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- EMDB-25719: Cryo-EM structure of TRPV5 in nanodiscs with PI(4,5)P2 at pH6 state 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-25719
TitleCryo-EM structure of TRPV5 in nanodiscs with PI(4,5)P2 at pH6 state 1
Map data
Sample
  • Complex: Tetrameric assembly of TRPV5 ion channel reconstituted into lipid nanodiscs
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 5
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
KeywordsCalcium / Ion channel / Kidney / TRANSPORT 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: 2.8 Å
AuthorsFluck EC / Yazici AT
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM103899 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM129357 United States
CitationJournal: Cell Rep / Year: 2022
Title: Structural basis of TRPV5 regulation by physiological and pathophysiological modulators.
Authors: Edwin C Fluck / Aysenur Torun Yazici / Tibor Rohacs / Vera Y Moiseenkova-Bell /
Abstract: Transient receptor potential vanilloid 5 (TRPV5) is a kidney-specific Ca-selective ion channel that plays a key role in Ca homeostasis. The basal activity of TRPV5 is balanced through activation by ...Transient receptor potential vanilloid 5 (TRPV5) is a kidney-specific Ca-selective ion channel that plays a key role in Ca homeostasis. The basal activity of TRPV5 is balanced through activation by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and inhibition by Ca-bound calmodulin (CaM). Parathyroid hormone (PTH), the key extrinsic regulator of Ca homeostasis, increases the activity of TRPV5 via protein kinase A (PKA)-mediated phosphorylation. Metabolic acidosis leads to reduced TRPV5 activity independent of PTH, causing hypercalciuria. Using cryoelectron microscopy (cryo-EM), we show that low pH inhibits TRPV5 by precluding PI(4,5)P activation. We capture intermediate conformations at low pH, revealing a transition from open to closed state. In addition, we demonstrate that PI(4,5)P is the primary modulator of channel gating, yet PKA controls TRPV5 activity by preventing CaM binding and channel inactivation. Our data provide detailed molecular mechanisms for regulation of TRPV5 by two key extrinsic modulators, low pH and PKA.
History
DepositionDec 14, 2021-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25719.png

Downloads & links

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Map

FileDownload / File: emd_25719.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.278
Minimum - Maximum-1.6178606 - 2.7901373
Average (Standard dev.)0.006508442 (±0.08071168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Tetrameric assembly of TRPV5 ion channel reconstituted into lipid...

EntireName: Tetrameric assembly of TRPV5 ion channel reconstituted into lipid nanodiscs
Components
  • Complex: Tetrameric assembly of TRPV5 ion channel reconstituted into lipid nanodiscs
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 5
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Supramolecule #1: Tetrameric assembly of TRPV5 ion channel reconstituted into lipid...

SupramoleculeName: Tetrameric assembly of TRPV5 ion channel reconstituted into lipid nanodiscs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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 / Details: Rabbit TRPV5 with c-terminal 1D4 epitope tag / 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: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 2 / Number of copies: 4 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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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

BufferpH: 6
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
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 grids imaged: 1 / Number real images: 7006 / 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: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1536734
Startup modelType of model: OTHER / Details: cryoSPARC ab-initio
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 63746
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3.2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7t6m:
Cryo-EM structure of TRPV5 in nanodiscs with PI(4,5)P2 at pH6 state 1

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