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- EMDB-19191: REEL analysis reconstructions of Ryanodine Receptor 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-19191
TitleREEL analysis reconstructions of Ryanodine Receptor 1
Map dataREEL-EM reference reconstruction of RyR1 generated from elastic bright-field images which were extracted from STEM-EELS spectral images.
Sample
  • Complex: Ryanodine Receptor 1
Keywordsstem-eels / eels / reel-em / energy-loss / single-particle / elemental mapping / elastic bright field / reconstructed energy loss / METAL BINDING PROTEIN
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.3 Å
AuthorsPfeil-Gardiner O / Murphy BJ
Funding support Germany, European Union, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
European Research Council (ERC)101116848European Union
CitationJournal: Nat Methods / Year: 2024
Title: Elemental mapping in single-particle reconstructions by reconstructed electron energy-loss analysis.
Authors: Olivia Pfeil-Gardiner / Higor Vinícius Dias Rosa / Dietmar Riedel / Yu Seby Chen / Dominique Lörks / Pirmin Kükelhan / Martin Linck / Heiko Müller / Filip Van Petegem / Bonnie J Murphy /
Abstract: For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and ...For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here we combine electron energy-loss spectroscopy with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, reconstructed electron energy-loss (REEL) analysis, allows a three-dimensional reconstruction of electron energy-loss spectroscopy data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localize abundant elements. We discuss the current limitations of the method and potential future developments.
History
DepositionDec 19, 2023-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19191.png

Downloads & links

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Map

FileDownload / File: emd_19191.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationREEL-EM reference reconstruction of RyR1 generated from elastic bright-field images which were extracted from STEM-EELS spectral images.
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.65 Å/pix.
x 120 pix.
= 438. Å		3.65 Å/pix.
x 120 pix.
= 438. Å		3.65 Å/pix.
x 120 pix.
= 438. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.65 Å
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Contour LevelBy AUTHOR: 0.00548
Minimum - Maximum-0.0021110408 - 0.011709052
Average (Standard dev.)0.00053117075 (±0.0018666589)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 438.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Background subtracted carbon map generated by per-voxel background...

Fileemd_19191_additional_1.map
AnnotationBackground subtracted carbon map generated by per-voxel background fitting and subtraction of gaussian-filtered REEL-EM spectral volumes and summing at the carbon edge.
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Additional map: Background subtracted nitrogen map generated by per-voxel background...

Fileemd_19191_additional_2.map
AnnotationBackground subtracted nitrogen map generated by per-voxel background fitting and subtraction of gaussian-filtered REEL-EM spectral volumes and summing at the nitrogen edge.
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Additional map: Energy-loss map at the carbon edge generated by...

Fileemd_19191_additional_3.map
AnnotationEnergy-loss map at the carbon edge generated by summing REEL-EM maps in the energy range between and 283.2 eV to 287.8 eV. Best displayed with lowpass filtering.
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Additional map: Energy-loss map at the nitrogen K-edge generated by...

Fileemd_19191_additional_4.map
AnnotationEnergy-loss map at the nitrogen K-edge generated by summing REEL-EM volumes in the energy range between and 403.3 eV to 407.9 eV. Best displayed with lowpass filtering.
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Additional map: Energy-loss map at the oxygen K-edge generated by...

Fileemd_19191_additional_5.map
AnnotationEnergy-loss map at the oxygen K-edge generated by summing REEL-EM maps in the energy range between and 540.4 eV to 545.0 eV. Best displayed with lowpass filtering.
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Additional map: Energy-loss map before the carbon K-edge generated by...

Fileemd_19191_additional_6.map
AnnotationEnergy-loss map before the carbon K-edge generated by summing REEL-EM maps in the energy range between and 237.5 eV to 242.1 eV. Best displayed with lowpass filtering.
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Additional map: Carbon halfmap 2. Sum of halfmaps in the...

Fileemd_19191_additional_7.map
AnnotationCarbon halfmap 2. Sum of halfmaps in the energy-loss region of the carbon K-edge.
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Additional map: Carbon halfmap 1. Sum of halfmaps in the...

Fileemd_19191_additional_8.map
AnnotationCarbon halfmap 1. Sum of halfmaps in the energy-loss region of the carbon K-edge.
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Half map: Halfmap 1 from the refinement of the reference reconstruction.

Fileemd_19191_half_map_1.map
AnnotationHalfmap 1 from the refinement of the reference reconstruction.
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Half map: Halfmap 2 from the refinement of the reference reconstruction.

Fileemd_19191_half_map_2.map
AnnotationHalfmap 2 from the refinement of the reference reconstruction.
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Sample components

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Entire : Ryanodine Receptor 1

EntireName: Ryanodine Receptor 1
Components
  • Complex: Ryanodine Receptor 1

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Supramolecule #1: Ryanodine Receptor 1

SupramoleculeName: Ryanodine Receptor 1 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle

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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

Concentration12 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: CEOS CEFID
Details: energy filter used in spectroscopy mode, so without a slit
DetailsThe data were collected as STEM-EELS spectral images.
Image recordingFilm or detector model: DECTRIS ELA (1k x 0.5k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1142 / Average electron dose: 92.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.0 µm / Calibrated defocus min: 0.0 µm / Illumination mode: OTHER / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 80000
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

DetailsSpectral images were segmented into energy-loss micrographs. Micrographs from the zeroloss region were used for reference reconstruction. Energy-loss reconstructions along the full spectrum were reconstructed with the reconstruction parameters determined with the reference reconstruction.
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 24.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0beta) / Number images used: 460651
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta)
Details: Stochastic gradient descent by Relion 3D initial model
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta)
FSC plot (resolution estimation)

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