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- EMDB-11957: TRPC4 in complex with inhibitor GFB-8749 -

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Basic information

Entry
Database: EMDB / ID: EMD-11957
TitleTRPC4 in complex with inhibitor GFB-8749
Map data
Sample
  • Complex: TRPC4 in complex with inhibitor GFB-8749
    • Protein or peptide: Transient receptor potential cation channel subfamily c member 4a
  • Ligand: 4-[4-[[4,4-bis(fluoranyl)cyclohexyl]methyl]-3-oxidanylidene-piperazin-1-yl]-5-chloranyl-1~{H}-pyridazin-6-one
  • Ligand: CALCIUM ION
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate
KeywordsION CHANNEL / TRPC4 / INHIBITOR / COMPLEX / MEMBRANE PROTEIN / TRANSPORT PROTEIN
Function / homology
Function and homology information


store-operated calcium channel activity / cation channel complex / inositol 1,4,5 trisphosphate binding / monoatomic cation transport / monoatomic cation channel activity / regulation of cytosolic calcium ion concentration / calcium ion transmembrane transport / plasma membrane
Similarity search - Function
Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor ion channel II / Transient receptor potential channel, canonical / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily ...Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor ion channel II / Transient receptor potential channel, canonical / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat 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 c member 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsVinayagam D / Quentin D
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Elife / Year: 2020
Title: Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.
Authors: Deivanayagabarathy Vinayagam / Dennis Quentin / Jing Yu-Strzelczyk / Oleg Sitsel / Felipe Merino / Markus Stabrin / Oliver Hofnagel / Maolin Yu / Mark W Ledeboer / Georg Nagel / Goran ...Authors: Deivanayagabarathy Vinayagam / Dennis Quentin / Jing Yu-Strzelczyk / Oleg Sitsel / Felipe Merino / Markus Stabrin / Oliver Hofnagel / Maolin Yu / Mark W Ledeboer / Georg Nagel / Goran Malojcic / Stefan Raunser /
Abstract: Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca signaling. Inhibition of TRPCs by small molecules was shown to be promising in ...Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.
History
DepositionNov 18, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0093
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0093
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7b05
  • Surface level: 0.0093
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11957.png

Downloads & links

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Map

FileDownload / File: emd_11957.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.)
0.91 Å/pix.
x 288 pix.
= 262.08 Å		0.91 Å/pix.
x 288 pix.
= 262.08 Å		0.91 Å/pix.
x 288 pix.
= 262.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0093 / Movie #1: 0.0093
Minimum - Maximum-0.025400542 - 0.04339399
Average (Standard dev.)0.00019152283 (±0.0016873146)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 262.08002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z262.080262.080262.080
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0250.0430.000

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Supplemental data

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Sample components

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Entire : TRPC4 in complex with inhibitor GFB-8749

EntireName: TRPC4 in complex with inhibitor GFB-8749
Components
  • Complex: TRPC4 in complex with inhibitor GFB-8749
    • Protein or peptide: Transient receptor potential cation channel subfamily c member 4a
  • Ligand: 4-[4-[[4,4-bis(fluoranyl)cyclohexyl]methyl]-3-oxidanylidene-piperazin-1-yl]-5-chloranyl-1~{H}-pyridazin-6-one
  • Ligand: CALCIUM ION
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

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Supramolecule #1: TRPC4 in complex with inhibitor GFB-8749

SupramoleculeName: TRPC4 in complex with inhibitor GFB-8749 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 108 KDa

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Macromolecule #1: Transient receptor potential cation channel subfamily c member 4a

