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- PDB-7b0s: TRPC4 in complex with inhibitor GFB-8438 -

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Basic information

Entry
Database: PDB / ID: 7b0s
TitleTRPC4 in complex with inhibitor GFB-8438
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 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
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / Chem-S9Q / Transient receptor potential cation channel subfamily c member 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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 21, 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)424,29616
Polymers421,1154
Non-polymers3,18112
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The structure confirms the tetrameric assembly
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area33270 Å2
ΔGint-295 kcal/mol
Surface area127550 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 29 through 55 or (resid 56...
d_2ens_1(chain "B" and (resid 29 through 55 or (resid 56...
d_3ens_1chain "C"
d_4ens_1(chain "D" and (resid 29 through 55 or (resid 56...

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_144E44ED
d_21ens_1LEULYSE1 - 643
d_22ens_1LIGLIGF
d_23ens_1CACAG
d_24ens_144E44EH
d_31ens_1LEULYSI1 - 643
d_32ens_1LIGLIGJ
d_33ens_1CACAK
d_34ens_144E44EL
d_41ens_1LEULYSM1 - 643
d_42ens_1LIGLIGN
d_43ens_1CACAO
d_44ens_144E44EP

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Components

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


Mass: 105278.781 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: trpc4b, trpc4a / Plasmid: pEG BacMam / Cell line (production host): HEK293S / Organ (production host): embryonic kidney cells / Production host: Homo sapiens (human) / References: UniProt: U3N7D8
#2: Chemical
ChemComp-S9Q / 5-chloranyl-4-[3-oxidanylidene-4-[[2-(trifluoromethyl)phenyl]methyl]piperazin-1-yl]-1~{H}-pyridazin-6-one


Mass: 386.756 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C16H14ClF3N4O2 / 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-44E / (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate


Mass: 368.360 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C15H29O8P / 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-8438 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.108 MDa / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish) / Cellular location: plasma membrane
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GnTI- / Plasmid: pEG BacMam
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRISC4H11NO31
2150 mMsodium chlorideNACL1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
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 FIELD / Nominal magnification: 105000 X / Calibrated magnification: 55000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 66.58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 60
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4.1.10CTF correction
7Coot0.8.8model fitting
9SPHIRE1.3initial Euler assignmentMeridien
10SPHIRE1.3final Euler assignmentMeridien
11RELION3.0.4classification
12SPHIRE1.33D reconstructionMeridien
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 577045
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42524 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 67.33 / 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: 67.33 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008321576
ELECTRON MICROSCOPYf_angle_d0.717429235
ELECTRON MICROSCOPYf_chiral_restr0.04453276
ELECTRON MICROSCOPYf_plane_restr0.0043619
ELECTRON MICROSCOPYf_dihedral_angle_d23.01917821
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.0169431061222
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.00846379438893
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.000712323157452

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