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- EMDB-33117: Subtomogram average of 70S ribosome (30S aligned) -

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Basic information

Entry
Database: EMDB / ID: EMD-33117
TitleSubtomogram average of 70S ribosome (30S aligned)
Map data
Sample
  • Complex: 70S ribosome (30S aligned)
Keywords70S / bacterial / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 6.5 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_33117.png

Downloads & links

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Map

FileDownload / File: emd_33117.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 200 pix.
= 432. Å		2.16 Å/pix.
x 200 pix.
= 432. Å		2.16 Å/pix.
x 200 pix.
= 432. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.6
Minimum - Maximum-2.6668684 - 10.523778
Average (Standard dev.)-0.000006134973 (±0.6345521)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33117_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_33117_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33117_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 70S ribosome (30S aligned)

EntireName: 70S ribosome (30S aligned)
Components
  • Complex: 70S ribosome (30S aligned)

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Supramolecule #1: 70S ribosome (30S aligned)

SupramoleculeName: 70S ribosome (30S aligned) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: DH5alpha

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration1.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMHEPES-KOHHEPES-KOH
10.0 mMMgCl2Magnesium chloride
30.0 mMNH4ClAmmonium chloride
150.0 mMKClPotassium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 2100F
DetailsMicroscope was actually a JEOL JEM-F200
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 2.1 sec. / Average electron dose: 5.4 e/Å2
Details: Tilt series were recorded in parallel using beam image shift
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 40.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number subtomograms used: 10540
ExtractionNumber tomograms: 25 / Number images used: 53982 / Software - Name: crYOLO
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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