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- EMDB-18505: Human 80S ribosome structure from pFIB-lamellae reprocessed with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-18505
TitleHuman 80S ribosome structure from pFIB-lamellae reprocessed with TomoBEAR
Map dataFocused refinement of LSU, filtered to local resolution, calibrated apix 1.9
Sample
  • Cell: 80S human ribosome
Keywordscryo-ET / tomography / StA / subtomogram averaging / tilt series / subnanometer resolution / pFIB / FIB-milling / plasma FIB / cell / RIBOSOME
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 6.2 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
DepositionSep 25, 2023-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18505.png

Downloads & links

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Map

FileDownload / File: emd_18505.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused refinement of LSU, filtered to local resolution, calibrated apix 1.9
Voxel sizeX=Y=Z: 1.9 Å
Density
Contour LevelBy AUTHOR: 0.0379
Minimum - Maximum-0.2195296 - 0.28307843
Average (Standard dev.)0.0000009114718 (±0.016452841)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18505_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18505_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18505_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 : 80S human ribosome

EntireName: 80S human ribosome
Components
  • Cell: 80S human ribosome

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Supramolecule #1: 80S human ribosome

SupramoleculeName: 80S human ribosome / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 5.75
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior the PF, the cells media has been replaced with ...Details: 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior the PF, the cells media has been replaced with the complete media with 10% glycerol (v/v)...

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 2.5 µm / Calibrated magnification: 64000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 5.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 179 / Number images used: 179000 / Reference model: EMD-15636 / Method: Template Matching in TomoBEAR / Software - Name: Dynamo
Final 3D classificationSoftware - Name: RELION (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number subtomograms used: 7575
FSC plot (resolution estimation)

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