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- PDB-8tkd: Human Type 3 IP3 Receptor - Preactivated State (+IP3/ATP) -

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Basic information

Entry
Database: PDB / ID: 8tkd
TitleHuman Type 3 IP3 Receptor - Preactivated State (+IP3/ATP)
ComponentsInositol 1,4,5-trisphosphate receptor type 3
KeywordsTRANSPORT PROTEIN / Ion Channel / Calcium Channel / Endoplasmic Reticulum
Function / homology
Function and homology information


DAG and IP3 signaling / sensory perception of bitter taste / inositol 1,3,4,5 tetrakisphosphate binding / sensory perception of umami taste / inositol 1,4,5-trisphosphate-gated calcium channel activity / sensory perception of sweet taste / platelet dense tubular network membrane / Effects of PIP2 hydrolysis / Elevation of cytosolic Ca2+ levels / PLC beta mediated events ...DAG and IP3 signaling / sensory perception of bitter taste / inositol 1,3,4,5 tetrakisphosphate binding / sensory perception of umami taste / inositol 1,4,5-trisphosphate-gated calcium channel activity / sensory perception of sweet taste / platelet dense tubular network membrane / Effects of PIP2 hydrolysis / Elevation of cytosolic Ca2+ levels / PLC beta mediated events / inositol hexakisphosphate binding / inositol 1,4,5 trisphosphate binding / nuclear outer membrane / CLEC7A (Dectin-1) induces NFAT activation / transport vesicle membrane / cytoplasmic side of endoplasmic reticulum membrane / brush border / intracellularly gated calcium channel activity / Role of phospholipids in phagocytosis / calcium ion homeostasis / Ion homeostasis / release of sequestered calcium ion into cytosol / FCERI mediated Ca+2 mobilization / phosphatidylinositol binding / FCGR3A-mediated IL10 synthesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / secretory granule membrane / VEGFR2 mediated cell proliferation / sarcoplasmic reticulum / Regulation of insulin secretion / long-term synaptic potentiation / memory / platelet activation / response to calcium ion / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / sensory perception of taste / apical part of cell / Ca2+ pathway / positive regulation of cytosolic calcium ion concentration / protein homotetramerization / receptor complex / G protein-coupled receptor signaling pathway / neuronal cell body / calcium ion binding / endoplasmic reticulum membrane / nucleolus / endoplasmic reticulum / nucleoplasm / membrane / plasma membrane / cytoplasm
Similarity search - Function
Inositol 1,4,5-trisphosphate receptor / RyR/IP3 receptor binding core, RIH domain superfamily / RyR/IP3R Homology associated domain / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / RyR and IP3R Homology associated / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / : / MIR motif ...Inositol 1,4,5-trisphosphate receptor / RyR/IP3 receptor binding core, RIH domain superfamily / RyR/IP3R Homology associated domain / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / RyR and IP3R Homology associated / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / : / MIR motif / MIR domain / MIR domain profile. / Domain in ryanodine and inositol trisphosphate receptors and protein O-mannosyltransferases / Mir domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE / Inositol 1,4,5-trisphosphate-gated calcium channel ITPR3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPaknejad, N. / Sapuru, V. / Hite, R.K.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM13230704 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F31CA243235 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural titration reveals Ca-dependent conformational landscape of the IP receptor.
Authors: Navid Paknejad / Vinay Sapuru / Richard K Hite /
Abstract: Inositol 1,4,5-trisphosphate receptors (IPRs) are endoplasmic reticulum Ca channels whose biphasic dependence on cytosolic Ca gives rise to Ca oscillations that regulate fertilization, cell division ...Inositol 1,4,5-trisphosphate receptors (IPRs) are endoplasmic reticulum Ca channels whose biphasic dependence on cytosolic Ca gives rise to Ca oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IPR-mediated Ca responses, the structural underpinnings of the biphasic Ca dependence that underlies Ca oscillations are incompletely understood. Here, we collect cryo-EM images of an IPR with Ca concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for relative conformational free energy, we demonstrate that Ca binding at a high-affinity site allows IPRs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca concentrations, IPRs preferentially enter an inhibited state stabilized by a second, low-affinity Ca binding site. Together, these studies provide a mechanistic basis for the biphasic Ca-dependence of IPR channel activity.
History
DepositionJul 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Inositol 1,4,5-trisphosphate receptor type 3
B: Inositol 1,4,5-trisphosphate receptor type 3
C: Inositol 1,4,5-trisphosphate receptor type 3
D: Inositol 1,4,5-trisphosphate receptor type 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,221,92516
Polymers1,217,9554
Non-polymers3,97112
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "A"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11SERSERARGARGDD5 - 26575 - 2657
d_12ZNZNZNZNDN3001
d_13I3PI3PI3PI3PDO3002
d_14ATPATPATPATPDP3003
d_21SERSERARGARGBB5 - 26575 - 2657
d_22ZNZNZNZNBH3001
d_23I3PI3PI3PI3PBI3002
d_24ATPATPATPATPBJ3003
d_31SERSERARGARGCC5 - 26575 - 2657
d_32ZNZNZNZNCK3001
d_33I3PI3PI3PI3PCL3002
d_34ATPATPATPATPCM3003
d_41SERSERARGARGAA5 - 26575 - 2657
d_42ZNZNZNZNAE3001
d_43I3PI3PI3PI3PAF3002
d_44ATPATPATPATPAG3003

