|Entry||Database: EMDB / ID: 6134|
|Title||Negative stain random conical tilt reconstructions of E. coli ribosomal 30S subunit assembly intermediates|
|Map data||Group II 30S assembly intermediate from E. coli rimP deletion strain (volume 5 Figure 5D)|
|Sample||Group II 30S assembly intermediate from E. coli rimP deletion strain (volume 5 of 10):|
|Keywords||Ribosome assembly / 30S subunit / Assembly intermediate|
|Source||Escherichia coli BW25113 (bacteria)|
|Method||single particle reconstruction / negative staining / 55 Å resolution|
|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
|Date||Deposition: Oct 8, 2014 / Header (metadata) release: Oct 22, 2014 / Map release: Oct 22, 2014 / Last update: Dec 3, 2014|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6134.map.gz (map file in CCP4 format, 43905 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.0504 Å|
CCP4 map header:
-Entire Group II 30S assembly intermediate from E. coli rimP deletion str...
|Entire||Name: Group II 30S assembly intermediate from E. coli rimP deletion strain (volume 5 of 10)|
Details: Group II particles from heterogeneous sample taken from the center of the 30S sucrose gradient peak of E. coli rimP deletion strain lysate
Number of components: 1
|Mass||Theoretical: 800 kDa|
Measured by: Sedimentation and calculation of MW of known components
-Component #1: ribosome-prokaryote, 30S assembly intermediate
|Ribosome-prokaryote||Name: 30S assembly intermediate|
a.k.a: 30S ribosomal subunitProkaryotic small ribosomal subunit
Details: Particles from center of 30S sucrose gradient peak / Prokaryote: SSU 30S, PSR16s / Recombinant expression: No
|Mass||Theoretical: 800 kDa|
|Source||Species: Escherichia coli BW25113 (bacteria)|
|External references||Gene Ontology: ribosome|
|Specimen||Specimen state: particle / Method: negative staining|
|Sample solution||Specimen conc.: 0.015 mg/ml|
Buffer solution: 20 mM Tris, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM 2-mercaptoethanol
|Support film||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|
|Staining||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.|
|Vitrification||Instrument: NONE / Cryogen name: NONE|
-Electron microscopy imaging
Model: Tecnai Spirit / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI SPIRIT / Date: Apr 22, 2013|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 52000 X (nominal), 52000 X (calibrated)|
Astigmatism: Objective lens astigmatism was corrected at 52,000 times magnification using a live image of the power spectrum.
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - 2500 nm
|Specimen Holder||Model: SIDE ENTRY, EUCENTRIC / Tilt Angle: -50 - 0 deg. / Temperature: K ( 298 - K)|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Image acquisition||Number of digital images: 1360|
Details: 680 tilt pairs were collected at -50 and 0 degrees.
|Processing||Method: single particle reconstruction / Number of class averages: 1 / Applied symmetry: C1 (asymmetric) / Number of projections: 544|
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.
|3D reconstruction||Algorithm: Random-conical tilt reconstruction / Software: Spider, Appion / CTF correction: Each micrograph / Details: RCT reconstruction / Resolution: 55 Å / Resolution method: FSC 0.5, semi-independent|
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