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- EMDB-5789: Cryo-electron microscopy of an immature 50S ribosomal subunit (45... -

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Basic information

Entry
Database: EMDB / ID: EMD-5789
TitleCryo-electron microscopy of an immature 50S ribosomal subunit (45S particle) from Bacillus depleted of RbgA (YlqF), conformation 1
Map data3D reconstruction of an immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
Sample
  • Sample: Immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
  • Complex: 45 subunit
KeywordsRibosome assembly / 50S subunit / RbgA / YlqF / GTPase / 45S subunit
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsJomaa A / Jain N / Davis JH / Williamson JR / Britton RA / Ortega J
CitationJournal: Nucleic Acids Res / Year: 2014
Title: Functional domains of the 50S subunit mature late in the assembly process.
Authors: Ahmad Jomaa / Nikhil Jain / Joseph H Davis / James R Williamson / Robert A Britton / Joaquin Ortega /
Abstract: Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages ...Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.
History
DepositionNov 10, 2013-
Header (metadata) releaseDec 25, 2013-
Map releaseJan 8, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5789.tif

Downloads & links

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Map

FileDownload / File: emd_5789.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of an immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 128 pix.
= 325.12 Å		2.54 Å/pix.
x 128 pix.
= 325.12 Å		2.54 Å/pix.
x 128 pix.
= 325.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.54 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-3.86161613 - 11.15494728
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 325.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z325.120325.120325.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-3.86211.1550.000

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Supplemental data

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Sample components

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Entire : Immature 50S ribosomal subunit (45S particle) from Bacillus subti...

EntireName: Immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
Components
  • Sample: Immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
  • Complex: 45 subunit

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Supramolecule #1000: Immature 50S ribosomal subunit (45S particle) from Bacillus subti...

SupramoleculeName: Immature 50S ribosomal subunit (45S particle) from Bacillus subtilis depleted of RbgA (YlqF), conformation 1
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 1.6 MDa

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Supramolecule #1: 45 subunit

SupramoleculeName: 45 subunit / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI
Ribosome-details: ribosome-prokaryote: LSU 50S, LSU RNA 23S, LSU RNA 5S
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: RB419 / Location in cell: cytoplasm
Molecular weightTheoretical: 1.6 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: 10 mM Tris-HCl, pH 7.5, 10 mM magnesium acetate, 60 mM ammonium chloride, 3 mM 2-mercaptoethanol
GridDetails: 400 mesh holey carbon grids with an additional layer (5-10 nm) of thin carbon. Grids were glow discharged at 5 mA for 15 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III
Method: Grids were blotted twice, for 7 seconds each time, before being plunged into liquid ethane.

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureMin: 77 K / Max: 77 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 300,000 times magnification
DateFeb 15, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 300 / Average electron dose: 20 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1 mm / Nominal defocus max: 3.9 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan 914 cryo-holder / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: ctFFIND
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: Xmipp / Number images used: 20655

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