[English] 日本語
Yorodumi- PDB-6wot: Cryo-EM structure of recombinant rabbit Ryanodine Receptor type 1... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6wot | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of recombinant rabbit Ryanodine Receptor type 1 mutant R164C in complex with FKBP12.6 | ||||||||||||||||||||||||
Components |
| ||||||||||||||||||||||||
Keywords | TRANSPORT PROTEIN/ISOMERASE / Ryanodine receptor / Calcium channel / Mutation / NTDA mutation / RyR1 / malignant hyperthermia / central core disease / TRANSPORT PROTEIN-ISOMERASE complex | ||||||||||||||||||||||||
Function / homology | Function and homology information ATP-gated ion channel activity / positive regulation of sequestering of calcium ion / cyclic nucleotide binding / negative regulation of insulin secretion involved in cellular response to glucose stimulus / terminal cisterna / ryanodine receptor complex / negative regulation of release of sequestered calcium ion into cytosol / ryanodine-sensitive calcium-release channel activity / neuronal action potential propagation / insulin secretion involved in cellular response to glucose stimulus ...ATP-gated ion channel activity / positive regulation of sequestering of calcium ion / cyclic nucleotide binding / negative regulation of insulin secretion involved in cellular response to glucose stimulus / terminal cisterna / ryanodine receptor complex / negative regulation of release of sequestered calcium ion into cytosol / ryanodine-sensitive calcium-release channel activity / neuronal action potential propagation / insulin secretion involved in cellular response to glucose stimulus / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / ossification involved in bone maturation / cell communication by electrical coupling involved in cardiac conduction / response to redox state / protein maturation by protein folding / 'de novo' protein folding / skin development / negative regulation of heart rate / negative regulation of phosphoprotein phosphatase activity / FK506 binding / positive regulation of axon regeneration / cellular response to caffeine / outflow tract morphogenesis / intracellularly gated calcium channel activity / organelle membrane / : / smooth muscle contraction / toxic substance binding / smooth endoplasmic reticulum / negative regulation of ryanodine-sensitive calcium-release channel activity / response to vitamin E / voltage-gated calcium channel activity / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / skeletal muscle fiber development / protein peptidyl-prolyl isomerization / striated muscle contraction / T cell proliferation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / release of sequestered calcium ion into cytosol / Ion homeostasis / regulation of ryanodine-sensitive calcium-release channel activity / sarcoplasmic reticulum membrane / calcium channel complex / muscle contraction / regulation of cytosolic calcium ion concentration / cellular response to calcium ion / sarcoplasmic reticulum / peptidylprolyl isomerase / peptidyl-prolyl cis-trans isomerase activity / calcium ion transmembrane transport / calcium channel activity / response to hydrogen peroxide / Stimuli-sensing channels / sarcolemma / Z disc / intracellular calcium ion homeostasis / disordered domain specific binding / positive regulation of cytosolic calcium ion concentration / protein refolding / protein homotetramerization / transmembrane transporter binding / calmodulin binding / signaling receptor binding / calcium ion binding / ATP binding / identical protein binding / membrane / cytoplasm / cytosol Similarity search - Function | ||||||||||||||||||||||||
Biological species | Oryctolagus cuniculus (rabbit) Homo sapiens (human) | ||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å | ||||||||||||||||||||||||
Authors | Iyer, K.A. / Hu, Y. / Kurebayashi, N. / Murayama, T. / Samso, M. | ||||||||||||||||||||||||
Funding support | United States, 7items
| ||||||||||||||||||||||||
Citation | Journal: Sci Adv / Year: 2020 Title: Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM. Authors: Kavita A Iyer / Yifan Hu / Ashok R Nayak / Nagomi Kurebayashi / Takashi Murayama / Montserrat Samsó / Abstract: Mutations in ryanodine receptors (RyRs), intracellular Ca channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains ...Mutations in ryanodine receptors (RyRs), intracellular Ca channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect. | ||||||||||||||||||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6wot.cif.gz | 5.1 MB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb6wot.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 6wot.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6wot_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 6wot_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 6wot_validation.xml.gz | 403.4 KB | Display | |
Data in CIF | 6wot_validation.cif.gz | 620.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wo/6wot ftp://data.pdbj.org/pub/pdb/validation_reports/wo/6wot | HTTPS FTP |
-Related structure data
Related structure data | 21860MC 6wouC 6wovC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
#1: Protein | Mass: 565737.500 Da / Num. of mol.: 4 / Mutation: R164C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: RYR1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P11716 #2: Protein | Mass: 11667.305 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: FKBP1B, FKBP12.6, FKBP1L, FKBP9, OTK4 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: P68106, peptidylprolyl isomerase #3: Chemical | ChemComp-ZN / Has ligand of interest | N | |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
| |||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 2.26 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) |
| |||||||||||||||||||||||||
Source (recombinant) |
| |||||||||||||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||||||||||||
Buffer component |
| |||||||||||||||||||||||||
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3.49 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4923 |
EM imaging optics | Energyfilter slit width: 20 eV |
-Processing
Software |
| ||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| ||||||||||||||||||||||||||||||||
CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 581283 | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C4 (4 fold cyclic) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 385324 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5TB0 Accession code: 5TB0 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 223.74 Å2 | ||||||||||||||||||||||||||||||||
Refine LS restraints |
|