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- EMDB-47336: Structure of PKA phosphorylated human RyR2-R2474S in the open sta... -

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Basic information

Entry
Database: EMDB / ID: EMD-47336
TitleStructure of PKA phosphorylated human RyR2-R2474S in the open state in the presence of Calmodulin - focused map on xanthine
Map dataStructure of PKA phosphorylated human RyR2-R2474S in the open state in the presence of Calmodulin - focused map on xanthine
Sample
  • Complex: Complex of RyR2-R2474S, Calstabin-2, and Calmodulin
    • Complex: Ryanodine receptor 2
    • Complex: Peptidyl-prolyl cis-trans isomerase FKBP1B (E.C.5.2.1.8), Calmodulin-1
KeywordsRyanodine receptor 2 / calcium channel / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.38 Å
AuthorsMiotto MC / Marks AR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL145473 United States
CitationJournal: Sci Adv / Year: 2022
Title: Structural analyses of human ryanodine receptor type 2 channels reveal the mechanisms for sudden cardiac death and treatment.
Authors: Marco C Miotto / Gunnar Weninger / Haikel Dridi / Qi Yuan / Yang Liu / Anetta Wronska / Zephan Melville / Leah Sittenfeld / Steven Reiken / Andrew R Marks /
Abstract: Ryanodine receptor type 2 (RyR2) mutations have been linked to an inherited form of exercise-induced sudden cardiac death called catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT ...Ryanodine receptor type 2 (RyR2) mutations have been linked to an inherited form of exercise-induced sudden cardiac death called catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT results from stress-induced sarcoplasmic reticular Ca leak via the mutant RyR2 channels during diastole. We present atomic models of human wild-type (WT) RyR2 and the CPVT mutant RyR2-R2474S determined by cryo-electron microscopy with overall resolutions in the range of 2.6 to 3.6 Å, and reaching local resolutions of 2.25 Å, unprecedented for RyR2 channels. Under nonactivating conditions, the RyR2-R2474S channel is in a "primed" state between the closed and open states of WT RyR2, rendering it more sensitive to activation that results in stress-induced Ca leak. The Rycal drug ARM210 binds to RyR2-R2474S, reverting the primed state toward the closed state. Together, these studies provide a mechanism for CPVT and for the therapeutic actions of ARM210.
History
DepositionOct 17, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47336.png

Downloads & links

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Map

FileDownload / File: emd_47336.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of PKA phosphorylated human RyR2-R2474S in the open state in the presence of Calmodulin - focused map on xanthine
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 424.96 Å		0.83 Å/pix.
x 512 pix.
= 424.96 Å		0.83 Å/pix.
x 512 pix.
= 424.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.9906223 - 1.817873
Average (Standard dev.)0.0035103862 (±0.031707898)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 424.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_47336_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_47336_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of RyR2-R2474S, Calstabin-2, and Calmodulin

EntireName: Complex of RyR2-R2474S, Calstabin-2, and Calmodulin
Components
  • Complex: Complex of RyR2-R2474S, Calstabin-2, and Calmodulin
    • Complex: Ryanodine receptor 2
    • Complex: Peptidyl-prolyl cis-trans isomerase FKBP1B (E.C.5.2.1.8), Calmodulin-1

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Supramolecule #1: Complex of RyR2-R2474S, Calstabin-2, and Calmodulin

SupramoleculeName: Complex of RyR2-R2474S, Calstabin-2, and Calmodulin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3, #1-#2

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Supramolecule #2: Ryanodine receptor 2

SupramoleculeName: Ryanodine receptor 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Peptidyl-prolyl cis-trans isomerase FKBP1B (E.C.5.2.1.8), Calmodulin-1

SupramoleculeName: Peptidyl-prolyl cis-trans isomerase FKBP1B (E.C.5.2.1.8), Calmodulin-1
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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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

Concentration2.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
230.0 mMNaClsodium chloride
10.0 mMHEPES
0.4 %CHAPS
1.0 mMEGTA
0.5 mMTCEP
0.001 %DOPC
10.0 mMATP
0.2 mMcAMP
0.65 mMCalcium
0.5 mMXanthine

Details: Xanthine was made fresh to avoid aggregation. Xanthine stock solution was 10 mM in NaOH 0.5 N.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details40 uM of Calmodulin was added to the final sample.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: CryoSPARC ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 109064
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

7ua3


Chain - Source name: PDB / Chain - Initial model type: experimental model

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