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- PDB-7b1g: TRPC4 in complex with Calmodulin -

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Basic information

Entry
Database: PDB / ID: 7b1g
TitleTRPC4 in complex with Calmodulin
Components
  • Calmodulin-1
  • Transient receptor potential cation channel subfamily c member 4a
KeywordsTRANSPORT PROTEIN / ION CHANNEL / TRPC4 / COMPLEX / MEMBRANE PROTEIN / CALMODULIN
Function / homology
Function and homology information


CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) / Activation of RAC1 downstream of NMDARs / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / CLEC7A (Dectin-1) induces NFAT activation / Synthesis of IP3 and IP4 in the cytosol ...CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) / Activation of RAC1 downstream of NMDARs / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / CLEC7A (Dectin-1) induces NFAT activation / Synthesis of IP3 and IP4 in the cytosol / RHO GTPases activate PAKs / Calmodulin induced events / Inactivation, recovery and regulation of the phototransduction cascade / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Calcineurin activates NFAT / eNOS activation / Ion transport by P-type ATPases / Unblocking of NMDA receptors, glutamate binding and activation / Protein methylation / RAF activation / VEGFR2 mediated vascular permeability / RAS processing / Smooth Muscle Contraction / Ca2+ pathway / FCERI mediated Ca+2 mobilization / RAF/MAP kinase cascade / RHO GTPases activate IQGAPs / Extra-nuclear estrogen signaling / store-operated calcium channel activity / PKA activation / Platelet degranulation / cation channel complex / Stimuli-sensing channels / Ion homeostasis / type 3 metabotropic glutamate receptor binding / inositol 1,4,5 trisphosphate binding / organelle localization by membrane tethering / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / nitric-oxide synthase binding / protein phosphatase activator activity / monoatomic cation transport / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / monoatomic cation channel activity / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / sperm midpiece / calcium channel complex / regulation of cytosolic calcium ion concentration / adenylate cyclase activator activity / regulation of heart rate / nitric-oxide synthase regulator activity / protein serine/threonine kinase activator activity / sarcomere / regulation of cytokinesis / spindle microtubule / calcium ion transmembrane transport / positive regulation of receptor signaling pathway via JAK-STAT / spindle pole / cellular response to type II interferon / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / growth cone / transmembrane transporter binding / protein domain specific binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / nucleoplasm / plasma membrane / cytosol / cytoplasm
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 / EF-hand domain pair / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ankyrin repeats / Ankyrin repeat ...Transient receptor ion channel II / Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor potential channel, canonical / Ankyrin repeat / EF-hand domain pair / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / Calmodulin-1 / Transient receptor potential cation channel subfamily c member 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Mus musculus (house mouse)
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 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily c member 4a
E: Calmodulin-1
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)439,30513
Polymers437,6715
Non-polymers1,6348
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37140 Å2
ΔGint-313 kcal/mol
Surface area119140 Å2
MethodPISA

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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 line (production host): HEK293S GnTI- / Organ (production host): KIDNEY / Production host: Homo sapiens (human) / References: UniProt: U3N7D8
#2: Protein Calmodulin-1 /


Mass: 16852.545 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Calm1, Calm, Cam, Cam1 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 codon plus RIL cells / References: UniProt: P0DP26
#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 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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1TRPC4 membrane protein in complex with calmodulinORGANELLE OR CELLULAR COMPONENTTRPC4 was solubilised in LMNG detergent#1-#20MULTIPLE SOURCES
2Transient receptor potential cation channel subfamily c member 4aORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3Calmodulin-1ORGANELLE OR CELLULAR COMPONENT#21RECOMBINANT
Molecular weightValue: 0.126 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
22Danio rerio (zebrafish)7955plasma membrane
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
22Homo sapiens (human)9606HEK 293 GnTI-pEG BACMAM
33Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRISC4H11NO31
2150 mMsodium chlorideNaClSodium chloride1
30.003 percentLMNGC47H88O261
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 59000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1500 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: 88.52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7972
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Spherical aberration corrector: Cs corrector first generation
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 80 / Used frames/image: 1-80

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategoryDetails
1crYOLOparticle selection
2EPUimage acquisition
4CTFFIND4.1.10CTF correction
7Coot0.8.8model fitting
10SPHIREfinal Euler assignmentMERIDIEN
11SPHIRE1.3classificationSORT3D
12SPHIRE3D reconstructionMERIDIEN
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 678331
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160829 / Algorithm: FOURIER SPACE / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingB value: 58.04 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-ID
16GIK

6gik

1
22LV6

2lv6

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.92 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006322178
ELECTRON MICROSCOPYf_angle_d0.711630089
ELECTRON MICROSCOPYf_chiral_restr0.0453402
ELECTRON MICROSCOPYf_plane_restr0.00463748
ELECTRON MICROSCOPYf_dihedral_angle_d24.57797951

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