EAAPELAKEP ALCEPFVGQT ALHIAVMNQN LNLVRALLAR GASVSARATG AAFRRSPHNL I YYGEHPLS ...String:
MGACPPKAKG PWAQLQKLLI SWPVGEQDWE QYRDRVNMLQ QERIRDSPLL QAAKENDLRL LKILLLNQSC DFQQRGAVGE TALHVAALY DNLEAATLLM EAAPELAKEP ALCEPFVGQT ALHIAVMNQN LNLVRALLAR GASVSARATG AAFRRSPHNL I YYGEHPLS FAACVGSEEI VRLLIEHGAD IRAQDSLGNT VLHILILQPN KTFACQMYNL LLSYDEHSDH LQSLELVPNH QG LTPFKLA GVEGNTVMFQ HLMQKRKHVQ WTCGPLTSTL YDLTEIDSWG EELSFLELVV SSKKREARQI LEQTPVKELV SFK WKKYGR PYFCVLASLY ILYMICFTTC CIYRPLKLRD DNRTDPRDIT ILQQKLLQEA YVTHQDNIRL VGELVTVTGA VIIL LLEIP DIFRVGASRY FGQTILGGPF HVIIITYASL VLLTMVMRLT NMNGEVVPLS FALVLGWCSV MYFARGFQML GPFTI MIQK MIFGDLMRFC WLMAVVILGF ASAFHITFQT EDPNNLGEFS DYPTALFSTF ELFLTIIDGP ANYSVDLPFM YCITYA AFA IIATLLMLNL FIAMMGDTHW RVAQERDELW RAQVVATTVM LERKMPRFLW PRSGICGYEY GLGDRWFLRV ENHHDQN PL RVLRYVEAFK CSDKEDGQEQ LSEKRPSTVE SGMLSRASVA FQTPSLSRTT SQSSNSHRGW EILRRNTLGH LNLGLDLG E GDGEEVYHFT ETSQVAPA

UniProtKB: Transient receptor potential cation channel subfamily V member 5

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

MacromoleculeName: ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2)
type: ligand / ID: 2 / 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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Macromolecule #3: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 3 / Number of copies: 8 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

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Macromolecule #4: ERGOSTEROL

MacromoleculeName: ERGOSTEROL / type: ligand / ID: 4 / Number of copies: 8 / Formula: ERG
Molecular weightTheoretical: 396.648 Da
Chemical component information

ChemComp-ERG:
ERGOSTEROL

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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

Concentration3.5 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 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 real images: 9237 / 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 / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1640882
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 39976
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 3 / Avg.num./class: 21904 / Software - Name: cryoSPARC (ver. 3.3.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8ffn:
RR-bound wildtype rabbit TRPV5 in nanodiscs

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