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- EMDB-3300: Electron cryo-microscopy of a human pre-ribosomal (pre-40S) particle -

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Basic information

Entry
Database: EMDB / ID: EMD-3300
TitleElectron cryo-microscopy of a human pre-ribosomal (pre-40S) particle
Map dataReconstruction of human pre-40S particle purified by using a HASt-tagged version of the ribosome biogenesis factor LTV1 as bait. the map has been filtered and masked using the RELION1.3 postprocess option.
Sample
  • Sample: pre-40S particles purified from human cells by using the tagged ribosome biogenesis factor HASt_LTV1 as bait.
  • Complex: cytoplasmic precursor of the human small ribosomal subunit
Keywordsribosome / small subunit precursor / human cells
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.4 Å
AuthorsLarburu N / Montellese C / O'Donohue M-F / Kutay U / Gleizes P-E / Plisson-Chastang C
CitationJournal: Nucleic Acids Res / Year: 2016
Title: Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing.
Authors: Natacha Larburu / Christian Montellese / Marie-Françoise O'Donohue / Ulrike Kutay / Pierre-Emmanuel Gleizes / Célia Plisson-Chastang /
Abstract: Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects ...Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3'-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.
History
DepositionJan 14, 2016-
Header (metadata) releaseJan 27, 2016-
Map releaseSep 21, 2016-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3300.tif

Downloads & links

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Map

FileDownload / File: emd_3300.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human pre-40S particle purified by using a HASt-tagged version of the ribosome biogenesis factor LTV1 as bait. the map has been filtered and masked using the RELION1.3 postprocess option.
Voxel sizeX=Y=Z: 2.27 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.04256002 - 0.22263312
Average (Standard dev.)0.00185307 (±0.01161947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 435.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.272.272.27
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z435.840435.840435.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0430.2230.002

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Supplemental data

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Sample components

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Entire : pre-40S particles purified from human cells by using the tagged r...

EntireName: pre-40S particles purified from human cells by using the tagged ribosome biogenesis factor HASt_LTV1 as bait.
Components
  • Sample: pre-40S particles purified from human cells by using the tagged ribosome biogenesis factor HASt_LTV1 as bait.
  • Complex: cytoplasmic precursor of the human small ribosomal subunit

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Supramolecule #1000: pre-40S particles purified from human cells by using the tagged r...

SupramoleculeName: pre-40S particles purified from human cells by using the tagged ribosome biogenesis factor HASt_LTV1 as bait.
type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightTheoretical: 1.5 MDa

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Supramolecule #1: cytoplasmic precursor of the human small ribosomal subunit

SupramoleculeName: cytoplasmic precursor of the human small ribosomal subunit
type: complex / ID: 1 / Name.synonym: pre-40S particle / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Embryonic Kidney / Cell: HEK 293
Molecular weightTheoretical: 1.5 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

Concentration0.02 mg/mL
BufferpH: 7.6 / Details: 10 mM Tris-HCl, 100 mM KCl, 2 mM MgCl2
GridDetails: Glow discharged Quantifoil R2/1 on Cu 300 Mesh grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: LEICA EM GP / Method: 1.8 - 2.1'' blot before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.02 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 100 K
DateJan 23, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 3812 / Average electron dose: 30 e/Å2
Details: Every image has been acquired in 1s by the direct electron detector (no movies recorded)
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.4 Å / Resolution method: OTHER / Software - Name: RELION1.3 / Number images used: 54436
DetailsParticles were picked with e2boxer.py fro EMAN2 Reconstruction was carried out with Relion 1.3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4v6x

SoftwareName: Chimera
DetailsThe 40S components of pdb model 4V6X were fitted into the pre-40S map by rigid body docking.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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