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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
Keywordsnanodisc / nanobody / FKBP12.6 / RyR2 / MEMBRANE PROTEIN
Function / homology
Function and homology information


cyclic nucleotide binding / 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 / Ion homeostasis / left ventricular cardiac muscle tissue morphogenesis / suramin binding / regulation of AV node cell action potential ...cyclic nucleotide binding / 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 / Ion homeostasis / left ventricular cardiac muscle tissue morphogenesis / suramin binding / regulation of AV node cell action potential / regulation of SA node cell action potential / positive regulation of axon regeneration / regulation of ventricular cardiac muscle cell action potential / ventricular cardiac muscle cell action potential / positive regulation of sequestering of calcium ion / embryonic heart tube morphogenesis / cardiac muscle hypertrophy / negative regulation of insulin secretion involved in cellular response to glucose stimulus / neuronal action potential propagation / negative regulation of release of sequestered calcium ion into cytosol / calcium ion transport into cytosol / ryanodine-sensitive calcium-release channel activity / response to redox state / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / insulin secretion / negative regulation of heart rate / protein peptidyl-prolyl isomerization / cellular response to caffeine / response to vitamin E / negative regulation of ryanodine-sensitive calcium-release channel activity / FK506 binding / response to muscle activity / protein kinase A regulatory subunit binding / protein kinase A catalytic subunit binding / positive regulation of the force of heart contraction / smooth endoplasmic reticulum / regulation of ryanodine-sensitive calcium-release channel activity / smooth muscle contraction / detection of calcium ion / regulation of cytosolic calcium ion concentration / response to glucose / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / T cell proliferation / striated muscle contraction / positive regulation of heart rate / calcium channel inhibitor activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / response to muscle stretch / release of sequestered calcium ion into cytosol / cellular response to epinephrine stimulus / calcium channel complex / sarcoplasmic reticulum membrane / regulation of heart rate / sarcoplasmic reticulum / peptidylprolyl isomerase / peptidyl-prolyl cis-trans isomerase activity / response to hydrogen peroxide / establishment of localization in cell / sarcolemma / Z disc / intracellular calcium ion homeostasis / positive regulation of cytosolic calcium ion concentration / transmembrane transporter binding / calmodulin binding / response to hypoxia / signaling receptor binding / intracellular membrane-bounded organelle / calcium ion binding / identical protein binding / membrane / 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 / 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 / FKBP-type peptidyl-prolyl cis-trans isomerase / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / Peptidyl-prolyl cis-trans isomerase domain superfamily / EF-hand domain pair / EF-hand calcium-binding domain profile. / 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 speciesBos taurus (domestic 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: 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
DepositionMay 23, 2020-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing 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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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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 200 pix.
= 560. Å		2.8 Å/pix.
x 200 pix.
= 560. Å		2.8 Å/pix.
x 200 pix.
= 560. Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
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

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Half map: half map 1

Fileemd_11072_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_11072_half_map_2.map
Annotationhalf map 2
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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 (domestic 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 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
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 149707
Startup modelType of model: OTHER
Details: Initial model was generated in sx_rviper of sphire package
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
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5go9


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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