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- EMDB-6127: Negative stain random conical tilt reconstructions of E. coli rib... -

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Basic information

Entry
Database: EMDB / ID: EMD-6127
TitleNegative stain random conical tilt reconstructions of E. coli ribosomal 30S subunit assembly intermediates
Map dataGroup III 30S assembly intermediate from wild-type E. coli (Figure 2C)
Sample
  • Sample: Group III 30S ribosomal subunit assembly intermediate from wild-type E. coli
  • Complex: 30S assembly intermediate
KeywordsRibosome assembly / 30S subunit / Assembly intermediate
Biological speciesEscherichia coli BW25113 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 55.0 Å
AuthorsSashital DG / Greeman CA / Lyumkis D / Potter CS / Carragher B / Williamson JR
CitationJournal: Elife / Year: 2014
Title: A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli.
Authors: Dipali G Sashital / Candacia A Greeman / Dmitry Lyumkis / Clinton S Potter / Bridget Carragher / James R Williamson /
Abstract: Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory ...Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3' domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3'-domain is unanchored and the 5'-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.
History
DepositionOct 8, 2014-
Header (metadata) releaseOct 22, 2014-
Map releaseOct 22, 2014-
UpdateDec 3, 2014-
Current statusDec 3, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6127.gif

Downloads & links

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Map

FileDownload / File: emd_6127.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGroup III 30S assembly intermediate from wild-type E. coli (Figure 2C)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.05 Å/pix.
x 224 pix.
= 459.29 Å		2.05 Å/pix.
x 224 pix.
= 459.29 Å		2.05 Å/pix.
x 224 pix.
= 459.29 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.0504 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.50806522 - 3.5357151
Average (Standard dev.)-0.00480572 (±0.23969674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 459.2896 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.05040178571432.05040178571432.0504017857143
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z459.290459.290459.290
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-1.5083.536-0.005

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Supplemental data

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Sample components

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Entire : Group III 30S ribosomal subunit assembly intermediate from wild-t...

EntireName: Group III 30S ribosomal subunit assembly intermediate from wild-type E. coli
Components
  • Sample: Group III 30S ribosomal subunit assembly intermediate from wild-type E. coli
  • Complex: 30S assembly intermediate

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Supramolecule #1000: Group III 30S ribosomal subunit assembly intermediate from wild-t...

SupramoleculeName: Group III 30S ribosomal subunit assembly intermediate from wild-type E. coli
type: sample / ID: 1000
Details: Group III particles from heterogeneous sample taken from the center of the 30S sucrose gradient peak
Number unique components: 1
Molecular weightTheoretical: 800 KDa
Method: Sedimentation and calculation of MW of known components

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Supramolecule #1: 30S assembly intermediate

SupramoleculeName: 30S assembly intermediate / type: complex / ID: 1 / Name.synonym: 30S ribosomal subunit / Details: Particles from center of 30S sucrose gradient peak / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: SSU 30S, PSR16s
Ref GO0: GO:0005840
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 800 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 7.5
Details: 20 mM Tris, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM 2-mercaptoethanol
StainingType: NEGATIVE
Details: Negative stain grids were prepared by applying the sample (3 uL) to the grid for 1 min, then blotting from the side to remove excess sample. The grid was washed immediately with 3 uL Buffer ...Details: Negative stain grids were prepared by applying the sample (3 uL) to the grid for 1 min, then blotting from the side to remove excess sample. The grid was washed immediately with 3 uL Buffer A, then blotted from the side. Concurrent with blotting, 3 uL of fresh 2% uranyl formate was applied to the grid, then blotted from the side. This step was repeated twice, then the grid was allowed to dry for at least 10 minutes.
GridDetails: C-flat grids (Protochips) with 2 micron diameter holes coated with a thin (2-5 nm) layer of continuous carbon support, plasma-cleaned for 5s
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
TemperatureMin: 298 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 52,000 times magnification using a live image of the power spectrum.
DateMay 27, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 900 / Average electron dose: 20 e/Å2
Details: 450 tilt pairs were collected at -50 and 0 degrees.
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.79 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -50
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

DetailsImage tilt pairs were collected (-50 and 0 degrees) and particle tilt pairs were identified and extracted as two separate stacks. The untilted stack was aligned and classified iteratively, and RCT volumes were created for a single class average by applying alignment parameters to the corresponding tilt pairs.
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 55.0 Å / Resolution method: OTHER / Software - Name: Spider, Appion / Details: RCT reconstruction / Number images used: 502
Final two d classificationNumber classes: 1

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