|Entry||Database: EMDB / ID: EMD-5502|
|Title||Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM|
|Sample||Immature ribosomal small subunit from rimm gene deleted E.coli strain:|
|Keywords||Ribosome biogenesis / 30S subunit assembly / RimM|
|Biological species||Escherichia coli (E. coli)|
|Method||single particle reconstruction / cryo EM / Resolution: 13.1 Å|
|Authors||Guo Q / Goto S / Chen Y / Muto A / Himeno H / Deng H / Lei J / Gao N|
|Citation||Journal: Nucleic Acids Res / Year: 2013|
Title: Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process.
Authors: Qiang Guo / Simon Goto / Yuling Chen / Boya Feng / Yanji Xu / Akira Muto / Hyouta Himeno / Haiteng Deng / Jianlin Lei / Ning Gao /
Abstract: Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo ...Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3'-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3'-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3'-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_5502.map.gz / Format: CCP4 / Size: 7.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.76 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Immature ribosomal small subunit from rimm gene deleted E.coli strain
|Entire||Name: Immature ribosomal small subunit from rimm gene deleted E.coli strain|
Number of components: 1
|Mass||Theoretical: 800 kDa / Experimental: 800 kDa|
-Component #1: ribosome-prokaryote, Small subunit from rimm gene deleted E.coli ...
|Ribosome-prokaryote||Name: Small subunit from rimm gene deleted E.coli strain / a.k.a: immature 30S / Prokaryote: SSU 30S / Recombinant expression: No|
|Mass||Theoretical: 800 kDa / Experimental: 800 kDa|
|Source||Species: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Buffer solution: 150mM NH4Cl,10mM Tris-HCL,10mM MgCl2 / pH: 7.5|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Method: Blot for 1 seconds before plunging|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Jan 1, 2012|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 80000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 1300 - 4000 nm|
|Specimen Holder||Model: GATAN LIQUID NITROGEN|
|Camera||Detector: GENERIC GATAN (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 30262 |
Details: This is a classification volume (No. 3) using ML3D methods.
Applied symmetry: C1 (asymmetric)
|3D reconstruction||Algorithm: Reference projections / Software: Spider / CTF correction: Weiner filter / Resolution: 13.1 Å / Resolution method: FSC 0.5|
-Atomic model buiding
|Modeling #1||Software: MDFF / Refinement protocol: flexible / Target criteria: Cross-correlation / Refinement space: REAL|
Details: Protocol: Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref: Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible ...Details: Protocol: Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref: Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure, 16, 673-683
Input PDB model: 3OFA
Chain ID: A
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