MacromoleculeName: Transient receptor potential cation channel subfamily c member 4a
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 105.204641 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSQLYFRRTD NSSYRDRIPL RIVRAESELS TQEKSYLSAV EKGDYASVKL ALEEAEIYFK ININCIDPLG RTALLIAIEN ENLEIIELL LSFNVYVGDA LLHAIRKEVV GAVELLLNHK KPSGEKQVPP ILLDKQFSDF TPDITPIILA AHTNNYEIIK M LVQKGVSV ...String:
GSQLYFRRTD NSSYRDRIPL RIVRAESELS TQEKSYLSAV EKGDYASVKL ALEEAEIYFK ININCIDPLG RTALLIAIEN ENLEIIELL LSFNVYVGDA LLHAIRKEVV GAVELLLNHK KPSGEKQVPP ILLDKQFSDF TPDITPIILA AHTNNYEIIK M LVQKGVSV PQPHEVRCNC VECVSSSDVD SLRHSRSRLN IYKALASPSL IALSSEDPFL TAFQLSWELQ ELSKVENEFK AE YEELSHQ CKHFAKDLLD QTRSSRELEL ILNFRDDMNL LQDEANNELA RLKLAIKYRQ KEFVAQPNCQ QLLASRWYDE FPG WRRRHW AGKLITCVFI GLMFPLLSLC YLVAPKSRYG LFIRKPFIKF ICHTASYLTF LFLLLLASQH IVSNNPDRQG PKPT TVEWM ILPWVLGFIW TEIKQMWDGG FQDYIHDWWN LMDFVMNSLY LATISLKIVA YVKYSGCKPR DTWEMWHPTL VAEAV FAIA NIFSSLRLIS LFTANSHLGP LQISLGRMLL DILKFLFIYC LVLLAFANGL NQLYFYYENS EGMTCKGIRC ERQNNA FST LFETLQSLFW SIFGLISLYV TNVKADHKFT EFVGATMFGT YNVISLVVLL NMLIAMMNNS YQHIADHADI EWKFART KL WMSYFEEGGT LPPPFNIIPS PKSICYLITW IKVHVFKRRS KRTETFGTLG RRAAENVRLN HQYQEVLRNL VKRYVAAM I RDAKTEEGLT EENFKELKQD ISSFRYEVIG MMKGNRKSTR ANKSDTSASD VSHPEGSLQY SSALKQNSKL HLYDVTTAL QQQNSEEAKA SLGCLANGSA VVLTEPILKD KARSDFPKDF TDFGLFPKKQ NPNKIYSLAE EATESDPDIL DWGKEDKPLA GKVEQDVNE SKCLMEEDER VLEEQEMEHI ASSHEH

UniProtKB: Transient receptor potential cation channel subfamily c member 4a

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Macromolecule #2: 4-[4-[[4,4-bis(fluoranyl)cyclohexyl]methyl]-3-oxidanylidene-piper...

MacromoleculeName: 4-[4-[[4,4-bis(fluoranyl)cyclohexyl]methyl]-3-oxidanylidene-piperazin-1-yl]-5-chloranyl-1~{H}-pyridazin-6-one
type: ligand / ID: 2 / Number of copies: 4 / Formula: SJQ
Molecular weightTheoretical: 360.787 Da
Chemical component information

ChemComp-SJQ:
4-[4-[[4,4-bis(fluoranyl)cyclohexyl]methyl]-3-oxidanylidene-piperazin-1-yl]-5-chloranyl-1~{H}-pyridazin-6-one

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

MacromoleculeName: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / type: ligand / ID: 4 / Number of copies: 4 / Formula: 44E
Molecular weightTheoretical: 368.36 Da
Chemical component information

ChemComp-44E:
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / phospholipid*YM

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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

Concentration0.4 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC4H11NO3TRIS
5.0 percentC3H8O3Glycerol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 160.0 K / Max: 190.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 1290 / Average exposure time: 3.0 sec. / Average electron dose: 72.0 e/Å2
Details: Images were collected in a movie mode with 60 frames in total
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 0.86 µm / Calibrated magnification: 54945 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 105000
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: 304244
Startup modelType of model: EMDB MAP
EMDB ID:

4339

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE (ver. 1.3) / Number images used: 44989
Initial angle assignmentType: NOT APPLICABLE / Software - Name: SPHIRE (ver. 1.3) / Software - details: MERIDIEN / Details: used model from the existing map form EMD-4339
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.3) / Software - details: MERIDIEN
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0.4)

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Atomic model buiding 1

Initial modelPDB ID:

6gik


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 74.22 / Target criteria: Correlation coefficient
Output model

PDB-7b05:
TRPC4 in complex with inhibitor GFB-8749

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