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- EMDB-6142: Cryo-EM reconstructions of E. coli ribosomal 30S subunit assembly... -

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Basic information

Entry
Database: EMDB / ID: EMD-6142
TitleCryo-EM reconstructions of E. coli ribosomal 30S subunit assembly intermediates
Map dataReconstruction of Group I intermediate from cryo-EM analysis of affinity-purified 30S assembly intermediates (Frealign model 1 of 4)
Sample
  • Sample: Early (Group I) 30S ribosomal subunit assembly intermediate, class 1
  • Complex: 30S assembly intermediate
KeywordsRibosome assembly / 30S subunit / Assembly intermediate
Biological speciesEscherichia coli BW25113 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 27.6 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
  • Download
  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6142.gif

Downloads & links

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Map

FileDownload / File: emd_6142.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Group I intermediate from cryo-EM analysis of affinity-purified 30S assembly intermediates (Frealign model 1 of 4)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.84 Å/pix.
x 72 pix.
= 348.48 Å		4.84 Å/pix.
x 72 pix.
= 348.48 Å		4.84 Å/pix.
x 72 pix.
= 348.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.05261171 - 2.73427486
Average (Standard dev.)-0.00815924 (±0.22979906)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 348.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.844.844.84
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z348.480348.480348.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-1.0532.734-0.008

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Supplemental data

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Sample components

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Entire : Early (Group I) 30S ribosomal subunit assembly intermediate, class 1

EntireName: Early (Group I) 30S ribosomal subunit assembly intermediate, class 1
Components
  • Sample: Early (Group I) 30S ribosomal subunit assembly intermediate, class 1
  • Complex: 30S assembly intermediate

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Supramolecule #1000: Early (Group I) 30S ribosomal subunit assembly intermediate, class 1

SupramoleculeName: Early (Group I) 30S ribosomal subunit assembly intermediate, class 1
type: sample / ID: 1000
Details: Group I particles from early 30S assembly intermediates affinity-purified from E. coli rimP deletion strain
Number unique components: 1
Molecular weightTheoretical: 500 KDa / Method: 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 / 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: 500 KDa

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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

Concentration0.03 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
GridDetails: C-flat grids (Protochips) with 2 micron diameter holes coated with a thin (2 nm) layer of continuous carbon support, plasma-cleaned for 5s
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 120 K / Instrument: GATAN CRYOPLUNGE 3
Method: Sample was applied to grid for 1 minute, then blotted for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 80 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using a live image of the power spectrum.
DateAug 29, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 1253 / Average electron dose: 33.67 e/Å2
Details: The dose was fractionated over 30 frames (200 ms each) and image frames were aligned using a program generously provided by Yifan Cheng and Xueming Li.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsImage frame sets (30 frames, 200 ms each) were aligned prior to image processing. Particles were selected using DoG picker in Appion. Group I particles were identified following extensive alignment and classification.
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 27.6 Å / Resolution method: OTHER / Software - Name: Xmipp, Frealign
Details: Group I particles were sorted into 4 groups and reconstructed using Frealign 9.
Number images used: 3407

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