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- PDB-6wov: Cryo-EM structure of recombinant mouse Ryanodine Receptor type 2 ... -

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Basic information

Entry
Database: PDB / ID: 6wov
TitleCryo-EM structure of recombinant mouse Ryanodine Receptor type 2 wild type in complex with FKBP12.6
Components
  • Peptidyl-prolyl cis-trans isomerase FKBP1B
  • Ryanodine receptor 2
KeywordsTRANSPORT PROTEIN/ISOMERASE / Ryanodine receptor / Calcium channel / RyR2 / CICR / TRANSPORT PROTEIN-ISOMERASE complex / excitation-contraction coupling
Function / homology
Function and homology information


establishment of protein localization to endoplasmic reticulum / manganese ion transmembrane transport / : / type B pancreatic cell apoptotic process / Purkinje myocyte to ventricular cardiac muscle cell signaling / regulation of atrial cardiac muscle cell action potential / left ventricular cardiac muscle tissue morphogenesis / suramin binding / regulation of AV node cell action potential / sarcoplasmic reticulum calcium ion transport ...establishment of protein localization to endoplasmic reticulum / manganese ion transmembrane transport / : / type B pancreatic cell apoptotic process / Purkinje myocyte to ventricular cardiac muscle cell signaling / regulation of atrial cardiac muscle cell action potential / left ventricular cardiac muscle tissue morphogenesis / suramin binding / regulation of AV node cell action potential / sarcoplasmic reticulum calcium ion transport / regulation of SA node cell action potential / calcium-induced calcium release activity / calcium ion transport into cytosol / A band / embryonic heart tube morphogenesis / Stimuli-sensing channels / ventricular cardiac muscle cell action potential / negative regulation of calcium-mediated signaling / cardiac muscle hypertrophy / Ion homeostasis / regulation of ventricular cardiac muscle cell action potential / ryanodine-sensitive calcium-release channel activity / negative regulation of release of sequestered calcium ion into cytosol / response to redox state / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / negative regulation of heart rate / response to caffeine / regulation of cardiac muscle contraction by calcium ion signaling / extrinsic component of cytoplasmic side of plasma membrane / response to muscle activity / cellular response to caffeine / 'de novo' protein folding / FK506 binding / calcium ion transmembrane import into cytosol / protein kinase A regulatory subunit binding / protein kinase A catalytic subunit binding / negative regulation of cytosolic calcium ion concentration / positive regulation of the force of heart contraction / smooth endoplasmic reticulum / response to muscle stretch / intracellularly gated calcium channel activity / regulation of ryanodine-sensitive calcium-release channel activity / response to magnesium ion / detection of calcium ion / regulation of cardiac muscle contraction / regulation of cytosolic calcium ion concentration / positive regulation of heart rate / cardiac muscle contraction / calcium channel inhibitor activity / release of sequestered calcium ion into cytosol / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Ion homeostasis / regulation of heart rate / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / cellular response to epinephrine stimulus / calcium channel complex / sarcoplasmic reticulum membrane / calcium-mediated signaling / peptidylprolyl isomerase / sarcomere / sarcoplasmic reticulum / striated muscle contraction / peptidyl-prolyl cis-trans isomerase activity / calcium channel regulator activity / protein maturation / response to calcium ion / protein refolding / sarcolemma / intracellular calcium ion homeostasis / Stimuli-sensing channels / Z disc / calcium ion transmembrane transport / calcium channel activity / calcium ion transport / nuclear envelope / protein folding / scaffold protein binding / monoatomic ion transmembrane transport / response to hypoxia / transmembrane transporter binding / calmodulin binding / signaling receptor binding / calcium ion binding / protein kinase binding / enzyme binding / protein-containing complex / membrane / identical protein binding / cytoplasm
Similarity search - Function
Ryanodine receptor, SPRY domain 2 / : / Ryanodine receptor junctional solenoid repeat / 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, SPRY domain 2 / : / Ryanodine receptor junctional solenoid repeat / 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 / FKBP-type peptidyl-prolyl cis-trans isomerase / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / Peptidyl-prolyl cis-trans isomerase domain superfamily / EF-hand domain pair / EF-hand domain / Ion transport domain / Ion transport protein / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Ryanodine receptor 2 / Peptidyl-prolyl cis-trans isomerase FKBP1B
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsIyer, K.A. / Hu, Y. / Nayak, A.R. / 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
Revision 1.3May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ryanodine receptor 2
B: Ryanodine receptor 2
C: Ryanodine receptor 2
D: Ryanodine receptor 2
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)2,309,07512
Polymers2,308,8138
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26300 Å2
ΔGint-137 kcal/mol
Surface area743840 Å2

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Components

#1: Protein
Ryanodine receptor 2 / RyR2 / Cardiac muscle ryanodine receptor / Cardiac muscle ryanodine receptor-calcium release ...RyR2 / Cardiac muscle ryanodine receptor / Cardiac muscle ryanodine receptor-calcium release channel / Type 2 ryanodine receptor


Mass: 565536.000 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ryr2 / Production host: Homo sapiens (human) / References: UniProt: E9Q401
#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 mouse Ryanodine Receptor type 2, RyR2, wild type in complex with FKBP12.6COMPLEX#1-#20MULTIPLE SOURCES
2recombinant mouse Ryanodine Receptor type 2, RyR2, wild typeCOMPLEX#11RECOMBINANT
3FKBP12.6COMPLEX#21RECOMBINANT
Molecular weightValue: 2.26 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
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
20.4 MSodium ChlorideNaCl1
32 mMDithiothreitolDTT1
40.02 %Tween 20Tween201
52 mMEGTAEGTA1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was coated with Carbon using a Denton Vacuum Evaporator 502B prior to vitrification.
Grid 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 14 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4470
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17_3644refinement
PHENIX1.17_3644refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
7UCSF Chimeramodel fitting
9RELION1.4initial Euler assignment
10SPIDER17.05final Euler assignment
12FREALIGN3D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 240724
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103845 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5L1D

5l1d


Accession code: 5L1D / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 348.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0063127772
ELECTRON MICROSCOPYf_angle_d1.0169172724
ELECTRON MICROSCOPYf_chiral_restr0.04919156
ELECTRON MICROSCOPYf_plane_restr0.006122336
ELECTRON MICROSCOPYf_dihedral_angle_d10.133117272

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