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- EMDB-43077: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in ... -

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Entry
Database: EMDB / ID: EMD-43077
TitleCryo-EM structure of the Mycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP) (Structure 6)
Map dataMycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP). Map
Sample
  • Complex: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP) (Structure 6)
Keywordscryo-EM / mycobacteria / hibernation / Msmeg1130 / Balon / MsmegEF-Tu / RIBOSOME
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsRybak MY / Helena-Bueno K / Hill CH / Melnikov SV / Gagnon MG
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136936 United States
Welch FoundationH-2032-20230405 United States
Other governmentJames W. McLaughlin Postdoctoral Fellowship
CitationJournal: Nature / Year: 2024
Title: A new family of bacterial ribosome hibernation factors.
Authors: Karla Helena-Bueno / Mariia Yu Rybak / Chinenye L Ekemezie / Rudi Sullivan / Charlotte R Brown / Charlotte Dingwall / Arnaud Baslé / Claudia Schneider / James P R Connolly / James N Blaza / ...Authors: Karla Helena-Bueno / Mariia Yu Rybak / Chinenye L Ekemezie / Rudi Sullivan / Charlotte R Brown / Charlotte Dingwall / Arnaud Baslé / Claudia Schneider / James P R Connolly / James N Blaza / Bálint Csörgő / Patrick J Moynihan / Matthieu G Gagnon / Chris H Hill / Sergey V Melnikov /
Abstract: To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage. In bacteria, two families of ...To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.
History
DepositionDec 8, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43077.png

Downloads & links

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Map

FileDownload / File: emd_43077.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP). Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 512 pix.
= 435.2 Å		0.85 Å/pix.
x 512 pix.
= 435.2 Å		0.85 Å/pix.
x 512 pix.
= 435.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.37194967 - 0.7777852
Average (Standard dev.)0.0010366343 (±0.03046054)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 435.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43077_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: Mycobacterium smegmatis 70S ribosome in complex with hibernation...

Fileemd_43077_additional_1.map
AnnotationMycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP). Sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mycobacterium smegmatis 70S ribosome in complex with hibernation...

Fileemd_43077_half_map_1.map
AnnotationMycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP). Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mycobacterium smegmatis 70S ribosome in complex with hibernation...

Fileemd_43077_half_map_2.map
AnnotationMycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP). Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in ...

EntireName: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP) (Structure 6)
Components
  • Complex: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP) (Structure 6)

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Supramolecule #1: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in ...

SupramoleculeName: Cryo-EM structure of the Mycobacterium smegmatis 70S ribosome in complex with hibernation factor Msmeg1130 (Balon) and MsmegEF-Tu(GDP) (Structure 6)
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 2.6 MDa

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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
Details: 20 mM HEPES-KOH, 60 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 295 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 10161 / Average electron dose: 40.12 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 101909
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)

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Atomic model buiding 1

Initial modelPDB ID:

5o61


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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