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- PDB-7b16: TRPC4 in complex with inhibitor GFB-9289 -

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Basic information

Entry
Database: PDB / ID: 7b16
TitleTRPC4 in complex with inhibitor GFB-9289
ComponentsTransient receptor potential cation channel subfamily c member 4a
KeywordsTRANSPORT PROTEIN / ION CHANNEL / TRPC4 / INHIBITOR / MEMBRANE 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 II / Transient receptor ion channel domain / 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 II / Transient receptor ion channel domain / 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
Chem-LPP / Chem-SKQ / Transient receptor potential cation channel subfamily c member 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsVinayagam, D. / Quentin, D. / Sistel, O. / Merino, F. / Stabrin, M. / Hofnagel, O. / Ledeboer, M.W. / Malojcic, G. / Raunser, S.
Funding support Germany, 1items
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 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily c member 4a
B: Transient receptor potential cation channel subfamily c member 4a
C: Transient receptor potential cation channel subfamily c member 4a
D: Transient receptor potential cation channel subfamily c member 4a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)424,81816
Polymers420,8194
Non-polymers3,99912
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, tetrameric organisation confirmed by the structure
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEULYSA1 - 643
d_12ens_1LIGLIGB
d_13ens_1CACAC
d_14ens_1LPPLPPD
d_21ens_1LEULYSE1 - 643
d_22ens_1LIGLIGF
d_23ens_1CACAG
d_24ens_1LPPLPPH
d_31ens_1LEULYSI1 - 643
d_32ens_1LIGLIGJ
d_33ens_1CACAK
d_34ens_1LPPLPPL
d_41ens_1LEULYSM1 - 643
d_42ens_1LIGLIGN
d_43ens_1CACAO
d_44ens_1LPPLPPP

NCS oper:
IDCodeMatrixVector
1given(-1), (-1), (1)138.32, 138.32
2given(1), (-1), (1)138.32
3given(-1), (1), (1)138.32

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Components

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


Mass: 105204.641 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: trpc4b, trpc4a / Plasmid: pEG MaM / Cell (production host): GnTI- / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: U3N7D8
#2: Chemical
ChemComp-SKQ / 5-chloranyl-4-(4-cyclohexyl-3-oxidanylidene-piperazin-1-yl)-1~{H}-pyridazin-6-one / GFB-9289


Mass: 310.779 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H19ClN4O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#4: Chemical
ChemComp-LPP / 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE / 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / L-B,G-DIPALMITOYL-A-PHOSPHATIDIC ACID DISODIUM SALT / 3-SN-PHOSPHATIDIC ACID / 1,2-DIPALMITOYLDISODIUM SALT


Mass: 648.891 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C35H69O8P / Comment: phospholipid*YM
Has ligand of interestY

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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: TRPC4 in complex with inhibitor GFB-9289 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.108 MDa / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK292S GnTI- / Plasmid: BacMam
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTRISC4H11NO31
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was prepared in Amphipols
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 55000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1600 nm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 190 K / Temperature (min): 160 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 65.45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2970
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5740 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisitionAFIS
4CTFFIND4.1.10CTF correctionCTF estimation
5RELION3.0.4CTF correctionCTF correction
8Coot0.8.8model fitting
11RELION3.0.4final Euler assignment
12RELION3.0.4classification
13RELION3.0.43D reconstruction
14PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 434945
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65811 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 46.76 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6G1K

6g1k


Accession code: 6G1K / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.71 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00921652
ELECTRON MICROSCOPYf_angle_d0.792229300
ELECTRON MICROSCOPYf_chiral_restr0.0463276
ELECTRON MICROSCOPYf_plane_restr0.0053620
ELECTRON MICROSCOPYf_dihedral_angle_d22.02777952
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.0495446107581
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.0501391613056
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.00113702683907

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