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- EMDB-16556: 21S ribosomal precursors induced by heat shock. -

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Basic information

Entry
Database: EMDB / ID: EMD-16556
Title21S ribosomal precursors induced by heat shock.
Map data21S cryo-EM map, postprocessed and masked
Sample
  • Complex: 21S ribosomal precursors induced by heat shock.
    • Complex: 16S ribosomal RNA
    • Complex: 21S Ribosomal proteins
Keywords21S / DnaK / Heat shock / Ribosome assembly / RIBOSOME
Biological speciesEscherichia coli str. K-12 substr. MG1655 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsGiudice E / Georgeault S / Lavigne R / Pineau C / Trautwetter A / Ermel G / Blanco C / Gillet R
Funding support France, 2 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-14-ASTR-0001 France
Other privateFRM EQU202203014663
CitationJournal: Int J Mol Sci / Year: 2023
Title: Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.
Authors: Emmanuel Giudice / Sylvie Georgeault / Régis Lavigne / Charles Pineau / Annie Trautwetter / Gwennola Ermel / Carlos Blanco / Reynald Gillet /
Abstract: Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out ...Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.
History
DepositionJan 26, 2023-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16556.png

Downloads & links

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Map

FileDownload / File: emd_16556.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation21S cryo-EM map, postprocessed and masked
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 400 pix.
= 444. Å		1.11 Å/pix.
x 400 pix.
= 444. Å		1.11 Å/pix.
x 400 pix.
= 444. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.11 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.047801584 - 0.15277559
Average (Standard dev.)0.00035005197 (±0.0042180666)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 444.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16556_msk_1.map
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Mask #2

Fileemd_16556_msk_2.map
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Mask #3

Fileemd_16556_msk_3.map
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Additional map: 21S cryo-EM map (not postprocessed)

Fileemd_16556_additional_1.map
Annotation21S cryo-EM map (not postprocessed)
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Additional map: multibody analysis : 21S Body first half map

Fileemd_16556_additional_2.map
Annotationmultibody analysis : 21S Body first half map
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Additional map: multibody analysis : 21S Body, postprocessed and masked

Fileemd_16556_additional_3.map
Annotationmultibody analysis : 21S Body, postprocessed and masked
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Additional map: multibody analysis : 21S Body (not postprocessed)

Fileemd_16556_additional_4.map
Annotationmultibody analysis : 21S Body (not postprocessed)
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Additional map: multibody analysis : 21S Body second half map

Fileemd_16556_additional_5.map
Annotationmultibody analysis : 21S Body second half map
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Additional map: multibody analysis : 21S Head (not postprocessed)

Fileemd_16556_additional_6.map
Annotationmultibody analysis : 21S Head (not postprocessed)
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Additional map: multibody analysis : 21S Head, postprocessed and masked

Fileemd_16556_additional_7.map
Annotationmultibody analysis : 21S Head, postprocessed and masked
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Additional map: multibody analysis : 21S Head first half map

Fileemd_16556_additional_8.map
Annotationmultibody analysis : 21S Head first half map
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Additional map: multibody analysis : 21S Head second half map

Fileemd_16556_additional_9.map
Annotationmultibody analysis : 21S Head second half map
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Half map: 21S first half map

Fileemd_16556_half_map_1.map
Annotation21S first half map
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Half map: 21S second half map

Fileemd_16556_half_map_2.map
Annotation21S second half map
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Sample components

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Entire : 21S ribosomal precursors induced by heat shock.

EntireName: 21S ribosomal precursors induced by heat shock.
Components
  • Complex: 21S ribosomal precursors induced by heat shock.
    • Complex: 16S ribosomal RNA
    • Complex: 21S Ribosomal proteins

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Supramolecule #1: 21S ribosomal precursors induced by heat shock.

SupramoleculeName: 21S ribosomal precursors induced by heat shock. / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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Supramolecule #2: 16S ribosomal RNA

SupramoleculeName: 16S ribosomal RNA / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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Supramolecule #3: 21S Ribosomal proteins

SupramoleculeName: 21S Ribosomal proteins / type: complex / ID: 3 / Parent: 1
Details: uS3 uS4 uS5 bS6 uS7 uS8 uS9 uS10 uS11 uS12 uS13 uS14 uS15 bS16 uS17 bS18 uS19 bS20
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
5.0 mMC8H19N2O5SKHEPES-KOH
50.0 mMKClKCl
9.0 mMMgOAcMgOAc
10.0 mMNH4ClNH4Cl
1.0 mMC4H10O2S2Dithiothreitol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa / Details: 30mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK III / Details: blot for 2s before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 922 / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.7 µm / Calibrated defocus min: 0.4 µm / Calibrated magnification: 120443 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0023 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
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

Particle selectionNumber selected: 174502
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4v4q


Details: Only the 30S portion was used, and low-pass filtered to a resolution of 40A.
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 48873
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0.6)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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