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- PDB-6fg3: Structure of Ryanodine receptor 1 in nanodiscs in the presence of... -

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Basic information

Entry
Database: PDB / ID: 6fg3
TitleStructure of Ryanodine receptor 1 in nanodiscs in the presence of calcium, ATP and ryanodine
ComponentsRyanodine receptor 1
KeywordsMEMBRANE PROTEIN / calcium release channel / nanodiscs
Function / homology
Function and homology information


ATP-gated ion channel activity / terminal cisterna / ryanodine receptor complex / ryanodine-sensitive calcium-release channel activity / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / ossification involved in bone maturation / skin development / cellular response to caffeine / intracellularly gated calcium channel activity / outflow tract morphogenesis ...ATP-gated ion channel activity / terminal cisterna / ryanodine receptor complex / ryanodine-sensitive calcium-release channel activity / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / ossification involved in bone maturation / skin development / cellular response to caffeine / intracellularly gated calcium channel activity / outflow tract morphogenesis / organelle membrane / toxic substance binding / voltage-gated calcium channel activity / skeletal muscle fiber development / release of sequestered calcium ion into cytosol / sarcoplasmic reticulum membrane / cellular response to calcium ion / sarcoplasmic reticulum / muscle contraction / calcium ion transmembrane transport / calcium channel activity / intracellular calcium ion homeostasis / disordered domain specific binding / protein homotetramerization / transmembrane transporter binding / calmodulin binding / calcium ion binding / ATP binding / membrane / identical protein binding
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. / Ion transport domain / Ion transport protein / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Ryanodine receptor 1
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsWillegems, K. / Efremov, R.G.
Funding support Belgium, 2items
OrganizationGrant numberCountry
IWT131261 Belgium
FWOG.0266.15N Belgium
CitationJournal: Structure / Year: 2018
Title: Influence of Lipid Mimetics on Gating of Ryanodine Receptor.
Authors: Katrien Willegems / Rouslan G Efremov /
Abstract: Understanding gating principles of ion channels at high resolution is of great importance. Here we investigate the conformational transition from closed to open state in ryanodine receptor 1 (RyR1) ...Understanding gating principles of ion channels at high resolution is of great importance. Here we investigate the conformational transition from closed to open state in ryanodine receptor 1 (RyR1) reconstituted into lipid nanodiscs. RyR1 is a homotetrameric giant ion channel that couples excitation of muscle cells to fast calcium release from the sarcoplasmic reticulum. Using single-particle cryo-EM we show that RyR1 reconstituted into lipid nanodiscs is stabilized in the open conformation when bound to the plant toxin ryanodine, but not in the presence of its physiological activators, calcium and ATP. Further, using ryanodine binding assays we show that membrane mimetics influence RyR1 transition between closed and open-channel conformations. We find that all detergents, including fluorinated detergents added to nanodiscs, stabilize closed state of RyR1. Our biochemical results correlate with available structural data and suggest optimal conditions for structural studies of RyR1 gating.
History
DepositionJan 9, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 8, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 15, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 10, 2018Group: Data collection / Database references / Structure summary
Category: citation / entity
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _entity.formula_weight
Revision 1.3Nov 6, 2019Group: Data collection / Refinement description / Structure summary
Category: entity / software / Item: _entity.formula_weight / _software.name

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,128,55412
Polymers2,128,1324
Non-polymers4228
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20890 Å2
ΔGint-301 kcal/mol
Surface area863800 Å2
MethodPISA

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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: 532033.062 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Details: Poly Unk stretches correspond to polypeptide regions where the register is not assigned.
Source: (natural) Oryctolagus cuniculus (rabbit) / Strain: New Zealand white rabbit / Tissue: skeletal muscle / References: UniProt: P11716
#2: Chemical
ChemComp-ZN / ZINC ION


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


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

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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: Ryanodine receptor 1 reconstituted in lipid nanodiscs in the presence of calcium, ATP and ryanodine
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 2.2 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Strain: New Zealand white rabbit / Cellular location: sarcoplasmic reticulum / Tissue: skeletal muscle
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM3-morpholinopropane-1-sulfonic acidC7H15NO4S1
2200 mMsodium chlorideNaClSodium chloride1
37 mMadenosine triphosphateC10H16N5O13P31
41 mMDithiothreitolC4H10O2S21
50.2 mMEGTAC14H24N2O101
60.25 mMcalcium chlorideCaCl21
72 mg/mLfluorinated octyl maltosideC20H25F13O111
80.02 mMryanodineC25H35NO91
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharged in ELMO plasma cleaner at current of 3 mA for 1 min.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 293.15 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: 59000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 28 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3078
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096 / Movie frames/image: 7 / Used frames/image: 1-7

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Processing

Software
NameVersionClassification
PHENIX1.12rc2_2821refinement
PHENIX1.12rc2_2821refinement
EM software
IDNameVersionCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7PHENIX1.12model fitting
9PHENIX1.12model refinement
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 75794
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61773 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00447246034396119676
ELECTRON MICROSCOPYf_angle_d0.925838009769163228
ELECTRON MICROSCOPYf_chiral_restr0.048677161676719112
ELECTRON MICROSCOPYf_plane_restr0.0069167650801421496
ELECTRON MICROSCOPYf_dihedral_angle_d9.7948062609571404

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