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- PDB-7s88: Open apo-state cryo-EM structure of human TRPV6 in glyco-diosgeni... -

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Basic information

Entry
Database: PDB / ID: 7s88
TitleOpen apo-state cryo-EM structure of human TRPV6 in glyco-diosgenin detergent
ComponentsTransient receptor potential cation channel subfamily V member 6
KeywordsMEMBRANE PROTEIN / Transient Receptor Potential V Family Member 6 / TRP / channel / apo / open / human / TRPV6
Function / homology
Function and homology information


parathyroid hormone secretion / regulation of calcium ion-dependent exocytosis / TRP channels / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / response to calcium ion / calcium ion transport ...parathyroid hormone secretion / regulation of calcium ion-dependent exocytosis / TRP channels / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / response to calcium ion / calcium ion transport / calmodulin binding / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 6 / 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 6 / 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
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Chem-POV / CHOLESTEROL HEMISUCCINATE / Transient receptor potential cation channel subfamily V member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsNeuberger, A. / Nadezhdin, K.D. / Sobolevsky, A.I.
Funding support Germany, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA206573 Germany
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS083660 Germany
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS107253 Germany
National Science Foundation (NSF, United States)1818086 Germany
German Research Foundation (DFG)464295817 Germany
CitationJournal: Nat Commun / Year: 2021
Title: Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole.
Authors: Arthur Neuberger / Kirill D Nadezhdin / Alexander I Sobolevsky /
Abstract: TRPV6 is a calcium-selective ion channel implicated in epithelial Ca uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, ...TRPV6 is a calcium-selective ion channel implicated in epithelial Ca uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel's periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel's selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.
History
DepositionSep 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 6
B: Transient receptor potential cation channel subfamily V member 6
C: Transient receptor potential cation channel subfamily V member 6
D: Transient receptor potential cation channel subfamily V member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)359,63169
Polymers313,6854
Non-polymers45,94665
Water2,594144
1


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

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Transient receptor potential cation channel subfamily V member 6 / TrpV6 / CaT-like / CaT-L / Calcium transport protein 1 / CaT1 / Epithelial calcium channel 2 / ECaC2


Mass: 78421.328 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPV6, ECAC2 / Production host: Homo sapiens (human) / References: UniProt: Q9H1D0

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Non-polymers , 5 types, 209 molecules

#2: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C31H50O4
#3: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#4: Chemical...
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 32 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 144 / Source method: isolated from a natural source / Formula: H2O

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Details

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: sample 1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMtris(hydroxymethyl)aminomethane1
31 mMbeta-Mercaptoethanol2-Mercaptoethanol1
40.01 %glyco-diosgenin1
50.001 %cholesteryl hemisuccinate1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: human TRPV6 in glyco-diosgenin detergent
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 3 sec. / Electron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7904
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4RELION3.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11cryoSPARC3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14cryoSPARC3.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3117068
3D reconstructionResolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 276688 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7K4A

7k4a

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