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- EMDB-11442: Human pre-40S particle purified using RIO1(kd)-StHA as bait - Str... -

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Basic information

Entry
Database: EMDB / ID: EMD-11442
TitleHuman pre-40S particle purified using RIO1(kd)-StHA as bait - Structural State B, Body
Map data
Samplehuman cytoplasmic late precursor to the small ribosomal subunit, purified using RIO1(kd)-StHA as bait. Structural state A (pre 18S rRNA maturation)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsPlassart L / Shayan R / Plisson-Chastang C
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR 16-CE11-0029 France
CitationJournal: Elife / Year: 2021
Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock.
Authors: Laura Plassart / Ramtin Shayan / Christian Montellese / Dana Rinaldi / Natacha Larburu / Carole Pichereaux / Carine Froment / Simon Lebaron / Marie-Françoise O'Donohue / Ulrike Kutay / ...Authors: Laura Plassart / Ramtin Shayan / Christian Montellese / Dana Rinaldi / Natacha Larburu / Carole Pichereaux / Carine Froment / Simon Lebaron / Marie-Françoise O'Donohue / Ulrike Kutay / Julien Marcoux / Pierre-Emmanuel Gleizes / Celia Plisson-Chastang /
Abstract: Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, ...Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.
History
DepositionJul 24, 2020-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateMay 12, 2021-
Current statusMay 12, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_11442.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 384 pix.
= 399.36 Å
1.04 Å/pix.
x 384 pix.
= 399.36 Å
1.04 Å/pix.
x 384 pix.
= 399.36 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.12156527 - 0.207952
Average (Standard dev.)1.3922728e-05 (±0.004331762)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 399.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z399.360399.360399.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1220.2080.000

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Supplemental data

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Half map: #2

Fileemd_11442_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_11442_half_map_2.map
Projections & Slices
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Sample components

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Entire human cytoplasmic late precursor to the small ribosomal subunit, ...

EntireName: human cytoplasmic late precursor to the small ribosomal subunit, purified using RIO1(kd)-StHA as bait. Structural state A (pre 18S rRNA maturation)
Number of components: 1

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Component #1: protein, human cytoplasmic late precursor to the small ribosomal ...

ProteinName: human cytoplasmic late precursor to the small ribosomal subunit, purified using RIO1(kd)-StHA as bait. Structural state A (pre 18S rRNA maturation)
Recombinant expression: No
MassTheoretical: 1.4 MDa
SourceSpecies: Homo sapiens (human) / Strain: HEK 293

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.6
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 291 K / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 29.4 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 9494

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 276012
3D reconstructionSoftware: RELION / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Input PDB model: 6EK0

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