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- EMDB-8449: Structure of bL17-depleted large ribosomal subunit assembly inter... -

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Basic information

Entry
Database: EMDB / ID: EMD-8449
TitleStructure of bL17-depleted large ribosomal subunit assembly intermediate - Class D4
Map dataClass D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate
Sample
  • Complex: bL17-depleted large ribosomal subunit assembly intermediate - Class D4
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsTan Y / Davis JH / Lyumkis D
Funding support United States, Singapore, 7 items
OrganizationGrant numberCountry
Jane Coffin Childs Foundation post-doctoral fellowship United States
Leona M. and Harry B. Helmsley Charitable Trust Grant2012-PG-MED002 United States
National Institute of Aging K99 transitional awardAG050749 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM053757 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Agency for Science, Technology and Research Singapore Singapore
Simons Foundation349247 United States
CitationJournal: Cell / Year: 2016
Title: Modular Assembly of the Bacterial Large Ribosomal Subunit.
Authors: Joseph H Davis / Yong Zi Tan / Bridget Carragher / Clinton S Potter / Dmitry Lyumkis / James R Williamson /
Abstract: The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as ...The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.
History
DepositionOct 25, 2016-
Header (metadata) releaseNov 16, 2016-
Map releaseDec 14, 2016-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0317
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0317
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8449.png

Downloads & links

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Map

FileDownload / File: emd_8449.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClass D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0317 / Movie #1: 0.0317
Minimum - Maximum-0.056272082 - 0.14997143
Average (Standard dev.)-0.0013506218 (±0.007150554)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0560.150-0.001

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Supplemental data

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Mask #1

Fileemd_8449_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Class D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate -...

Fileemd_8449_additional.map
AnnotationClass D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate - Unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Class D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate -...

Fileemd_8449_half_map_1.map
AnnotationClass D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate - Halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Class D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate -...

Fileemd_8449_half_map_2.map
AnnotationClass D4 bL17-Depleted 50S Ribosomal Biogenesis Intermediate - Halfmap 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 : bL17-depleted large ribosomal subunit assembly intermediate - Class D4

EntireName: bL17-depleted large ribosomal subunit assembly intermediate - Class D4
Components
  • Complex: bL17-depleted large ribosomal subunit assembly intermediate - Class D4

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Supramolecule #1: bL17-depleted large ribosomal subunit assembly intermediate - Class D4

SupramoleculeName: bL17-depleted large ribosomal subunit assembly intermediate - Class D4
type: complex / ID: 1 / Parent: 0
Details: E. coli large ribosome subunit purified from bL17-depleted cells.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: NCM3722 rplQ::cat, pHSL-rplQ

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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
50.0 mMC4H11NO3Tris Hydrochloride
100.0 mMNH4ClAmmonium Chloride
10.0 mMMgCl2Magnesium Chloride
0.5 mMC10H16N2O8Ethylenediaminetetraacetic Acid
6.0 mMC2H6OSBeta-mercaptoethanol2-Mercaptoethanol

Details: Most of the sucrose was removed by spin concentration
GridModel: C-Flat CF-1.2/1.3-4Au / Material: GOLD / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / Details: 3 microliter of the sample was added..
DetailsFraction was obtained directly from a sucrose gradient, and was spin-concentrated in a 100 kDa MW-cutoff filter.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsIn order to account for highly preferred orientation of the specimen, data was acquired using tilts ranging from 10-60 degrees, in addition to untilted images at 0 degrees. The CTF was estimated on a per-particle basis to account for the gradient of CTF values across individual micrographs
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 2 / Number real images: 2479 / Average exposure time: 10.0 sec. / Average electron dose: 34.27 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 447017
Details: 84028 particles were initially picked with DoG picker. These particles were put through 2D classification. Good class 2D class averages then served as templates for FindEM. FindEM picked a ...Details: 84028 particles were initially picked with DoG picker. These particles were put through 2D classification. Good class 2D class averages then served as templates for FindEM. FindEM picked a total of 447017 particles.
CTF correctionSoftware: (Name: CTFFIND (ver. 3), CTFTILT, Gctf (ver. 0.5), Appion)
Startup modelType of model: INSILICO MODEL
In silico model: Ab initio model was produced from the 2D class averages using Optimod in the Appion pipeline.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: Xmipp
Final 3D classificationNumber classes: 4 / Avg.num./class: 6600 / Software - Name: FREALIGN (ver. 9.07)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.07)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.07) / Number images used: 5343
FSC plot (resolution estimation)

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