NCS oper:
IDCodeMatrixVector
1given(-0.999999187341, -0.00121433805119, -0.000388200882386), (0.0012130502556, -0.999993817444, 0.00330054292549), (-0.000392206457176, 0.0033000693361, 0.999994477843)423.414875344, 421.561683488, -0.437828839842
2given(-0.000689615336508, 0.999997645367, -0.00205759348962), (-0.999999307742, -0.000687651879258, 0.000954804258226), (0.000953387101983, 0.0020582505129, 0.999997427326)0.877546563227, 422.946344022, -0.655545267111
3given(-0.00367696385103, -0.999990882703, 0.002171278033), (0.999984472357, -0.00366783064087, 0.00419548124988), (-0.00418747911833, 0.00218667095106, 0.999988841682)423.268984527, -0.568722216457, 0.580584044201

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Components

#1: Protein
Inositol 1,4,5-trisphosphate receptor type 3 / IP3 receptor isoform 3 / InsP3R3 / Type 3 inositol 1 / 4 / 5-trisphosphate receptor / Type 3 InsP3 receptor


Mass: 304488.688 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ITPR3 / Production host: Homo sapiens (human) / References: UniProt: Q14573
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-I3P / D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE


Mass: 420.096 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H15O15P3
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY

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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: Human Type 3 IP3 Receptor / Type: COMPLEX
Details: In the presence of saturating IP3, ATP, and Ca2+ titrated from 1 nM to 10 um
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.2 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: 120 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.02% LMNG, 2 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, 2 mM BAPTA, 2 mM HEDTA, 1 mM ATP, 500 uM fluorinated fos-choline-8, 3 mM free Mg2+, 1-10000 nM free Ca2+
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K
Details: Custom-made calcium free blotting paper (see publication for details).

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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: 29000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 700 nm / Calibrated defocus max: 4300 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 6 / Num. of real images: 17733
Details: Images were collected at 0.826 A/px magnification on an FEI Krios with Gatan K3 detector at 15 e-/pix/sec with 3 sec exposure (0.05 sec/frame) for a total dose of 66 e-/A2 in automated ...Details: Images were collected at 0.826 A/px magnification on an FEI Krios with Gatan K3 detector at 15 e-/pix/sec with 3 sec exposure (0.05 sec/frame) for a total dose of 66 e-/A2 in automated fashion using SerialEM. Five datasets were collected during the same session for each Ca2+ concentration on a series of grids that were prepared sequentially resulting in 637 movies at 1 nM, 2150 movies at 10 nM, 6126 movies at 100 nM, 1372 movies at 1 uM, and 3136 movies at 10 uM. A sixth dataset of 4312 movies collected at nominal 100 nM free Ca2+ from a grid prepared later in the sequence was collected as a technical replicate to assess experimental error.

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
9PHENIXdev_4761:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 186210 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Map to Model FSC
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.81 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001973644
ELECTRON MICROSCOPYf_angle_d0.452999580
ELECTRON MICROSCOPYf_chiral_restr0.034211428
ELECTRON MICROSCOPYf_plane_restr0.003412652
ELECTRON MICROSCOPYf_dihedral_angle_d3.12779672
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DDELECTRON MICROSCOPYNCS constraints1.10143433543E-12
ens_1d_3DDELECTRON MICROSCOPYNCS constraints4.22675908552E-10
ens_1d_4DDELECTRON MICROSCOPYNCS constraints1.60771838589E-12

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