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- EMDB-17272: Structure of RyR1 obtained from SR vesicles tilt series (EMPIAR-1... -

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Entry
Database: EMDB / ID: EMD-17272
TitleStructure of RyR1 obtained from SR vesicles tilt series (EMPIAR-10452), reprocessed with TomoBEAR
Map datathe nominal apix is 1.8, the calibrated value was found to be 1.7 on that microscope with used mag
Sample
  • Organelle or cellular component: Sarcoplasmic reticulum
KeywordsSR / RyR1 / Ryanodine receptor type 1 / skeletal muscle / sarcoplasmic reticulum / cryo-ET / tomography / StA / subtomogram averaging / tilt series / subnanometer resolution / native membrane / MEMBRANE PROTEIN
Biological speciesOryctolagus cuniculus (rabbit)
Methodsubtomogram averaging / cryo EM / Resolution: 8.9 Å
AuthorsBalyschew N / Yushkevich A / Mikirtumov V / Sanchez RM / Sprink T / Kudryashev M
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)KU 3222/2-1 Germany
German Research Foundation (DFG)KU3222/3-1 Germany
German Research Foundation (DFG)INST 335/588-1 FUGG Germany
Alexander von Humboldt FoundationSofja Kovalevskaja Award to MK Germany
Citation
Journal: Nat Commun / Year: 2023
Title: Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR.
Authors: Nikita Balyschew / Artsemi Yushkevich / Vasilii Mikirtumov / Ricardo M Sanchez / Thiemo Sprink / Mikhail Kudryashev /
Abstract: Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to ...Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR-a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent ("white box") approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.
#1: Journal: bioRxiv / Year: 2023
Title: Streamlined Structure Determination by Cryo-Electron Tomography and Subtomogram Averaging using TomoBEAR
Authors: Balyschew N / Yushkevich A / Mikirtumov V / Sanchez RM / Sprink T / Kudryashev M
History
DepositionMay 3, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17272.png

Downloads & links

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Map

FileDownload / File: emd_17272.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationthe nominal apix is 1.8, the calibrated value was found to be 1.7 on that microscope with used mag
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.7 Å/pix.
x 320 pix.
= 544. Å		1.7 Å/pix.
x 320 pix.
= 544. Å		1.7 Å/pix.
x 320 pix.
= 544. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.827
Minimum - Maximum-1.1972286 - 2.6745455
Average (Standard dev.)-0.000082491395 (±0.15448742)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 544.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17272_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_17272_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17272_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 : Sarcoplasmic reticulum

EntireName: Sarcoplasmic reticulum
Components
  • Organelle or cellular component: Sarcoplasmic reticulum

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Supramolecule #1: Sarcoplasmic reticulum

SupramoleculeName: Sarcoplasmic reticulum / type: organelle_or_cellular_component / 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
Processingsubtomogram averaging
Aggregation statetissue

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Sample preparation

BufferpH: 7.1
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.5 µm / Calibrated defocus min: 3.5 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 52 / Number images used: 5200 / Reference model: emd-10840 / Method: template matching / Software - Name: Dynamo / Details: Dynamo GPU template matching in TomoBEAR
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. relion 4 beta)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 3169
FSC plot (resolution estimation)

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