|Entry||Database: EMDB / ID: EMD-6127|
|Title||Negative stain random conical tilt reconstructions of E. coli ribosomal 30S subunit assembly intermediates|
|Map data||Group III 30S assembly intermediate from wild-type E. coli (Figure 2C)|
|Keywords||Ribosome assembly / 30S subunit / Assembly intermediate|
|Biological species||Escherichia coli BW25113 (bacteria)|
|Method||single particle reconstruction / negative staining / Resolution: 55.0 Å|
|Authors||Sashital DG / Greeman CA / Lyumkis D / Potter CS / Carragher B / Williamson JR|
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_6127.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Annotation||Group III 30S assembly intermediate from wild-type E. coli (Figure 2C)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.0504 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire : Group III 30S ribosomal subunit assembly intermediate from wild-t...
|Entire||Name: Group III 30S ribosomal subunit assembly intermediate from wild-type E. coli|
-Supramolecule #1000: Group III 30S ribosomal subunit assembly intermediate from wild-t...
|Supramolecule||Name: 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 weight||Theoretical: 800 KDa|
Method: Sedimentation and calculation of MW of known components
-Supramolecule #1: 30S assembly intermediate
|Supramolecule||Name: 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 GO||divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...|
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kGO3A00058 40ampajax1 classpoptr giGO000584 0ispandiv
|Source (natural)||Organism: Escherichia coli BW25113 (bacteria)|
|Molecular weight||Theoretical: 800 KDa|
|Processing||single particle reconstruction|
|Buffer||pH: 7.5 |
Details: 20 mM Tris, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM 2-mercaptoethanol
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.
|Grid||Details: 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|
|Vitrification||Cryogen name: NONE / Instrument: OTHER|
|Microscope||FEI TECNAI SPIRIT|
|Electron beam||Acceleration voltage: 120 kV / Electron source: LAB6|
|Electron optics||Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.79 µm / Nominal magnification: 52000|
|Sample stage||Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -50|
|Temperature||Min: 298 K|
|Alignment procedure||Legacy - Astigmatism: Objective lens astigmatism was corrected at 52,000 times magnification using a live image of the power spectrum.|
|Date||May 27, 2013|
|Image recording||Category: 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 max||0|
Model: Tecnai Spirit / Image courtesy: FEI Company
|CTF correction||Details: Each micrograph|
|Final two d classification||Number classes: 1|
|Final reconstruction||Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 55.0 Å / Resolution method: OTHER / Software - Name: Spider, Appion / Details: RCT reconstruction / Number images used: 502|
|Details||Image 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.|
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