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- PDB-8rrs: Structure of mouse RyR2 solubilised in detergent in open state in... -

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Basic information

Entry
Database: PDB / ID: 8rrs
TitleStructure of mouse RyR2 solubilised in detergent in open state in complex with Ca2+, ATP, caffeine and Nb9657.
Components
  • Nanobody 9657
  • Ryanodine receptor 2
KeywordsTRANSPORT PROTEIN / Ion channel / Ca2+ / tetramer
Function / homology
Function and homology information


establishment of protein localization to endoplasmic reticulum / 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 / regulation of SA node cell action potential / : / Stimuli-sensing channels ...establishment of protein localization to endoplasmic reticulum / 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 / regulation of SA node cell action potential / : / Stimuli-sensing channels / regulation of ventricular cardiac muscle cell action potential / ventricular cardiac muscle cell action potential / Ion homeostasis / positive regulation of sequestering of calcium ion / embryonic heart tube morphogenesis / cardiac muscle hypertrophy / calcium ion transport into cytosol / ryanodine-sensitive calcium-release channel activity / response to caffeine / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / response to redox state / calcium ion transmembrane import into cytosol / response to muscle activity / protein kinase A regulatory subunit binding / protein kinase A catalytic subunit binding / cellular response to caffeine / positive regulation of the force of heart contraction / intracellularly gated calcium channel activity / detection of calcium ion / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / smooth endoplasmic reticulum / positive regulation of heart rate / response to muscle stretch / sarcoplasmic reticulum membrane / cellular response to epinephrine stimulus / calcium channel complex / regulation of heart rate / sarcoplasmic reticulum / sarcomere / establishment of localization in cell / calcium-mediated signaling / calcium ion transmembrane transport / calcium channel activity / Z disc / intracellular calcium ion homeostasis / calcium ion transport / response to hypoxia / calmodulin binding / calcium ion binding / protein kinase binding / enzyme binding / protein-containing complex / identical protein binding / membrane
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 / 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 / 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
ADENOSINE-5'-TRIPHOSPHATE / CAFFEINE / Ryanodine receptor 2
Similarity search - Component
Biological speciesVicugna pacos (alpaca)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi, C. / Efremov, R.G.
Funding support Belgium, European Union, 3items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
European Research Council (ERC)726436European Union
CitationJournal: J Biol Chem / Year: 2024
Title: Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.
Authors: Chenyao Li / Katrien Willegems / Tomasz Uchański / Els Pardon / Jan Steyaert / Rouslan G Efremov /
Abstract: Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of ...Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.
History
DepositionJan 23, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ryanodine receptor 2
B: Nanobody 9657
C: Ryanodine receptor 2
D: Nanobody 9657
E: Ryanodine receptor 2
F: Ryanodine receptor 2
G: Nanobody 9657
I: Nanobody 9657
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,325,87324
Polymers2,322,6468
Non-polymers3,22716
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein / Antibody , 2 types, 8 molecules ACEFBDGI

#1: Protein
Ryanodine receptor 2 / RYR-2 / RyR2 / Cardiac muscle ryanodine receptor / Cardiac muscle ryanodine receptor-calcium ...RYR-2 / 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 natural source / Source: (natural) Mus musculus (house mouse) / References: UniProt: E9Q401
#2: Antibody
Nanobody 9657


Mass: 15125.495 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 16 molecules

#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-CFF / CAFFEINE / 3,7-DIHYDRO-1,3,7-TRIMETHYL-1H-PURINE-2,6-DIONE


Mass: 194.191 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H10N4O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Mouse ryanodine receptor 2 complex with nanobodyCOMPLEX#1-#20MULTIPLE SOURCES
2Mouse ryanodine receptor 2COMPLEX#11NATURAL
3Nanobody 9657COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Vicugna pacos (alpaca)30538
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3.0particle selectiontemplate picking was used for particle picking
4RELION4.1CTF correction
10cryoSPARC3initial Euler assignment
12RELION3.1classification
13cryoSPARC4.313D reconstruction
Image processingDetails: The movies were selected after MotionCor correction and CTF refinement with the parameters: total drift less than 30 angstrom and resolution better than 5 angstrom
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 791664
Details: Particles were picked by template picking in Cryosparc
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76852 / Symmetry type: POINT

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