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- EMDB-11072: Reconstruction of bovine Ryanodine receptor 2 dimer of tetramers ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11072
TitleReconstruction of bovine Ryanodine receptor 2 dimer of tetramers in complex with FKBP12.6 and nanobodies
Map data
Sample
  • Complex: Dimer of bovine Ryanodine receptor 2
Function / homology
Function and homology information


Stimuli-sensing channels / suramin binding / establishment of protein localization to endoplasmic reticulum / type B pancreatic cell apoptotic process / Purkinje myocyte to ventricular cardiac muscle cell signaling / Ion homeostasis / regulation of SA node cell action potential / regulation of atrial cardiac muscle cell action potential / left ventricular cardiac muscle tissue morphogenesis / regulation of AV node cell action potential ...Stimuli-sensing channels / suramin binding / establishment of protein localization to endoplasmic reticulum / type B pancreatic cell apoptotic process / Purkinje myocyte to ventricular cardiac muscle cell signaling / Ion homeostasis / regulation of SA node cell action potential / regulation of atrial cardiac muscle cell action potential / left ventricular cardiac muscle tissue morphogenesis / regulation of AV node cell action potential / positive regulation of ATPase-coupled calcium transmembrane transporter activity / calcium-induced calcium release activity / regulation of ventricular cardiac muscle cell action potential / ventricular cardiac muscle cell action potential / positive regulation of sequestering of calcium ion / cyclic nucleotide binding / embryonic heart tube morphogenesis / negative regulation of release of sequestered calcium ion into cytosol / negative regulation of insulin secretion involved in cellular response to glucose stimulus / cardiac muscle hypertrophy / ryanodine-sensitive calcium-release channel activity / neuronal action potential propagation / response to muscle activity / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / insulin secretion / calcium ion transport into cytosol / response to redox state / negative regulation of heart rate / positive regulation of heart rate / negative regulation of phosphoprotein phosphatase activity / FK506 binding / positive regulation of axon regeneration / cellular response to caffeine / protein kinase A regulatory subunit binding / smooth endoplasmic reticulum / protein kinase A catalytic subunit binding / positive regulation of the force of heart contraction / : / detection of calcium ion / smooth muscle contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / response to vitamin E / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / response to glucose / protein peptidyl-prolyl isomerization / T cell proliferation / chaperone-mediated protein folding / 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 / sarcoplasmic reticulum membrane / cellular response to epinephrine stimulus / calcium channel complex / regulation of cytosolic calcium ion concentration / response to muscle stretch / regulation of heart rate / sarcoplasmic reticulum / peptidylprolyl isomerase / response to organic substance / peptidyl-prolyl cis-trans isomerase activity / establishment of localization in cell / response to hydrogen peroxide / intracellular calcium ion homeostasis / Z disc / : / positive regulation of cytosolic calcium ion concentration / transmembrane transporter binding / response to hypoxia / calmodulin binding / signaling receptor binding / intracellular membrane-bounded organelle / calcium ion binding / membrane / identical protein binding / cytoplasm
Similarity search - Function
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 ...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 / EF-hand calcium-binding domain profile. / Ion transport domain / Ion transport protein / EF-hand domain / 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 speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsWillegems K / Efremov R
Funding support Belgium, 2 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)131261 Belgium
Research Foundation - Flanders (FWO)G.0266.15N Belgium
CitationJournal: To Be Published
Title: Rapid purification and reconstitution into lipid nanodiscs of cardiac Ryanodine Receptor using nanobodies.
Authors: Willegems K / Efremov RG
History
DepositionMay 23, 2020-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11072.png

Downloads & links

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Map

FileDownload / File: emd_11072.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.049299493 - 0.09309337
Average (Standard dev.)0.0023231034 (±0.00980667)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 560.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z560.000560.000560.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0490.0930.002

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Supplemental data

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Mask #1

Fileemd_11072_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_11072_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_11072_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer of bovine Ryanodine receptor 2

EntireName: Dimer of bovine Ryanodine receptor 2
Components
  • Complex: Dimer of bovine Ryanodine receptor 2

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Supramolecule #1: Dimer of bovine Ryanodine receptor 2

SupramoleculeName: Dimer of bovine Ryanodine receptor 2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Complex of Ryanodine receptor 2 with auxiliary protein FKBP12.6 and high affinity nanobody
Source (natural)Organism: Bos taurus (cattle) / Organ: heart / Tissue: ventricle / Organelle: Sarcoplasmic reticulum / Location in cell: Sarcoplasmic reticulum membrane
Molecular weightTheoretical: 2.2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMTris2-Amino-2-(hydroxymethyl)-1,3-propanediol
50.0 mMHEPES4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
200.0 mMNaClSodium chloridesodium chloride
5.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
0.06 %fOMfluorinated octyl maltoside
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE / Support film - #1 - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293.15 K / Instrument: GATAN CRYOPLUNGE 3
Details: .Grid was blotted manually from the back in CP3 cryoplunge (Gatan) for 2s.
DetailsThe RyR2 tetrameric channels formed channel-dimers with the nanobody at the dimer-interface bound to the repeat12 domain of the individual channels

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 4088 / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 149707
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: Initial model was generated in sx_rviper of sphire package
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 9977
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5go9

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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