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- PDB-6wot: Cryo-EM structure of recombinant rabbit Ryanodine Receptor type 1... -

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Basic information

Entry
Database: PDB / ID: 6wot
TitleCryo-EM structure of recombinant rabbit Ryanodine Receptor type 1 mutant R164C in complex with FKBP12.6
Components
  • Peptidyl-prolyl cis-trans isomerase FKBP1B
  • Ryanodine receptor 1
KeywordsTRANSPORT 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 release of sequestered calcium ion into cytosol / terminal cisterna / negative regulation of insulin secretion involved in cellular response to glucose stimulus / ryanodine receptor complex / 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 release of sequestered calcium ion into cytosol / terminal cisterna / negative regulation of insulin secretion involved in cellular response to glucose stimulus / ryanodine receptor complex / 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 / skin development / 'de novo' protein folding / negative regulation of heart rate / negative regulation of phosphoprotein phosphatase activity / FK506 binding / positive regulation of axon regeneration / cellular response to caffeine / intracellularly gated calcium channel activity / outflow tract morphogenesis / organelle membrane / : / toxic substance binding / smooth muscle contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / voltage-gated calcium channel activity / response to vitamin E / 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 / T cell proliferation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / release of sequestered calcium ion into cytosol / regulation of ryanodine-sensitive calcium-release channel activity / Ion homeostasis / sarcoplasmic reticulum membrane / calcium channel complex / regulation of cytosolic calcium ion concentration / cellular response to calcium ion / sarcoplasmic reticulum / muscle contraction / peptidylprolyl isomerase / peptidyl-prolyl cis-trans isomerase activity / calcium ion transmembrane transport / calcium channel activity / response to hydrogen peroxide / intracellular calcium ion homeostasis / Stimuli-sensing channels / Z disc / 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 / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
: / Ryanodine receptor junctional solenoid repeat / Ryanodine receptor, SPRY domain 2 / Ryanodine Receptor TM 4-6 / Ryanodine receptor / Ryanodine receptor, SPRY domain 1 / Ryanodine receptor, SPRY domain 3 / Ryanodine Receptor TM 4-6 / Ryanodine receptor Ryr / RyR domain ...: / Ryanodine receptor junctional solenoid repeat / Ryanodine receptor, SPRY domain 2 / Ryanodine Receptor TM 4-6 / Ryanodine receptor / Ryanodine receptor, SPRY domain 1 / Ryanodine receptor, SPRY domain 3 / Ryanodine Receptor TM 4-6 / Ryanodine receptor Ryr / RyR domain / 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 / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / SPRY domain / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase / Peptidyl-prolyl cis-trans isomerase domain superfamily / Ion transport domain / Ion transport protein / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Ryanodine receptor 1 / Peptidyl-prolyl cis-trans isomerase FKBP1B
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsIyer, K.A. / Hu, Y. / Kurebayashi, N. / Murayama, T. / Samso, M.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR068431 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL133182 United States
Other privateMDA 352845 United States
American Heart Association19POST34430178 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)HSSN26120080001E United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116789 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116790 United States
CitationJournal: 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
DepositionApr 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 5, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 31, 2021Group: Structure summary / Category: em_entity_assembly / Item: _em_entity_assembly.name
Revision 1.2Aug 3, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.page_last / _citation.pdbx_database_id_DOI ..._citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Ryanodine receptor 1
B: Ryanodine receptor 1
C: Ryanodine receptor 1
D: Ryanodine receptor 1
E: Peptidyl-prolyl cis-trans isomerase FKBP1B
F: Peptidyl-prolyl cis-trans isomerase FKBP1B
G: Peptidyl-prolyl cis-trans isomerase FKBP1B
H: Peptidyl-prolyl cis-trans isomerase FKBP1B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,309,88112
Polymers2,309,6198
Non-polymers2624
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40100 Å2
ΔGint-246 kcal/mol
Surface area811470 Å2

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Components

#1: Protein
Ryanodine receptor 1 / / RyR1 / Skeletal muscle calcium release channel / Skeletal muscle ryanodine receptor / Skeletal ...RyR1 / Skeletal muscle calcium release channel / Skeletal muscle ryanodine receptor / Skeletal muscle-type ryanodine receptor / Type 1 ryanodine receptor


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
Peptidyl-prolyl cis-trans isomerase FKBP1B / PPIase FKBP1B / 12.6 kDa FK506-binding protein / FKBP-12.6 / FK506-binding protein 1B / FKBP-1B / ...PPIase FKBP1B / 12.6 kDa FK506-binding protein / FKBP-12.6 / FK506-binding protein 1B / FKBP-1B / Immunophilin FKBP12.6 / Rotamase / h-FKBP-12


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 / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
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
IDNameTypeEntity IDParent-IDSource
1recombinant rabbit Ryanodine Receptor type 1, RyR1, with mutation R164C in complex with FKBP12.6COMPLEX#1-#20MULTIPLE SOURCES
2recombinant rabbit Ryanodine Receptor type 1, RyR1, with mutation R164CCOMPLEX#11RECOMBINANT
3FKBP12.6FKBP1BCOMPLEX#21RECOMBINANT
Molecular weightValue: 2.26 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Oryctolagus cuniculus (rabbit)9986
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK293
23Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPSMOPS1
20.9 MSodium ChlorideNaClSodium chloride1
32 mMDithiothreitolDTT1
42 mMEGTAEGTA1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
4cryoSPARC2.11CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC2.11initial Euler assignment
11cryoSPARC2.11final Euler assignment
13cryoSPARC2.113D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 581283
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 385324 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5TB0

5tb0

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 223.74 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007130801
ELECTRON MICROSCOPYf_angle_d1.0419177967
ELECTRON MICROSCOPYf_chiral_restr0.049820359
ELECTRON MICROSCOPYf_plane_restr0.007323242
ELECTRON MICROSCOPYf_dihedral_angle_d20.117818292

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