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- EMDB-51177: Nsa-Tap Pre-60S particle - cluster 3, treated -

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Basic information

Entry
Database: EMDB / ID: EMD-51177
TitleNsa-Tap Pre-60S particle - cluster 3, treated
Map data
Sample
  • Complex: Nucleolar pre-60S particle
Keywordspre-60S / Complex / biogenesis / RIBOSOME
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsGrundmann L / Haselbach D / Bergler H / Grishkovskaya I / Valentin VV
Funding support Austria, 1 items
OrganizationGrant numberCountry
Austrian Science Fund Austria
CitationJournal: Nat Commun / Year: 2024
Title: The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation.
Authors: Lisa Kofler / Lorenz Grundmann / Magdalena Gerhalter / Michael Prattes / Juliane Merl-Pham / Gertrude Zisser / Irina Grishkovskaya / Victor-Valentin Hodirnau / Martin Vareka / Rolf ...Authors: Lisa Kofler / Lorenz Grundmann / Magdalena Gerhalter / Michael Prattes / Juliane Merl-Pham / Gertrude Zisser / Irina Grishkovskaya / Victor-Valentin Hodirnau / Martin Vareka / Rolf Breinbauer / Stefanie M Hauck / David Haselbach / Helmut Bergler /
Abstract: The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of ...The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.
History
DepositionJul 26, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51177.png

Downloads & links

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Map

FileDownload / File: emd_51177.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 448 pix.
= 479.36 Å		1.07 Å/pix.
x 448 pix.
= 479.36 Å		1.07 Å/pix.
x 448 pix.
= 479.36 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.112
Minimum - Maximum-0.016516546 - 2.4414825
Average (Standard dev.)0.001623921 (±0.026252467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 479.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51177_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_51177_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_51177_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Nucleolar pre-60S particle

EntireName: Nucleolar pre-60S particle
Components
  • Complex: Nucleolar pre-60S particle

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Supramolecule #1: Nucleolar pre-60S particle

SupramoleculeName: Nucleolar pre-60S particle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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
20.0 mMHEPES/KOHHEPES
10.0 mMKClPotassium Chloride
2.5 mMMGCl2Magnesium Dichloride
1.0 mMEGTAEthylenediaminetetraacetic acid
1.0 mMDTTDithiothreitol
0.5 mMPMSFphenylmethylsulfonyl fluoride
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2000 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 74 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 7595 / Average electron dose: 60.0 e/Å2
Details: 3608 exposures for the usnic acid treated dataset and 3987 exposures collected for the untreated dataset.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
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: 1120165
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1.) / Number images used: 16408
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4.4.1.)
Final 3D classificationSoftware - Name: cryoDRGN (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial rigid body fitting was done using ChimeraX
RefinementProtocol: FLEXIBLE FIT

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