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- EMDB-19502: structure of RyR1 in complex with nanobody and FKBP12 -

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Basic information

Entry
Database: EMDB / ID: EMD-19502
Titlestructure of RyR1 in complex with nanobody and FKBP12
Map data
Sample
  • Tissue: RyR1 in complex with nanobody and FKBP12
Keywordsion channel / Ca2+ / tetramer / transport protein / MEMBRANE PROTEIN
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsLi C / Efremov RG
Funding support Belgium, European Union, 3 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
European Research Council (ERC)726436European Union
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
DepositionJan 29, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19502.png

Downloads & links

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Map

FileDownload / File: emd_19502.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.49 Å/pix.
x 336 pix.
= 500.64 Å		1.49 Å/pix.
x 336 pix.
= 500.64 Å		1.49 Å/pix.
x 336 pix.
= 500.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.49 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.0560617 - 1.874798
Average (Standard dev.)0.0061778873 (±0.08495265)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 500.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19502_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_19502_half_map_1.map
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Half map: #1

Fileemd_19502_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : RyR1 in complex with nanobody and FKBP12

EntireName: RyR1 in complex with nanobody and FKBP12
Components
  • Tissue: RyR1 in complex with nanobody and FKBP12

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Supramolecule #1: RyR1 in complex with nanobody and FKBP12

SupramoleculeName: RyR1 in complex with nanobody and FKBP12 / type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7tzc

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 29246
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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