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- EMDB-33118: In situ subtomogram average of 80S ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-33118
TitleIn situ subtomogram average of 80S ribosome
Map data
Sample
  • Complex: 80S ribosome in situ
Keywordsin situ / 80S / eukaryotic / RIBOSOME
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 8.2 Å
AuthorsEisenstein F / Danev R
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)P20764 Japan
Japan Science and Technology18069571 Japan
Citation
Journal: Nat Methods / Year: 2023
Title: Parallel cryo electron tomography on in situ lamellae.
Authors: Fabian Eisenstein / Haruaki Yanagisawa / Hiroka Kashihara / Masahide Kikkawa / Sachiko Tsukita / Radostin Danev /
Abstract: In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native ...In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.
#1: Journal: Biorxiv / Year: 2022
Title: Parallel cryo electron tomography on in situ lamellae.
Authors: Eisenstein F / Yanagisawa H / Kashihara H / Kikkawa M / Tsukita S / Danev R
History
DepositionMar 24, 2022-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33118.png

Downloads & links

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Map

FileDownload / File: emd_33118.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy AUTHOR: 9.0
Minimum - Maximum-22.806985999999998 - 30.952589
Average (Standard dev.)-0.0011041955 (±1.8979613)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 524.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33118_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Half map: #2

Fileemd_33118_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33118_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : 80S ribosome in situ

EntireName: 80S ribosome in situ
Components
  • Complex: 80S ribosome in situ

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Supramolecule #1: 80S ribosome in situ

SupramoleculeName: 80S ribosome in situ / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: EpH4

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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: 7
GridModel: Homemade / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 80 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 0.5 sec. / Average electron dose: 3.55 e/Å2
Details: Tilt series were recorded in parallel using beam image shift
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number subtomograms used: 5262
ExtractionNumber tomograms: 21 / Number images used: 9447 / Software - Name: crYOLO
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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