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- PDB-7b0j: TRPC4 in LMNG detergent -

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Basic information

Entry
Database: PDB / ID: 7b0j
TitleTRPC4 in LMNG detergent
ComponentsTransient receptor potential cation channel subfamily c member 4a
KeywordsTRANSPORT PROTEIN / ION CHANNEL / TRPC4 / LMNG / MEMBRANE PROTEIN
Function / homology
Function and homology information


store-operated calcium channel activity / inositol 1,4,5 trisphosphate binding / cation channel complex / monoatomic cation transport / regulation of cytosolic calcium ion concentration / monoatomic cation channel activity / 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
Chem-LPP / Short transient receptor potential channel 4b
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 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 20, 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)423,57412
Polymers420,8194
Non-polymers2,7568
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The structure confirms the tetrameric assembly
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "A"
d_3ens_1(chain "C" and (resid 17 through 670 or (resid 671...
d_4ens_1chain "B"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALAMETJ1 - 682
d_12ens_1LPPLPPK
d_21ens_1ALAMETA1 - 682
d_22ens_1LPPLPPB
d_31ens_1ALAMETG1 - 682
d_32ens_1LPPLPPH
d_41ens_1ALAMETD1 - 682
d_42ens_1LPPLPPE

NCS oper:
IDCodeMatrixVector
1given(1), (-1), (1)0.85, 131.76
2given(-1), (1), (1)131.74, -0.86
3given(-1), (-1), (1)132.6, 130.9, -0.01

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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 Bacmam / Cell (production host): GNTI- / Cell line (production host): HEK293S / Organ (production host): KIDNEY / Production host: Homo sapiens (human) / References: UniProt: U3N7D8
#2: 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
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
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: TRPC4 membrane protein / Type: ORGANELLE OR CELLULAR COMPONENT / Details: solubilised in LMNG / 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: HEK 293 GnTI- / Plasmid: pEG BACMAM
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRISC4H11NO31
2150 mMsodium chlorideNaCl1
30.003 percentLMNGC47H88O261
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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 FIELD / Nominal magnification: 130000 X / Calibrated magnification: 59000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 0.001 mm / C2 aperture diameter: 70 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: 10 sec. / Electron dose: 88.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3079
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Spherical aberration corrector: BCOR Cs corrector first generation
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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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
10SPHIREfinal Euler assignmentMERIDIEN
12SPHIRE3D reconstructionMERIDIEN
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 393632
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126873 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 49.08 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6GIK

6gik


Accession code: 6GIK / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 51.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007722646
ELECTRON MICROSCOPYf_angle_d0.767630703
ELECTRON MICROSCOPYf_chiral_restr0.04783453
ELECTRON MICROSCOPYf_plane_restr0.00573804
ELECTRON MICROSCOPYf_dihedral_angle_d21.99753092
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000706170030849
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000709557908357
ens_1d_4BELECTRON MICROSCOPYNCS constraints0.000704540508539

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