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- EMDB-45107: Local refinement map of mink RyR3 in closed conformation using ma... -

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Basic information

Entry
Database: EMDB / ID: EMD-45107
TitleLocal refinement map of mink RyR3 in closed conformation using mask 1 (FKBP12.6/NTD/Nsol/SPRY/Repeat1&2)
Map dataauto-sharpened map with B-factor=74.9
Sample
  • Complex: mink RyR3 + FKBP12.6
KeywordsRyR3 / Ryanodine Receptor / calcium channel / ion channel / MEMBRANE PROTEIN
Biological speciesNeogale vison (American mink)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsChen YS / Van Petegem F
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-159601 Canada
Fonds de Recherche du Quebec - Sante (FRQS)BF7-310936 Canada
Other governmentRT-2023-3133
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM investigation of ryanodine receptor type 3.
Authors: Yu Seby Chen / Maricela Garcia-Castañeda / Maria Charalambous / Daniela Rossi / Vincenzo Sorrentino / Filip Van Petegem /
Abstract: Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in ...Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development. RyR3 has a unique functional profile with a very high sensitivity to activating ligands, enabling high gain in Ca-induced Ca release. Here we solve high-resolution cryo-EM structures of RyR3 in non-activating and activating conditions, revealing structural transitions that occur during channel opening. Addition of activating ligands yields only open channels, indicating an intrinsically high open probability under these conditions. RyR3 has reduced binding affinity to the auxiliary protein FKBP12.6 due to several sequence variations in the binding interface. We map disease-associated sequence variants and binding sites for known pharmacological agents. The N-terminal region contains ligand binding sites for a putative chloride anion and ATP, both of which are targeted by sequence variants linked to epileptic encephalopathy.
History
DepositionMay 29, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45107.png

Downloads & links

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Map

FileDownload / File: emd_45107.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationauto-sharpened map with B-factor=74.9
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 512 pix.
= 478.72 Å		0.94 Å/pix.
x 512 pix.
= 478.72 Å		0.94 Å/pix.
x 512 pix.
= 478.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.935 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0768
Minimum - Maximum-0.45016876 - 0.69072706
Average (Standard dev.)0.00055825786 (±0.012204805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 478.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45107_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened raw map

Fileemd_45107_additional_1.map
Annotationunsharpened raw map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2

Fileemd_45107_half_map_1.map
Annotationhalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1

Fileemd_45107_half_map_2.map
Annotationhalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mink RyR3 + FKBP12.6

EntireName: mink RyR3 + FKBP12.6
Components
  • Complex: mink RyR3 + FKBP12.6

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Supramolecule #1: mink RyR3 + FKBP12.6

SupramoleculeName: mink RyR3 + FKBP12.6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Neogale vison (American mink)
Molecular weightTheoretical: 2.25 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

Concentration5 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 250 mM NaCl, 2 mM DTT, 0.02% GDN, 5 mM EGTA, 1:1000 diluted protease inhibitor cocktail
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: blotted for 3 s (blot force of 7 to 10) using ashless blotting paper (Whatman).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 10545 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
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: 201998
Startup modelType of model: INSILICO MODEL
In silico model: Generated by stochastic gradient descent using ab initio reconstruction job type in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Details: using C4 symmetry expanded particles / Number images used: 438472
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
FSC plot (resolution estimation)

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